Aspidodera kinsellai n. sp. (Nematoda: Heterakoidea) from Nine-Banded Armadillos in Middle America with Notes on Phylogeny and Host–Parasite Biogeography Author(s): F. Agustín Jiménez, Ramón A. Carreno, and Scott L. Gardner Source: Journal of Parasitology, 99(6):1056-1061. 2013. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/13-196.1 URL: http://www.bioone.org/doi/full/10.1645/13-196.1
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J. Parasitol., 99(6), 2013, pp. 1056–1061 Ó American Society of Parasitologists 2013
ASPIDODERA KINSELLAI N. SP. (NEMATODA: HETERAKOIDEA) FROM NINE-BANDED ARMADILLOS IN MIDDLE AMERICA WITH NOTES ON PHYLOGENY AND HOST–PARASITE BIOGEOGRAPHY ´ A. Carreno*, and Scott L. Gardner† F. Agust´ın Jime´nez, Ramon Department of Zoology, Southern Illinois University, Carbondale, Illinois 62901. Correspondence should be sent to: [email protected]
Aspidodera kinsellai n. sp. (Heterakoidea: Aspidoderidae) from the 9-banded armadillo, Dasypus novemcinctus, is herein described. This nematode occurs from Costa Rica north through central Mexico where it can be found causing co-infections with Aspidodera sogandaresi. Aspidodera kinsellai n. sp. can be discriminated from this and all other species in the family based on 3 key features, including (1) conspicuous lateral grooves with no lateral alae starting immediately after the hood and terminating at the cloacal/anal region; (2) long hoods in both male (360 lm) and female (401 lm), and (3) a relatively long (152 lm) terminal spine or terminus that gradually tapers to a point from the last pair of papillae. This is the 18th recognized species of the family and the 3rd in the genus present outside of South America. A phylogenetic analysis of the species in the genus with the use of the mitochondrial partial genes cytochrome C oxidase subunit 1 (cox1), the ribosomal large subunit (rrnL), and the internal transcriber spacer (ITS) shows that 2 species of Aspidodera may have entered into North America from the south via 2 independent events.
The Aspidoderidae Skrjabin and Schikhobalova, 1947 (Ascaridida: Heterakoidea) currently includes 17 species divided among 5 genera (Jime´nez-Ruiz et al., 2008). The worms occur in the cecum and large intestine of mammals with southern Nearctic and general Neotropical distributions. The known host range for species in the family includes xenarthrans, didelphiomorphs, and hystricognath and sigmodontine rodents (Santos et al., 1990; Jime´nez-Ruiz et al., 2008). Species assigned to Aspidodera Railliet and Henry, 1912 are diagnosed by the distinctive structure of the hood on the anteriormost end of the nematode, which has anastomosing cordons. These cordons (Inglis, 1957) are cuticular grooves covered by a thin cuticle emerging from both edges. The conﬁguration of the cordons, the shape and length of the spicules, the shape of the caudal spine, and the number of caudal papillae (Santos et al., 1990) are characters used in combination to identify these nematodes to the species level. There are currently 9 species recognized in Aspidodera, although the group appears to be paraphyletic in the relative placement of 2 species of Nematomystes Sutton, Chabaud and Durette-Desset, 1980 (Jime´nez-Ruiz et al., 2008, 2012). A phylogenetic analysis of the family revealed that specimens collected in Mexico and previously determined as Aspidodera vazi Proenca, ¸ 1937 (Jime´nez-Ruiz et al., 2006) form a unique grouping with moderate support (Jime´nez et al., 2012). The species shows