A new species of Temnocephala (Platyhelminthes, Temnocephalida) and a description of T. axenos from Uruguay

In Uruguay, two species of Temnocephala Blanchard, 1849 have been reported on the anomuran crab Aegla Leach, 1920, namely Temnocephala axenos Monticelli, 1899 and Temnocephala talicei Dioni, 1967. A third species, described here as Temnocephala mertoni n. sp., has been found on Aegla platensis Schmitt, 1942 from southern Uruguay. The new species resembles T. talicei, but differs by having a penial stylet with a sinuous distal portion of the shaft; a small introvert with short, distal spines, and a discrete thickening in its wall. The penial stylet of T. mertoni n. sp. is more similar to that of T. axenos. In view of the importance of stylet structure and shape in the taxonomy of this group, a description of T. axenos is included, and a comprehensive study has been carried out to establish the differences between these species. Based upon this study, new characters are proposed for the taxonomy of the genus Temnocephala.

Unlike T. talicei, the taxonomy of T. axenos has proven difficult because of the poor original description by Monticelli (1899). Merton (1922) attempted to give a better description when he described Temnocephala brasiliensis (now a synonym of T. axenos), but he only had access to a single specimen in bad condition, and therefore he only pointed out a few characters to differentiate it from T. brasiliensis. Baer (1931) studied the type material of both T. axenos and T. brasiliensis, so he described and illustrated T. axenos better, and in the same work synonymized T. brasiliensis with the latter. More recently, Pérez-González (1949) described Temnocephala bresslaui, pointing out a few diagnostic characters in order to separate it from T. axenos, but some of these characters have been proven to have no taxonomic value (Dioni 1967a;Damborenea 1991). Finally, Dioni (1967a) synonymized T. bresslaui with T. axenos in the same work in which he described T. talicei.
While searching for material of T. axenos and T. talicei for a study on Uruguayan temnocephalids, a third species was found. This species does not correspond to any of the known species of Temnocephala, and therefore is here described as T. mertoni n. sp. This species resembles T. talicei, but the penial stylets of both species are clearly different. However, the penial stylet of T. mertoni n. sp. is morphometrically similar to that of T. axenos. Therefore, to avoid introducing more confusion in the taxonomy of the latter, a comprehensive study was carried out with material of both species, and therefore a detailed description of Uruguayan material of T. axenos is also given. Based upon the comparison of these species, new characters are proposed for the taxonomy of the species of Temnocephala. Hosts were brought alive to the laboratory, where all temnocephalids were removed with the aid of a stereomicroscope, examined and identified alive under a microscope, recovered and killed with hot Formalin-Acetic acid-Alcohol (FAA) and, after 24 h, transferred to 70% ethanol. Temnocephalid eggs were removed from the surface of the hosts and preserved in 70% ethanol. After removal of temnocephalids and their eggs, hosts were killed and preserved in 70% ethanol.

Specimens of
On Route 8, Km 238, three species of Temnocephala were found, namely T. talicei, T. axenos, and a previously unknown species. In the Colorado Stream only the latter two were found. Given the low number of specimens found at Colorado Stream, the morphometry of T. axenos and of the new species was determined with material from the Route 8 location. Temnocephalids were stained with acetic carmine and fast green, and mounted in Canada balsam. Some of the specimens were whole-mounted; others were dissected with the aid of a stereomicroscope, and mounts of the reproductive complex were made.
Eggs were removed from the uterus of previously identified gravid individuals and distinctive, qualitative characters were determined for the eggs of each species using transitory mounts in lactophenol. Eggs of the new species could not be separated qualitatively from the eggs of T. talicei, and therefore egg morphometry had to be carried out with material from Route 6, Km 35. Eggs taken from the surface of hosts from this location were classified using the forementioned characters, and were measured in water to avoid contraction.
Drawings were made with the aid of a camera lucida. Measurements are given as mean (range, standard deviation, n), in micrometres (mm).
In addition to the Uruguayan material, specimens of T. axenos deposited in the Facultad de Ciencias Naturales y Museo de la Universidad Nacional de La Plata (Argentina) were also examined.
Diagnosis. Small temnocephalid; sucker small, peduncle oblique with respect to body surface; elongate excretory syncytia, wider in area surrounding excretory pore; two paranephrocytes; intestine with central constriction not pronounced, and inconspicuous septa; one asymmetrical sphincter in female portion of atrium; eggs with subapical, almost lateral, medium-sized ornamentation, and opercular plates oblique to longitudinal axis of egg; penial stylet about 140 mm long; straight, with distal portion of shaft sinuous; small introvert with about 10 rows of small, distal spines, and a discrete thickening in its wall.

