Redescription and new records of freshwater Nematomorpha ( Gordiida ) from Chile , with the description of two new species Redescripción y nuevos registros de Nematomorpha ( Gordiida ) de Chile con la descripción de dos nuevas especies

Only four species of Gordiida (Nematomorpha) are known from Chile. This paper describes two new species Gordionus enigmaticus and Gordius austrinus. Gordionus enigmaticus is characterized by the variation in the shape of the areoles along the longitudinal ventral furrow and by the absence of the precloacal fields of bristles at the terminal end. Gordius austrinus can be distinguished from other Gordius species by the presence of the precloacal ledge bounding the cloacal area depression, by the cuticular prominence where the cloacal opening lies and by the distribution pattern of the bristles at the posterior end. Likewise, Gordius paranensis, Gordius robustus and Neochordodes meridionales are ultrastructuraly redescribed and the distribution of each of them in Chile is given.


INTRODUCTION
Gordiida (Nematomorpha) or horsehair worms, as they are commonly called, because of the popular notion that they arise from horse hair falling into water, have some affinities with Nematoda.In the traditional literature the name "Aschelminthes" was used in order to group the Rotifera, Acanthocephala, Nematomorpha, Nematoda, Gastrotricha, Kinorhyncha, Priapulida and Loricifera.Recent ultrastructural and morphological studies and the analyses of 18r DNA gene sequences (Winnepenninckx et al. 1995) suggest that "Aschelminthes" has a poliphyletic origin constituted by three different clades, and should not be considered a valid taxon (Kristensen 1995, Ehlers et al. 1996, Aguinaldo et al. 1997, Garey & Schmidt-Rhaesa 1998).At present, Nematomorpha and Nematoda constitute a sister group that was named Nematoidea by Schmidt-Rhaesa (1996).
To date about 300 species of Gordiida have been described.The taxonomy of this taxon is mainly based in male specimens, on the features of the posterior end and on the cuticular structure of the body wall.The most original descriptions were realised on light microscopy (LM).These descriptions in many cases, fail to give adequate diagnostic characters.New observation techniques, especially the scanning electron microscopy (SEM) allows to analyse fine structural details and have become a standard in Gordiida taxonomy.
The aim of this paper is to widen the information about the biodiversity of the Gordiida of Chile.Three species (Gordius paranensis, Gordius robustus and Neochordodes meridionales) are ultrastructuraly redescribed by SEM and two new species (Gordionus enigmaticus and Gordius austrinus) are described.The distribution of each of them in Chile is given.

MATERIAL AND METHODS
The specimens from Chile are from the collection of the Museum of Zoology, Concepción University (MZUC), Concepción, Chile.Body measurements of 41 specimens (23 male and 19 female) of gordiids were made with outstretched worms using a ruler.Diameters were measured under dissecting microscope using a calliper ruler.For examination by SEM, whole sections were taken from the middle and posterior end of the body.Fragments of all the worms studied were dehydrated in an increasing ethanol series, critical point-dried, mounted on bronze blocks and gold-sputter coated.Observations were performed using a JEOL SLM 1000 scanning electron microscope.enigmaticus n. sp. (Fig.1A,1B,1C,1D,1E) Gordionus enigmaticus n. sp.
Material investigated: holotype (SEM of midbody and posterior end).
Description: body color dark brown.In the anterior end a white cap and a dark collar are lacking.Posterior end bilobed (Fig. 1A).Tail lobes short (31.9 µm in length and 24.4 µm in wide).The cloacal opening is round, circumcloacal spines are completely absent.Numerous conic spines present posterior of the cloacal opening and extending onto the inner side of the tail lobes.Precloacal bristlefields are lacking completely.Specialized areoles called adhesive warts are present ventrolaterally in the midline anterior of the cloacal opening (Fig. 1B).These adhesive warts are oriented along the longitudinal axis of the body at a distance of 0.8 mm of the cloacal opening.They are oval, protuberant and their mid-line is keel-like (23.4 µm high).
The body cuticle is densely structured by areoles (Fig. 1C).The areoles are oval (50.7 µm in length, 36 µm wide and 20 µm high) with a smooth surface.The longitudinal axis of the areoles is parallel to the longitudinal axis of the body.Scattered between the areoles are structures that have a rounded basis with a very small tubercle on top which look like tubercle areoles (Fig. 1C).Along the body, at the level of the ventral furrow the areolar pattern differs, being limited at the mid posterior region by conic areoles (15.8 µm high) (Fig. 1D) while in the mid anterior region it is limited by the same areoles observed in the rest of the cuticle (Fig. 1E).
Dimensions: one male 105 mm length and 0.3 mm width.
Remarks: the presence of a bilobed terminal end, the adhesive warts and body cuticle with one type of areoles make Gordionus enigmaticus belong to the genus Gordionus.G. enigmaticus as described for Gordionus alascensis (Montgomery, 1907) (Schmidt-Rhaesa et al. 2003), G. longareolatus (Montgomery, 1898) (Schmidt-Rhaesa et al. 2003) and Gordionus sinepilosus Schmidt-Rhaesa, Hanelt andReeves, 2003 (Schmidt-Rhaesa et al. 2003) lacks the precloacal fields of bristles.The absence of these bristlefields is an unusual pattern which separates these last four species from other Gordionus species.Gordionus enigmaticus differs from G. alascensis in the presence of bristles or spines on the posterior end and in the absence of a parabolic integumentary ridge anterior of the cloacal opening.The cuticle of G. longareolatus contains scattered and elevated tubercles between the areoles (Montgomery 1898), while in the interareolar furrow of G. sinepilosus numerous bristles are found (Schmidt-Rhaesa et al. 2003).In G. enigmaticus the only interareolar structure is a rounded basis with a very small tubercle on top.These structures remain tubercle areoles of Beatogordius abbreviatus (Villot, 1874) (Schmidt-Rhaesa & De Villalobos 2002).Gordionus enigmaticus is distinguished from other species of the genus by the variation in the shape of the areoles along the longitudinal ventral furrow.Description.Holotype: body color dark brown.In the anterior end a white cap is absent.Distinct white spots are present in the cuticle of the body.Posterior end bilobed, the lobes are moderately long 0.21 mm and 0.12 mm wide and not diverging (Fig. 2A).The apex of the lobes forms a spoon shaped cavity in which small bristles can be observed.Posterior of the cloacal opening is a semicircular postcloacal crescent with a row of bristles on its lateral borders (Fig. 2A).Anterior to the post cloacal crescent there is a depression with a central cuticular protuberance where the cloacal opening lies (Fig. 2A, 2B).The cuticle in this depression is smooth.In the precloacal region there is a parabolic intergumentary ledge which bounds the cloacal depression.In the internal borders of the precloacal ledge and extending onto the inner side of the tail lobes there are bristles unbranched (Fig. 2B).These bristles are longer and more numerous than the bristles on the cuticle of the lateral borders of the lobes (Fig. 2A, 2B).

