“Revisiting the past”: a redescription of Physaloptera retusa (Nemata, Physalopteridae) from material deposited in museums and new material from Amazon lizards

Abstract Physaloptera Rudolphi, 1819 is a genus of nematodes that includes approximately 100 species parasitic in vertebrates around the world. From these, approximately 30 occur in the Neotropical region, with nine reported from neotropical reptiles. Physaloptera spp. are recognized by their distinct morphology of the apical end and characters of the reproductive system. However, despite the fact that the morphological characters for species diagnosis have been firmly established, we frequently find identification problems regarding poorly detailed descriptions and poorly preserved specimens. These may lead to taxonomic incongruencies. Physaloptera retusa (Rudolphi, 1819) is the most common species of the genus and has been reported from several species of neotropical reptiles. Based on our reexaminations of nematode specimens identified as P. retusa from different museum collections, we provide a detailed redescription including the type material, voucher specimens and new specimens recovered currently and showed in this study with new morphological data obtained using light and scanning electron microscopy tools.

Physaloptera spp. identification is based mainly on the number and disposition of teeth, the relative position of the excretory pore and deirids, the relative position of the vulva in females, the pattern and number of caudal papillae and the shape and size of the spicules in males (Ortlepp, 1922;Morgan, 1943;Skrjabin & Sobolev, 1964;Esteban et al., 1995;Lopes-Torres et al., 2009). However, many species in this genus were inaccurately or superficially described during the taxonomic history of the genus. The differential diagnosis of species was based on different morphological characters in the past years, which caused difficulties to establish which are taxonomically informative (Alves et al., 2022). Furthermore, problems regarding insufficiently detailed descriptions (especially in species described more than a century ago, limited by the technology of the time) and studies based on poorly preserved specimens, presenting technical artifacts, may lead to inaccurate identifications (Pereira et al., 2012(Pereira et al., , 2014Davis et al., 2016).
Physaloptera retusa (Rudolphi, 1819) was described in Tupinambis teguixin (Linnaeus, 1758) (Squamata, Teiidae) from Brazil. Then, Ortlepp (1922) redescribed P. retusa including material obtained from a specimen of T. teguixin found dead in the Gardens of the London Zoological Society. Alves et al. (2022) recently studied this species using material collected in the State of Minas Gerais, Brazil, and voucher specimens from the Helminthological Collection of the Oswaldo Cruz Institute. However, there is no recent studies regarding the morphology of type specimens.
We present a morphological redescription of P. retusa, indicating complementary characters for species differentiation, based on observations of the type series, and specimens identified as P. retusa deposited in different museum collections, and from new material obtained in this study using both light and scanning electron microscopy.

Materials and Methods
We collected 28 nematodes from 15 specimens of Ameiva ameiva Linnaeus, 1758 (commonly known as Giant Ameiva or Amazon Racerunner) from the "Osvaldo Rodrigues da Cunha" herpetological collection of Emílio Goeldi Museum (MPEG) collected from Caxiuanã National Forest (Flona Caxiuanã), Melgaço Municipality, Pará, Brazil. The hosts were previously fixed in 3% formaldehyde and stored in 70% ethanol and were dissected at the Laboratory of Cell Biology and Helminthology from the Federal University of Pará (LBCH/ICB/UFPA). The nematodes collected were stored in A.F.A solution (2% acetic acid, 3% formaldehyde and 95% alcohol 70%) and transferred to alcohol 70% after 24 hours.