characters different from those used in the diagnosis of A. vazi and directly observed in the type specimens from Brazil and vouchers collected from across South America. We describe herein this species collected from 9-banded armadillos Dasypus novemcinctus Linnaeus, 1758 in the northern Neotropics and southern Nearctic, which represent a small fraction of the distribution of this xenarthran, which ranges from central Argentina to the central United States. MATERIALS AND METHODS Thirteen 9-banded (or long-nosed) armadillos were collected from 2001 through 2004. Nine-banded armadillos were obtained from local hunters or excavated from their burrows in Morelos, Nayarit, Oaxaca, Veracruz Received 29 January 2013; revised 11 July 2013; accepted 31 July 2013. * Department of Zoology, Ohio Wesleyan University, Delaware, Ohio 43015. † The Harold W. Manter Laboratory of Parasitology, University of Nebraska State Museum, Lincoln, Nebraska 68588. DOI: 10.1645/13-196.1
(all 4 in Mexico), and Guanacaste, Costa Rica. Parasite collecting, ﬁxation, and preservation follow Jime´nez-Ruiz et al. (2006). Specimens prepared for scanning electron microscopy (SEM) were treated with the use of a modiﬁed osmium–thiocarbohydrazide–osmium–thiocarbohydrazide–osmium (OTOTO) technique (Friedman and Ellisman, 1981), dehydrated progressively in a graded ethanol series, dried to a nonliquid state by critical-point drying with CO2, attached to a SEM stub, and sputter coated with gold palladium. Specimens imaged with SEM were exposed to a beam between 3 and 20 kV on both Hitachie ﬁeld S-2460N and S-3000 scanning electron microscopes (Hitachi Ltd., Tokyo, Japan). Measurements are given in micrometers (lm). For each character studied, the range is given ﬁrst, followed by sample mean, and coefﬁcient of variation as a percent value (Sokal and Rohlf, 1995) in parentheses. Drawings were made with an Olympus BX50 (Olympus Optical Company, Ltd., Tokyo, Japan) equipped with a drawing tube. For extraction and isolation of DNA, the tail and anterior ends were excised from the body and mounted on temporary slides with alcohol and cleared in either glycerine or lactophenol to allow speciﬁc identiﬁcation. The rest of the body was used for extraction of DNA. Methods for DNA extraction, as well as primers and cycling conditions to amplify mitochondrial 16S rDNA (rrnL) and internal transcriber spacer (ITS) are described in Jime´nez et al. (2012). A fragment of about 700 base pairs (bp) of the mitochondrial gene cytochrome C oxidase subunit 1 (cox1) was ampliﬁed with the use of the primers NCOIf1 (5 0 -CCTACTATGATTGGTGGTTTTGGTAATTG-3 0 ) and NCOIr2 (5 0 -GTAGCAGCAGTAAAATAAGCAC-3 0 ) with the following cycling conditions 948C/60 sec (948C/10 sec, 608C/45 sec, 728C/60 sec) 3 35; 728C/10 min. Successfully ampliﬁed PCR products were puriﬁed with the use of ExoSap-IT (GE Healthcare, Cleveland, Ohio) following manufacturer’s recommendations. Puriﬁed products were processed with BigDye 3.2 (BigDyee Chemistry Perkin-Elmer Applied Biosystems, Foster City, California) and directly sequenced in a Base Station 51 DNA Fragment Analyzer (MJ Research, Inc., Watertown, Massachusetts). Sequences were deposited in GenBank with accession numbers KC470124–KC470137. Resulting amplicons were aligned with ClustalW (http://www.genome. jp/tools/clustalw/) with default settings. For the rrnL and ITS data sets, sites of low probability were detected and removed using the algorithms implemented in the program GBlocks, (http://molevol.cmima.csic.es/ castresana/Gblocks_server.html) with default settings (Castresana, 2000). The cured alignments resulted in matrices of 665 bp for rrnL and 595 bp for ITS. The model of evolution GTR þ G was selected for ITS whereas GTR þ G þ I was selected for both mitochondrial matrices with the use of the Akaike information criterion as implemented in JModeltest (Posada, 2008). Nuclear and mitochondrial data sets produced different topologies (Jime´nez et al., 2012), yet rrnL and cox1 appear to have the same phylogenetic signal. As a consequence they were analyzed in PAUP* with the use of parsimony and maximum likelihood as optimality criteria (Swofford, 2003), and in MrBayes 3.2.1 with the use of Bayesian-based inference to estimate posterior probabilities of the nodes (Ronquist and Huelsenbeck, 2003). In PAUP*, the phylogeny was reconstructed by means of a heuristic search with tree bisection reconnection (TBR) branch
JIME´NEZ ET AL.—A NEW SPECIES OF ASPIDODERA IN MESOAMERICA
swapping, 100 random additions of sequences, and 10 trees held at each replicate. One thousand bootstrap replicates were performed with the use of a heuristic search in PAUP*. For the Bayesian inference, 5 chains were set to run for 10 million generations with resampling every 1,000 iterations and a burn-in of 25% of the resulting trees. The remaining trees were used to reconstruct the consensus. Cured matrices for rrnL and ITS, as well as cox1, were used to reconstruct the phylogeny of species for the 10 taxa included with the use of the program BEAST* version 1.7 (Heled and Drummond, 2010). The species tree was reconstructed under a Yule model (Steel and McKenzie, 2001) with the following assumptions: constant population size; random local molecular clock with uniform rates across branches and a general time reversible (GTR) substitution model with gamma shape for the ITS matrix and a GTR substitution model with gamma shape plus invariant sites for the rrnL and cox1 data sets. Four gamma categories were allowed for all 3 matrices and MCMC chain length of 10 million, sampling every 1,000 iterations. Specimens examined were borrowed from and deposited into the ´ following collections: Coleca ¸ ˜ o Helmintologica do Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro, Brazil; Collection of Parasitology of the School of Veterinary Medicine of the University of Hokkaido (CPSVM), Sapporo, Japan, and the United States National Parasite Collection (USNPC), Beltsville, Maryland. Species used for comparisons included A. vazi CHIOC 9641, 14086, 18354; Aspidodera binansata Railliet & Henry, 1913 CHIOC 8260, 9637, 9639, 9643, 18351, and USNPC 58363; Aspidodera fasciata, CHIOC 4119, 11190, USNPC 8550, 26644, 59968, and CPSVM 2950; Aspidodera scoleciformis (Schneider, 1851) Railliet and Henry, 1912 CHIOC 10, 5681, 5809, 8387, 9668, 11408, 14551, 15073, 15257, 18355, 19494, 19628, 20046, 34557, and 34568, and A. sogandaresi Jime´nez-Ruiz, Gardner and Varela-Stokes, 2006 HWML 48179–85. Voucher specimens were deposited in the Harold W. Manter Laboratory of Parasitology of the University of Nebraska State Museum (Lincoln, Nebraska) and correlated; resulting sequences were uploaded to GenBank ID JN852753–JN852778, JQ995297–JQ995322, and resulting trees were uploaded to TreeBase (http://purl.org/phylo/treebase/phylows/study/ TB2:S11985?x-access-code¼44c612afe664950e8bb05cd469f4b8a5&format¼ html and http://purl.org/phylo/treebase/phylows/study/TB2:S12695? x-access-code¼a0d51406af8e3b4a5ff2c2143c7d1f5b&format¼html).