Gordius austrinus
The body cuticle shows transverse oriented slender folds with scattered short bristles (Fig. 2C).In the other specimens investigated the body color is variable, males from Chillán (MZUC 26324) are light yellowish but the others are dark brown.The anterior end in lightly colored specimens, with a white cap followed dark collar.A dark coloration is present on the postcloacal crescent in lightly colored specimens.Lobes vary (0.26 to 0.49 mm long), (0.16 to 0.25 mm width).The terminal end and body cuticle of the males examined showed the same features than the holotype.At the terminal end of one male (MZUC 26324) there was a sperm mass completely covering the depression where the cloacal opening is found (Fig. 3A, 3B).
Dimensions.Holotype: 263 mm in length and 0.6 mm in diameter.The length of the other 19 specimens measured varies between 133 mm and 438 mm with an average length of 331 mm.The diameters vary between 0.4 mm to 0.6 mm.
Female.The body color of 20 specimens observed ranged from dark brown (Valdivia, Cherchenco and Iquique specimens) to light brown (the other specimens).The white spots of the cuticle are lacking.Only the females with light color have a white calotte and dark collar at the anterior end.The body cuticle is smooth.The terminal end in all the females analyzed is covered by a mass of eggs (Fig. 3C).
Dimensions.Females measurements vary from 245 to 675 mm (average 407 mm) in length and from 0.6 to 1.2 mm in diameter.
Remarks.Gordius austrinus is separated from other Gordius species by the presence of the precloacal ledge bounding the cloacal area depression, by the prominence where the cloacal opening lies and by the distribution patterns of the bristles at the posterior end.Gordius austrinus specimens show variability in some characters.They exhibit differences in body color, varying from light yellow to dark brown, along the body and along the lobes of the terminal end of males.Likewise, the specimens with light color have a white calotte and a dark collar at the anterior end not observed in the dark brown specimens.Females differ from males not only at the posterior end but by the presence of a smooth cuticle without transverse oriented slender folds with scattered small bristles observed in the cuticle of males.This characteristic could be considered as a sexual dimorphism.
Taking into account that the new species as well as the one pointed for G. chilensis has a wide distribution for Chile, these specimens could have been assigned to G. chilensis.Nevertheless, it is very difficult to consider G. chilensis as a valid species due to the insufficient characteristics of its description by Blanchard (1849) as well as to its specimens which unfortunately seem to be lost.
Description.Male: body color brown with white spots on the cuticle.Anterior tip of the body rounded.A white calotte and a dark collar on the anterior end are not present.Posterior end is bilobed (Fig. 4A).Tail lobes are 0.29 mm long and 0.14 mm wide.Cloacal opening is round and without circumcloacal spines.Anterior of the cloacal opening is a semicircular row of bristles.The length of the bristles varies between 8 µm and 19 µm, some of them are bifurcated apically (Fig. 4A and  4B).Posterior of the cloacal opening is a semicircular crescent.Around the cloacal opening the cuticle is smooth.
Body cuticle is smooth and only structured by a rhomboidal pattern, which is due to underlying cuticular fibers arranged in cross layers (Fig. 4C).
Dimensions: 335 mm length and 0.4 mm in diameter.
Remarks.Schmidt-Rhaesa et al. ( 2000) redescribed ultrastructurally for the first time specimens of Gordius paranensis from New Zealand and from Chile.These specimens differ from this new specimen from Salto Quilpué, in the bristles which are arranged in a parabolic row anterior of the cloacal opening and are not furcated apically.This feature is important because no intraspecific variations for G. paranensis have been cited to date.The report of Gordius paranensis from North America by Chandler (1985) results from an erroneous citation of Montgomery (1898) (Schmidt-Rhaesa et al. 2003).The presence of a semicircular row of bristles was pointed by Smith (1994) for Gordius difficilis Smith, 1994 but this species differs from G. paranensis in the body cuticle which contains polygonal areoles and in the postcloacal crescent which is V-shaped.Montgomery (1898) cited this species from the locality of "Casabianca" which we changed to Casablanca, the correct name for Chile.Gordius robustus Leidy 1851 (Fig. 5A, 5B) Gordius robustus Leidy 1851, p. 266.
Description.Body color uniformly dark brown, without white spots.The Body measurement is 453 mm in length and 0.8 mm in diameter.The terminal end is bilobed, tail lobes are 0.32 mm in length and 0.25 mm in wide.The postcloacal crescent is semicircular (Fig. 5A).Body cuticle is smooth with scattered bristles (Fig. 5B).
Remarks.The specimen (one female) from Valparaíso described as Gordius aquaticus by Römers ( 1895 robustus is reported from Neartic region and also from several locations in Central and South America (Miralles 1976, Miralles & De Villalobos 1993, Schmidt-Rhaesa et al. 2003).
Description.Body color dark brown.Anterior end is tapering; without dark collar.Posterior end is rounded (Fig. 6A).Cloacal opening is oval and surrounded by long and unbranched circumcloacal spines.Cloacal opening is situated a long distance from the apex of terminal end (187 µm).Anterolateral of the cloacal opening are two narrow rows of scattered bristles.Cuticle (Fig. 6B) is densely structured by one type of variable size areoles randomly arranged or forming groups of 2, 3, 4 o 5. Generally two larger areoles are close  together and enclose a tubercle (megareolar pattern).Shape of areoles varies from rounded to oval and measure from 15.1 to 19.2 µm in diameter.Areolar surface is smooth.Interareolar furrow wide and covered by cuticular cords transversally arranged respect the corporal axis.Interareolar structures are represented by long tubercles (6.8 µm) and minute fine bristles.
Dimensions: 84 mm length and 0.15 mm in diameter.