Anatomical description
Temnocephalid of small size; body elliptic with maximum width at equatorial level (     Testes slightly lobed; two posterolateral to intestine and two larger, posterior to same organ and more central (Figure 1). Anterior and posterior testes of each side connected by short spermatic ducts. Vas efferens originate on inner portion of posterior testes; right long, left short, enter seminal vesicle adjacent to each other (Figures 1, 3 (12.7-16.3; 1.02; 15) maximum width, and 12.3 mm (10.9-12.7; 0.75; 15) minimum width; with about 10 rows of spines; wall with thickening between proximal end and base of proximal spines ( Figure 5). Ratio of body length without tentacles to distance between gonopore and base of tentacles: 1:0.57-0.64 (1:0.61). Gonopore glands surrounding gonopore and ventral to intestine, with conspicuous ducts (Figure 1). Monticelli,  Diagnosis. Large temnocephalid, body rounded; sucker large, with peduncle perpendicular with respect to body surface; elliptical excretory syncytia, small, extends from base of external tentacles to level of anterior portion of intestine; four paranephrocytes; intestine with central constriction pronounced, and conspicuous septa; two sphincters in the distal region of the metraterm, one more proximal, asymmetrical, and one more distal, symmetrical; eggs with subapical, medium-sized ornamentation, and opercular plates perpendicular to longitudinal axis of egg; penial stylet about 140 mm long; curved; small introvert with about 10 rows of small, distal spines.
Testes slightly lobed; two anterior, posterolateral to intestine, and two posterior to same organ and more central, larger (Figure 6). Anterior and posterior testes of each side connected by short spermatic ducts. Vas efferens originate on inner side of posterior testes; right long, left short ( spines ( Figure 10). Ratio of body length without tentacles to distance between gonopore and base of tentacles: 1:0.41-0.50 (1:0.47). Gonopore glands conspicuous, abundant around gonopore, and some ventral to intestine, with short ducts (Figure 6).

Taxonomic affinities
Temnocephala mertoni n. sp. is most similar to T. talicei. Based upon the characters used to distinguish species of Temnocephala, the features shared by both species separating them from T. axenos are the following. Firstly, both species are smaller than T. axenos despite some overlap in length. The length range reported here for T. mertoni n. sp. is 1.0-1.5 mm. Dioni (1967a) reported a range of body length of 1.3-1.5 mm for T. talicei from Uruguay, and, subsequently, Damborenea (1991Damborenea ( , 1992 reported a mean body length of 1.5 mm for Argentinian populations. Body length in T. axenos specimens has been reported to range from 1.1 to 5.0 mm, with most reports ranging from about 1.5 to 3.0 mm (Merton 1922;Baer 1931;Pérez-González 1949;Dioni 1967a;Damborenea 1991Damborenea , 1992; our sample, with an observed range of 1.2-2.7 mm, is in agreement with the latter. Secondly, in both T. talicei and T. mertoni n. sp., the acetabulum is small. The relation of total body length to acetabulum diameter is 6.7 for T. mertoni n. sp. Dioni (1967a) reported a value of 5 for T. talicei, which is in agreement with the measurements provided for Argentinian populations of T. talicei by Damborenea (1991Damborenea ( , 1992. In T. axenos, Dioni (1967a) reported this relation to be 3-4, and Damborenea (1992) reported it to be 3.9; in our sample, it has a value of 3.7. As for qualitative differences, in both T. talicei and T. mertoni n. sp. the intestine has a central constriction that is not as pronounced as in T. axenos, and the penial stylet is straight, whereas in T. axenos the central constriction of the intestine is quite pronounced and the stylet is generally curved. The eggs of T. mertoni n. sp. and T. talicei are qualitatively identical, both having subapical, almost lateral, medium-sized ornamentation, and opercular plates oblique to the longitudinal axis of the egg. Other characters could not be compared, because T. talicei was defined mainly by its distinctive penial stylet, and the original description by Dioni (1967a) lacks further details of the species' anatomy.
Despite the similarities observed, T. mertoni n. sp. can be clearly differentiated from T. talicei by the penial stylet. The stylet in T. mertoni n. sp., with a mean length of 138 mm and a width of 46 mm at the base, is somewhat similar in size to that of T. talicei, with a mean length of about 120 mm, and a width of 50 mm (Dioni 1967a;Damborenea 1992). However, the introverts in both species are completely different. The introvert is larger in T. talicei than in T. mertoni n. sp.; according to the illustration of the penial stylet by Dioni (1967a) the ratio of the length of the introvert to the penial stylet length would be 1:0.3, whereas in T. mertoni n. sp. this ratio is 1:0.2. Furthermore, in T. talicei, the spines extend throughout the whole length of the introvert, whereas in T. mertoni n. sp. they are only present in the distal portion. Dioni (1967a) reported these spines to measure 5-7 mm in length in T. talicei, but in T. mertoni n. sp. most spines measure 4-5 mm, and only the basal ones, which are the longest, measure 6 mm.
On the other hand, the stylet in T. mertoni n. sp. is morphometrically very similar to that of our sample of T. axenos, with a mean length of 141 mm, a width of 42 mm at the base, and a ratio of the length of the introvert to the penial stylet length of 1:0.2. These measurements are in agreement with those of Damborenea (1991Damborenea ( , 1992. What is more, in T. axenos the spines are also present only in the distal portion of the introvert. The differences between both stylets are merely qualitative: in T. axenos the stylet is curved, whereas in T. mertoni n. sp. it is always straight but with a sinuous distal portion of the shaft, and in T. mertoni n. sp. there is a thickening in the introvert wall which is absent in T. axenos. Therefore, in order to separate further these two species, and in addition to the characters mentioned above, the following differences should be considered: in T. axenos the excretory syncytia are elliptical, whereas in T. mertoni n. sp. they are elongate, and wider in the area surrounding the excretory pore; in T. axenos, the intestinal septa are very conspicuous, whereas in T. mertoni n. sp. they are barely visible; T. axenos has four paranephrocytes, and T. mertoni n. sp. has only two; in T. axenos there are two sphincters in the female portion of the atrium, but in T. mertoni n. sp. there is only one. Differences were also found between the eggs of both species, given that in T. axenos the eggs have a short, subterminal ornamentation, and opercular plates perpendicular to the longitudinal axis of the egg.