DESCRIPTION Aspidodera kinsellai n. sp. (Figs. 1–7, 12) General: Nemas with fusiform bodies, sharp tail, white when alive. Cephalic cap or hood with anastomosing cordons, 6 anteriad, and 6 posteriad loops; posteriad loops ﬂanking interlabium form acute angles; interlabium well developed, long, and slender (Fig. 6). Lips simple with blunt projections laterally. Interlocking structures between lateroventrals formed by blunt projection on both sinistroventral and dextroventral lips (Figs. 1, 3). Dorsal lip with 1 papilla on each side; lateroventral lips with 1 papillae and amphid on dorsal side and circular medial papilla (Fig. 4). Inner face of dorsal lip with 2 pairs of sockets (Fig. 3). Lateral ﬁelds forming grooves, not forming sharp lateral alae (Figs. 5, 12). Males (based on measurements of 18 specimens, unless otherwise indicated): Body length 4,538–7,312, 6,240, (13%); width at midbody 287–425, 342 (13%). Cephalic hood 325–389, 360 (6%) long, 155–219, 189 (11%) wide. Nerve ring and excretory pore located 437–640, 532 (n ¼ 16, 12%), and 675–955, 791 (n ¼ 16, 12%), respectively, from anterior end (Fig. 1). Stoma length 42–65, 55 (n ¼ 17, 13%). Length of esophagus including bulb 1,287–1,736, 1,422, (9%); pharynx 44–76, 61 (n ¼ 17, 17%) by 40–70, 54 (n ¼ 17, 12%); corpus of esophagus 1,050–1,532, 1,222, (11%) long; width of esophagus at level of nerve ring 51–84, 71 (n ¼ 15, 16%); esophageal bulb pyriform, 169–229, 202 (n ¼ 14, 9%) by 159–230, 194 (n ¼ 14, 12%) (Fig. 1). Spicules subequal, thick walled, length 675– 850, 748 (n ¼ 22, 6%), width at calomus 20–42, 34, (n ¼ 19, 19%); gubernaculum 115–164, 147 (n ¼ 15, 9%) long by 19–30, 24 (n ¼ 14, 13%) wide (Fig. 2); rim of sucker 74–115, 97 (11%) in diameter (Figs. 2, 7). With 27 pairs of caudal sessile papillae distributed in ﬁelds anterior, lateral, and posterior to cloaca. Anterior or precloacal papillae consisting of 3 subventral and 1 sublateral pairs, 1 single papilla ﬂanked by 2 papillae on posterior part of rim. Lateral or adcloacal papillae consisting of a total of 4 pairs, including 1 papilla each ﬂanking cloacal opening and 3 pairs sublateral to cloaca. Papillae distributed posterior to cloaca (postcloacal)
FIGURES 1, 2. Aspidodera kinsellai n. sp. (1) Detail of the anterior end showing relative size of the hood, esophagus, and cordons. Scale bar ¼ 300 lm. (2) Tail of male showing spicules, gubernaculum and a whip-like terminal spine or terminus. Scale bar ¼ 200 lm.
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FIGURES 3–11. Aspidodera kinsellai n. sp., A. sogandaresi and A. vazi. (3–7) Aspidodera kinsellai n. sp. (3) Lateral view, showing dorsal and dextroventral lip (lower left and right, respectively), and the inner part of the sinistroventral lip; scale bar ¼ 40 lm. (4) Detail of dorsal lip, showing the lateral digitiform projections and central lobe; scale bar ¼ 20 lm. (5) Detail of the lateral groove in a female; scale bar ¼ 400 lm. (6) View of the dextroventral (DV) and dorsal (Dors.) lips and hood, showing interlabium (*); scale bar ¼ 100 lm. (7) Tail of male showing the relative size of the tapering terminus or caudal spine; scale bar ¼ 200 lm. (8, 9) Aspidodera vazi. (8) Detail of hood showing the interlabium (*) separating sinistroventral and dorsal lips (labeled SV and Dorsal, respectively); scale bar ¼ 200 lm. (9) Tail of male showing an elongated terminus or spine attached to body by a constriction; left spicule is damaged, tip of gubernaculum is evident; scale bar ¼ 200 lm. (10, 11) Aspidodera sogandaresi. (10) Detail of the hood, showing the 3 lips and the interlabium (*) separating dorsal and sinistroventral (SV) lip; scale bar ¼ 100 lm. (11) Caudal end of male showing the needlelike terminus; scale bar ¼ 100 lm. consisting of 10 subventral and 9 sublateral pairs (Figs. 2, 7). Tail length 475–627, 533 (n ¼ 15; 10%) with tapering spine 125–185, 152, (n ¼ 15, 12%) (Figs. 2, 7, 12). Females (based on measurements of 17 individuals, unless otherwise indicated): Body length 5,307–6,906, 6,218 (10%), width at level of vulva 346–557, 429 (16%). Nerve ring and excretory pore 504–611, 562 (n ¼ 14, 7%) and 700–955, 836 (n ¼ 14, 11%), respectively, from anterior end. Cephalic hood 362–440, 401 (n ¼ 17, 7%) long, by 175–258, 208 (n ¼ 16, 14%) wide. Stoma 51–69, 62 (9%); total length of esophagus including bulb 1,324–1,640, 1,473 (n ¼ 15, 7%); pharynx 48–77, 62 (16%) long by 46–72, 57 (15%) wide; corpus length, 1,073–1,425, 1,251 (n ¼ 13, 9%), by 63–90, 74 (n ¼ 12, 13%); esophageal bulb 166–244, 208 (n ¼ 14, 12%) by 172–235, 198 (n ¼ 14, 10%). Vulva located 2,431–3,112, 2,860 (n ¼ 13, 8%) from anterior end. Uteri containing embryonated eggs 57–78, 68 (n ¼ 177, 11%) by 37–61, 48 (n ¼ 177, 12%). Tail 525–719, 631 (11%).