Remarks
The redescription of P. retusa by Alves et al. (2022) agrees with the description by Rudolphi (1819) regarding the spicules' morphology and the caudal papillae distribution pattern. However, the male specimens described by Alves et al. (2022) Alves, Couto & Pereira). Additionally, both the morphological and morphometric data of the specimens collected in this study from Ameiva ameiva agree with our observations of the type material of P. retusa.
Among the didelphic species group of the genus Physaloptera, 19 species were reported from Neotropical hosts and only 9 are parasitic in reptiles, namely: P. bainae; P. bonnie; P. liophis; P. lutzi; P. monodens; P. nordestina Matias, Morais & Ávila, 2020; P. obtusissima; P. retusa and P. tupinambae. Therefore, we compared our observations of type material of P. retusa only with the other parasitic species occurring in neotropical reptiles.
In comparison to P. retusa, P. lutzi is the most different regarding the morphology of oral structures (with a variable number of spikes in both inner and outer teeth), the position of the vulva (on the posterior third of the body, corresponding to 95% of the body length) and the length of spicules (the right spicule has half the length of the left, with a ratio of 1:1.8-2.0). Recently Alves et al. (2022) redescribed P. lutzi from T. torquatus and our observations of this species are congruent with these authors.
Physaloptera retusa also differs from P. bonnei and P. liophis by the vulva position (both subequatorial, 40% and 54.2% of body length respectively vs. 16% in P. retusa) and from P. liophis by the length of spicules (420-470 right and 410-490 left in P. retusa vs. 250 right and 260 left in P. liophis).
Physaloptera retusa can be easily differentiated from P. bainae and P. tupinambae when comparing the number of male caudal papillae; these are the only two species parasitic in neotropical reptiles with more than 21 papillae (23 and 22 respectively vs. 21 in P. retusa). Based on the morphology of oral structures, P. bainae is easily differentiated by having an outer tooth with four small spines in a cross-shaped pattern; and P. tupinambae differs by the presence of a bipartite internal tooth, while in P. retusa the outer tooth is triangular, and the inner tooth is tripartite.
Physaloptera retusa can be differentiated from P. monodens and P. obtusissima by the spicules' length and the oral structures' morphology. The spicules are larger in P. retusa (420-470 right and 410-490 left) than in P. monodens (362 right and 415 left) and P. obtusissima (385 right and 430 left); and the outer tooth in these species is conical, while that in P. retusa is triangular in shape.
Physaloptera retusa differs from P. nordestina by the shape of the outer tooth (triangular in P. retusa vs. conical in P nordestina), length of the spicules (420-470 right and 410-490 left in P. retusa vs. 195-376 right and 257-436 left in P. nordestina) and vulva ratio (16% of the body length in P. retusa vs. 5-26% of the body length in P. nordestina).
Physaloptera mucronata Leidy, 1856 was described based on specimens collected from Melanosuchus niger (Spix) (Alligatoridae) in Brazil (Diesing, 1851). The species was reported in Alligator mississippiensis (Daudin) (Alligatoridae) from the United States by Leidy (1856), which was considered synonymous with P. retusa (Walton, 1927). However, the species was renamed to Ascaris lanceolata by Molin (1860b) and posteriorly redescribed. Subsequently, the species was assigned to the genus Terranova by Sprent (1979) and, most recently, reassigned to Neoterranova lanceolata (Molin) by Moravec & Justine (2020). Thus, we did not compare this species with P. retusa. Pereira et al. (2012) suggested that the determination of the number of caudal papillae in the males of P. retusa in the study of Vicente et al. (1993) were quite different compared to the original description of Rudolphi (1819) and the subsequent studies of Ortlepp (1922) and Skrjabin & Sobolev (1964), which may have led and may in the future, lead to additional misidentifications. These references are essential keys for the taxonomic identification of P. retusa. Thus, researchers using these references for species identification should be careful.
We also observed differences regarding the morphology of P. liophis. This species is closely related to P. retusa but differ mainly by the number of caudal papillae (23 papillae in total, 8 pedunculate, and 15 sessile in P. liophis vs. 21 papillae in total, 8 pedunculate and 13 sessile in P. retusa). However, we did not observe these extra papillae in the type material of P. liophis, and according to the illustrations provided in the original description of P. liophis, we hypothesized that the authors probably included the phasmidial pores along with caudal papillae. Thus, we consider that P. liophis has 21 papillae (8 pedunculate and 13 sessile) instead of the 23 papillae previously indicated (Pereira et al., 2012(Pereira et al., , 2014. Also, the inner tooth morphology, not mentioned in the original description, remains unknown, and we could not observe it because of the poor preservation quality of the specimens.
Additional morphological and morphometric data of Physaloptera spp. parasites of reptiles from Neotropics are presented in Table 2.