DISCUSSION
In Gordiida the taxonomically important characters are almost entirely cuticular structures.These are the structures of the body cuticle and structures at the posterior end, especially in males.Additionally, the general shape of the posterior end is important (De Villalobos & Zanca 2001, Schmidt-Rhaesa 2002).Most of these features are visible with a scanning electron microscope (SEM).Both the coloration pattern and the body length are of questionable value, because these are likely to be variable characters (Schmidt-Rhaesa 1997).We could observe intraspecific variations between specimens of G. austrinus and G. paranenis.As mentioned before, these differences lack taxonomic value although it is important to mention them at the moment of describing a species.(Camerano 1897, Schmidt-Rhaesa 1997) We regard at present seven species to be valid for Chile: Beatogordius latastei, Gordionus enigmaticus, Gordius austrinus, G. paranenis, G. robustus, Neochordodes talensis and N. meridionalis.Taking into account that from the mentioned species de Chile, Beatogordius latastei, G. robustus, Neochordodes talensis and N. meridionalis have been also reported for Argentina (bordering country with Chile), we think that future collections of Gordiida in Chile will allow not only to widen the number of species but also to relate more adequately the Gordiida fauna in both countries.These data together with the data from other South American countries will give more information on the biodiversity of the Gordiida and will also allow to evaluate if a geographic barrier as the Andes could have an important role in Gordiida speciation events.
) must be renamed as Gordius robustus.Although G. aquaticus and G. robustus are characterized by the absence of diagnostic characters (Schmidt-Rhaesa et al. 2003), G. aquaticus is an entirely Paleartic species (see Schmidt-Rhaesa 1997) and G.