Taxonomic summary Symbiotype: Dasypus novemcinctus Linnaeus, 1758. Type locality: Nayarit, Carretera Tepic-Aguamilpa km 8, 21832 0 16 00 N, 104852 0 25 00 W, 79 m (16 June 2003). Prevalence in type locality: One of 2 specimens infected (50%). Date of collection of type specimens: 22 May 2001. Other localities: Mexico: Morelos, Teacalco, 18837 0 12 00 N, 99827 0 20 00 W ´ Presa Miguel (22 May 2001, prevalence 50%, 1/2). Oaxaca, Isla Limon Alema´n, 18817 0 07 00 N, 96834 0 55 00 W, 49 m (22 May 2001, prevalence 50%, 1/2). Piscifactoria Temazcal, 18815 0 30 00 N, 96825 0 14 00 W, 62 m (24 May 2001). Nayarit, Carretera Tepic–Aguamilpa km 8, 21832 0 16 00 N, 104852 0 25 00 W, 79 m (16 June 2003). Costa Rica: Santa Rosa, La Casona (17 June 2004). Specimens deposited: Holotype, male CNHE5358. Allotype, female CNHE8369; paratypes CNHE8376; HWML48181 through HWML48190, USNPC97134.
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Habitat: Attached to mucosa and in lumen of large intestine. Etymology: The species is named after Dr. John M. ‘‘Mike’’ Kinsella, a truly indefatigable American helminthologist, for his contributions to science. Remarks Aspidodera kinsellai is unique in showing a unique combination of the following characters: conspicuous lateral grooves with no lateral alae (Figs. 5, 12) that start immediately after the hood and terminate in the cloacal/anal region; long hoods 325–389 in males and 362–440 in females, and terminal spine or terminus that gradually tapers to a point from the last pair of papillae. The absence of lateral alae combined with the presence of the lateral grooves is a character shared with A. scoleciformis and A. fasciata. The absence of lateral alae is also characteristic of A. sogandaresi and was previously documented in some individuals of A. vazi. Yet lateral alae are known to occur in individuals of A. vazi, and as a consequence this character was considered polymorphic for this species. However, the presence of minute lateral alae has been detected in specimens of A. vazi showing lateral groove (Figs. 13–15), suggesting that the presence of lateral grooves does not preclude the presence of lateral alae. It also suggests that the groove in contracted organisms of A. vazi may have blocked the observation of the lateral alae. The lateral alae of A. vazi, when present, are ﬁne, narrow, and sharply edged (Fig. 14). Aspidodera kinsellai closely resemble A. vazi in the relative size of the hood and tail, and in the size and conﬁguration of the spicules. With the exception of A. vazi, specimens of A. kinsellai exhibit the longest hoods among species in the family. The dimensions of the hood of A. kinsellai are 325–389 in males and 362–440 in females, whereas the homologous structure in A. vazi are 330–480 in males and 420–530 in females. Although the dimensions overlap, there is a difference of about 90 lm between maximum lengths, and as a consequence in the average, of these structures in both species (Figs. 6, 8). The coefﬁcient of variation of the measurements of the hood of A. kinsellai suggests these structures are relatively homogeneous, and that these dimensions could be used in the species discrimination if used in a context of a sample consisting of several individuals. A very similar phenomenon is seen in the terminus of males of both species; the caudal spines of A. vazi are 100–190, whereas the homologous structures in A. kinsellai are 125–185. Yet the appearance of these structures is markedly different in that