Discussion
The presence of a cephalic collar at the anterior extremity, two lateral pseudolips, with an external tooth and an internal tripartite tooth, and the pattern of papillae of the male caudal region, namely, a caudal bursa ornamented with 21 caudal papillae are the main characters of the genus Physaloptera (Rudolphi, 1819;Ortlepp, 1922;Skrjabin & Sobolev, 1964;Chabaud, 2009). This genus includes more than 100 species widely distributed globally, of which several remain insufficiently described, hampering comparisons and species differentiation (Pereira et al., 2012(Pereira et al., , 2014. Physaloptera spp. females have a variable number of uterine branches. Ortlepp (1922) highlighted that this might be an essential character for species identification. Thus, several authors separated these species into groups according to the type of uteri: didelphic (two branches); tridelphic (three branches), or tetradelphic (four branches) (Ortlepp,1922;Ortlepp, 1937;Morgan, 1943;Skrjabin & Sobolev, 1964;Chabaud, 2009). In the Neotropical region, species of Physaloptera parasitic in reptiles typically have only two uterine branches. Thus, we compared our specimens with the 9 species of the didelphic group of these nematodes parasitic in Neotropical reptiles. Our scanning electron microscopy (SEM) analysis revealed ultrastructural details of important characters for species diagnosis. Using this method, we confirmed the details of both the inner and outer tooth morphology and the distribution of caudal papillae of P. retusa. The use of SEM as a tool for helminth taxonomy has been helping for a better comprehension of the morphology of several Physaloptera spp. (Marchiondo & Sawyer , 1978;Tiekotter, 1981;Mafra & Lanfredi, 1998;Lopes-Torres et al., 2009;Naem & Asadi, 2013;Chen et al., 2017;Ederli et al., 2018;Lopes-Torres et al., 2019Matias et al., 2020). But, studies of physalopterid nematodes using both light microscopy and SEM are scarce (Naem & Asadi, 2013) and we reinforce the fact that further studies of other physalopterid species using SEM may help to define and solidify the real taxonomic value of other characters as suggested by Lopes-Torres et al. (2009).
The naturalist Johann Natterer collected specimens of P. retusa from Cuiabá, Brazil, and later this material was sent to NHMW, and Rudolphi formally described the species in 1819 (see historical summary in Guerrero, 2021). However, the author did not give morphological details for P. retusa, and the species remained insufficiently described until the work of Ortlepp (1922). Our analysis on the type material of this species are congruent with the redescription of Ortlepp (1922) regarding the dimensions of males, the length of spicules, the distribution and pattern of male cloacal papillae, the morphology of the cloacal aperture, and the limits of the caudal bursa.
We observed differences in the morphology of the right spicule (this spicule is maleable, thin and weakly esclerotized, thus its morphology may varies according to the position of the nematode on the slide), as well as the length of the ovijector in females and other morphometric data. These differences might be associated with the limitation of bidimensional analyzes of internal structures with a complex morphological organization and/or intraspecific morphological variation. Lopes-Torres et al., (2019) conducted an integrative study presenting threedimensional information of morphological characters of Physaloptera mirandai Lent & Freitas, 1937. Despite the fact that we did not use three-dimensional techniques, our study includes new morphometric and morphological details of important characters obtained from recently collected specimens and type series that can be used to identify and differentiate P. retusa from its congeners. The combination of multidimensional techniques may help to better comprehend the morphological complexity of important taxonomic characters.
The differences observed between specimens of different hosts (T. teguxin and A. ameiva) might be related to different factors related to hosts, the helminths, and their host-parasite relationships. Some specimens or even species could have more equilibrated host-parasite relationships compared to the others and affect the parasite development. Chitwood (1957) and Haley (1962) listed some factors that may cause intraspecific variations in helminths' morphological and morphometric characters, such as host age, diet, metabolic and physical condition, number of parasites, presence of other parasite species, etc. Also, geographic, and ecological factors could influence species development and cause intraspecific variations (Chitwood, 1957). Some of these factors are difficult to evaluate and remain unstudied for several groups of parasites. Regarding nematode parasites, there are a small number of studies pointing and discussing their morphological variability in different host species and localities exist. Therefore, we think that differences in the physiology and ecology of the hosts and the phenotypic plasticity of the nematode might explain the variations observed.
Physaloptera retusa is the most common species of the genus in neotropical lizards, occurring in several host families Albuquerque et al., 2012). Therefore, all morphological and morphometrical variation observed suggest that P. retusa represents a set of cryptic species (or even a species complex) that still needs to be revised using the integrative taxonomy, exploring a combination of molecular and morphological studies. Pereira et al. (2012) described P. tupinambae with 22 papillae in total, resulting from an extra unpaired papilla in place of the "boss" between the last two sessile papillae. Drawings and photomicrographs provided by the authors indicate the presence of this character. The "boss" located between the last pair of sessile papillae seems to be a typical morphological character for some species, and the males of P. liophis, P. monodens and P. retusa have this structure. Nevertheless, it was not possible to identify an extra papilla in the descriptions of these species, which is congruent with our observations of the papillae distribution and ultrastructural details of the "boss" of P. retusa under SEM. Additionally, studies on Physaloptera spp. from mammals also observed the "boss" in the male caudal region, and this extra unpaired papilla is not present (Norman & Beveridge, 1999). Thus, P. bainae and P. tupinambae are the only species parasitic in reptiles with a different number of caudal papillae. However, additional morphological studies are necessary to confirm this character since it can be easily confused with the rough pattern present in the tail and cloacal aperture of these nematodes.
There are many problems in character definition relative to species delimitation, which may lead to error cascades as also pointed out by Bortolus (2008). Thus, once an error in measurement or interpretation enters the literature stream without correction, inaccurate information may be propagated down through time throughout the literature, which is particularly frequent in parasitology (see Van Bortel et al., 2001;Vink et al., 2012).
Use of archived museum specimens for reexamination of previously described taxa with more detailed descriptions and additional data are still necessary and may help solve taxonomic problems, increasing our knowledge of biodiversity and will improve and establish more accurate species identifications. Therefore, the problems within Physaloptera presented herein demonstrate the necessity of additional studies of both museum collections and new collections in the field that will serve to elucidate species diversity in the genus. Also, integrative approaches combining different sources of information and complementary perspectives are necessary to improve our comprehension of the morphological complexity of Physaloptera species and to improve helminth systematics.