Discussion
Some aspects of the taxonomy of T. axenos remain unclear. In particular, the reports of the length of the penial stylet are noteworthy: when Pérez-González (1949) described T. bresslaui, which she considered different from T. axenos, she reported stylet lengths of 280-304 mm. Subsequently, Dioni (1967a), considering that T. bresslaui was a synonym of T. axenos, reported stylet lengths of 125-250 mm for Uruguayan material of T. axenos; in a following work (Dioni 1967b), he stated that in populations from Argentina he had only found T. axenos with small stylets. Damborenea (1991Damborenea ( , 1992 reported stylets of around 125 mm in length for other Argentinian populations. Our results are similar to those of Damborenea, because we did not find any stylet longer than 163 mm. This discrepancy in the measurements of the penial stylet among populations of T. axenos casts doubts on the synonymization of T. bresslaui and T. axenos by Dioni (1967a). According to the report of Pérez-González (1949), T. bresslaui would be a species very similar to T. axenos, but with a penial stylet that is twice as long. Ribeiro Amato et al. (2003) recently described a species with those features, Temnocephala cyanoglandula from Aegla serrana Buckup and Rossi, 1977. Therefore, taking into account that T. bresslaui might indeed be a valid species, it would be desirable to make a comparison of material of T. axenos with the type material of T. bresslaui and of T. cyanoglandula, in order to establish the taxonomic status of the latter two species.
Another interesting aspect is that in T. axenos both Merton (1922) and Baer (1931) reported the presence of four seminal receptacles. We observed the same in only 10% of the individuals studied both alive and in dissected animals, whereas the rest of the specimens showed an expansion in the walls of the ootype, exactly where the seminal receptacles would normally be. This expansion was filled with sperm in some individuals, but completely empty in others, so we can confirm that this state does not result from the accumulation of sperm in this area. Pérez-González (1949) mentioned for T. bresslaui that the seminal receptacles were transitory; this statement seems therefore to apply to both T. axenos and T. mertoni n. sp.
The penial stylet remains the main taxonomic character for the identification of species of Temnocephala. However, as we demonstrate here, there are species whose stylet morphometry is similar to the extent that the only detectable differences are qualitative. In this context, it has become evident that in taxonomic descriptions of species of Temnocephala, authors not only must provide an accurate description of the penial stylet, they also must include a detailed morphological study including soft-part characters with taxonomic weight.
The comparison between T. mertoni n. sp., T. talicei, and T. axenos has shown that there are many characters of taxonomic weight which are not normally used in the taxonomy of Temnocephala, and whose inclusion in descriptions would facilitate species identification. We propose that the following characters are taken into account in addition to those already in use: shape and extension of the excretory syncytia, in agreement with Damborenea and Cannon (2001), who were the first to suggest that the excretory syncytia vary among species; presence/absence of conspicuous intestinal septa; number and position of paranephrocytes; number and shape of the sphincters in the female reproductive system; size and position of egg ornamentation, and disposition of opercular plates of the eggs; shape of the penial stylet; number, size, and position of the spines in the introvert; and presence/absence of thickenings in the wall of the introvert. We are confident that the use of these characters will facilitate the identification of species within this genus, in particular those which share their host, as is the case with T. mertoni n. sp., T. axenos, and T. talicei.