the terminus in A. kinsellai (Figs. 7, 12) tappers gradually to a point from the last pair of papillae. In contrasts, the terminus in A. vazi is slender and shows a constriction immediately after the last pair of caudal papillae (Figs. 9, 13). The shape of this feature may be more useful in the discrimination between these 2 species. Aspidodera kinsellai occurs concurrently with A. sogandaresi in the 9banded armadillo. These 2 species can be differentiated because the hood in the latter is shorter, 191–264 in males and 213–312 in females (Figs. 6, 10). In addition, the tail, terminal spine and spicules of A. sogandaresi are 268–393, 25–46, and 226–372, respectively; these structures are shorter than those present in A. kinsellai (Figs. 7, 11). The conﬁguration of lateral ﬁelds is a conspicuous character that enables differentiating both species; these structures form grooves in A. kinsellai, whereas the lateral ﬁelds in A. sogandaresi are ﬂat (see ﬁgure 3a in Jime´nez-Ruiz et al., 2006). Finally, because it is wider, the body of A. kinsellai appears to be fusiform relative to the more cylindrical body of A. sogandaresi. Aspidodera kinsellai can be readily differentiated from a group of 3 species that possess shorter tail lengths, shorter caudal spine lengths, and shorter extent of the hood. These species include Aspidodera ansirupta Proenca, ¸ 1937, A. scoleciformis, and Aspidodera subulata (Molin, 1860) Railliet and Henry, 1912, all of which have a length of their cephalic hood about 1/3 of the length of the homologous structure in A. kinsellai. In addition, the hoods in both A. scoleciformis and A. subulata show square anteriad anastomosing loops, which are relatively short, concordant to the relative length of their hoods. The sockets in the interlocking portions of the lips appear as deep indentations in these 3 species as compared to those in A. kinsellai, which have shallow indentations for the sockets. Finally, A. subulata is readily distinguished from A. kinsellai by the presence of straight spicules; in comparison, spicules in A. scoleciformis are twice as long (average 1,375 lm) as those in A. kinsellai, in turn, spicules in A. ansirupta are shorter (average 282 lm). Aspidodera lacombae Vicente, 1964, can be discriminated from A. kinsellai in the presence of uniformly spaced anteriad and posteriad loops
FIGURES 12–15. Aspidodera kinsellai n. sp. and Aspidodera vazi. (12) Aspidodera kinsellai n. sp. Lateral view of tail of male, showing lateral groove, scale bar ¼ 100 lm. (13–15) Aspidodera vazi. (13) Lateral view of tail of male, showing terminal end of lateral ala, scale bar ¼ 100 lm. (14) Cross section of a female showing the presence of the lateral alae and grooves, scale bar ¼ 50 lm. (15) Subventral view of a female showing lateral ala and groove, scale bar ¼ 400 lm. in the cordon of the hood, shorter hood (males 190–230, females 230–280) and the presence of a minuscule caudal spine in the males (9–10). Aspidodera kinsellai can be differentiated from Aspidodera raillieti by the relatively shorter hood (90–190 in both sexes), and shorter caudal spines (36–50) of the former. In addition the relative proportions of the digitiform lateral projections in the dorsal lips are larger in A. kinsellai. Finally, A. kinsellai can be distinguished from A. binansata in that the former shows a double anastomosing cordon on the surface of the dorsal plate of the hood. This results in the presence of 7 anastomosing anteriad loops present in A. binansata.
RESULTS Phylogeny The phylogeny based on the analysis of the combined mitochondrial gene regions rrnL and cox1 is shown in Figure 16. Paraspidodera uncinata split from the rest of the members in the family at the basal node. A set of specimens of the new species, previously identiﬁed as A. vazi, from Nayarit, Temazcal, and Costa Rica, appears to form a monophyletic grouping. This branch is part of a nonresolved clade that includes specimens of A. scoleciformis, A. raillieti, Nematomystes rodentiphilus, and Nematomystes scapteromi. The reconstruction of the species tree for 10 taxa in Aspidoderidae based on rrnL, cox1, and ITS, is shown in Figure 17; in this topology A. scoleciformis appears to be the sister to the species described below. These specimens, found in Costa Rica and Mexico, show a set of characteristics that are different from the type specimens from Brazil.
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FIGURE 16. Phylogenetic relationships among aspidoderid nematodes based on the concatenated mitochondrial genes cox1 and rrnL. Posterior probabilities for the nodes, as well as the bootstrap support, are labeled around the nodes.
The lack of fresh samples of A. vazi to extract and isolate DNA prevented us from including this species in the phylogenetic reconstruction of the family. The inclusion of this and the other 5 species missing (namely, A. ansirupta, A. fasciata, A. lacombae, A.
Most of the specimens herein described as A. kinsellai were previously reported in the literature as A. vazi (Jime´nez-Ruiz et al., 2006). This identiﬁcation was based on the overall size of the hood and spicules, caudal spine of males, and in the apparent conﬁguration of the lateral cuticular ﬁelds. In a previous examination of individual variation in morphological characters in A. vazi, Navone (1986) showed that lateral alae were present in some but not all of the individuals examined in Argentina. This apparent variability was judged as a polymorphic feature by Jime´nez-Ruiz et al. (2006), who made the decision of identifying these specimens as A. vazi without conspicuous alae. Our investigations allowed us to detect the presence of minute lateral alae in specimens of A. vazi showing lateral groove (Figs. 13–15). This suggests that lateral alae in A. vazi may have not been observed in organisms showing a strong body contraction resulting in the appearance of a groove. A ﬁxation without previous relaxation of the body of nematodes may have caused this artifact. However, structure of the cephalic hood, spicules, shape of caudal spine and lateral grooves are consistent among parasites collected across the northern hemisphere; therefore these structures are considered reliable morphological predictors FIGURE 17. Reconstruction of the species tree for 10 species of and can be used to identify and classify nematodes from Aspidoderidae based on the analysis of the mitochondrial ribosomal large armadillos. As discussed above, these characters facilitate the subunit –rrnL–, cytochrome oxidase 1 (cox1), and internal transcriber spacer (ITS) 1 and 2 and 5.8 rDNA. discrimination from A. vazi.
JIME´NEZ ET AL.—A NEW SPECIES OF ASPIDODERA IN MESOAMERICA
subulata, and Proencaia heterospiculata) should help to resolve the relationships among species of this apparent group of species that are evidently paraphyletic, as they are included in the genus Aspidodera (Figs. 1, 2) and clarify the origin of the putative 2 lineages of Aspidodera that dispersed north of Central America. ACKNOWLEDGMENTS Both work in the ﬁeld and writing of this paper were supported by NSF grants BSR-8612329, BSR-9024816, DEB-9496263, DEB-9631295, DBI0097019 to S.L.G. We thank Dely Noronha and Luis Muniz (CHIOC), Eric Hoberg (USNPC), Luis Garc ´ıa (CNHE), Judith Price (CMN), and Sumiya Ganzorig (CPSVM) for sending us material that they have under their care. We thank Ellis Greiner for providing specimens to complete this study and for reviewing an earlier version of this manuscript. Berenit Mendoza, Kit Lee, and Josephine Koltek assisted in the imaging of specimens in SEM. We also thank Daniel R. Brooks, coordinator of the ´ parasite inventory of the vertebrates from the Area de Conservacion Guanacaste, Costa Rica, and Calixto Moraga, Petrona R ´ıos, Elda Araya, Mar ´ıa Marta Chavarr ´ıa, and Duvalier Briceno ˜ for helping us with sample collection and sample processing. We are grateful for additional technical ´ and scientiﬁc assistance in Costa Rica from Roger Blanco Segura and Felipe Chavarr ´ıa.
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