Parasite material

Four dead Ph. brasilianus from a breeding colony of the Santa Elena lagoon, VIII Region, Chile (37°15′S, 72°28′W) were necropsied during 2006–2008. Specimens of C. chubutensis were collected from Ph. atriceps at Bahia Bustamante, Chubut Province, Argentina (45°11′S, 66°30′W). During necropsy, their entire digestive tracts were removed and frozen at −20 C until they could be examined. After thawing, their contents were washed with water on a sieve with a mesh of 0.25 mm and the sediment was placed in Petri dishes. Isolated nematodes were fixed and stored in 70% ethanol until morphological and genetic analyses could be undertaken.

Morphological study

Thirty adult nematodes (20 males and 10 females) of Contracaecum spp. collected from Ph. brasilianus were examined morphologically. For each adult specimen, the overall body length was measured directly. The middle part of the body was then separated from the rest of the body and used to genetically characterize the individual specimens by sequencing of the mtDNA cox2 gene. The anterior and posterior parts were then cleared and mounted in lactophenol (1∶1) for morphological studies. Specimens were studied using a compound microscope (×100–400) and a drawing apparatus. Measurements are presented in mm, except where indicated. Several characters considered diagnostic for anisakid nematodes (Fagerholm, 1989, 1991; Paggi et al., 2000) were analyzed, including interlabial structure, the pattern of distribution of male caudal papillae, spicule length and tip shape, and the size and pattern of the caudal papillae, all of which were labelled according to the nomenclature proposed by Fagerholm (1989). To consider allometric variation, spicule length measurements were related to either total body length or to tail length. In addition, a cecum to appendix ratio was obtained.

Some specimens were dried by the critical point method, then observed and photographed using an SEM (Jeol® JSV 6063 LV, Jeol Ltd., Akishma City, Tokyo, Japan). Holotype, allotype, and paratype specimens were stored in 70% ethanol and deposited in the Helminthological Collection of Museo de La Plata (CHMLP).

DNA amplification and sequencing

The 519-bp fragment of the mitochondrial cytochrome oxidase 2 gene (mtDNA cox-2) was analyzed from 6 specimens of C. chubutensis n. sp. and from 12 of the new species. A 470-bp fragment of the small subunit of the mitochondrial ribosomal RNA gene (rrnS) was analyzed in 6 specimens of C. chubutensis and in 6 of the new species. A 451-bp fragment of the ITS-1 and 284 bp of the ITS-2 regions were analyzed in 3 specimens of C. chubutensis and 3 of the new species. The total DNA was extracted from 2 mg of tissue from a single nematode using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin) or cetyltrithylammonium bromide (Valentini et al., 2006). The cox-2 gene from each specimen of Contracaecum was amplified according to the procedures as reported in Mattiucci et al. (2010) with the primers 211F 5′-TTTTCTAGTTATATAGATTGRTTYAT-3′ and 210R 5′-CACCAACTCTTAAAATTATC-3′ from Nadler and Hudspeth (2000) spanning the mtDNA nucleotide position 10,639–11,248 as defined in Ascaris suum (GenBank X54253).

Polymerase chain reaction (PCR)

Amplification was carried out in a volume of 50 µl containing 30 pmol of each primer, MgCl₂ 2.5 mM (Amersham Pharmacia Biotech Inc., Piscataway, New Jersey), PCR buffer 1× (Amersham), DMSO 0.08 mM, dNTPs 0.4 mM (Sigma-Aldrich, St. Louis, Missouri), 5 U of Taq Polymerase (Amersham), and 10 ng of total DNA. The mixture was denatured at 94 C for 3 min followed by 34 cycles at 94 C for 30 sec, 46 C for 1 min and 72 C for 1.5 min, followed by post-amplification at 72 C for 10 min. The PCR product was purified using PEG precipitation and automated DNA sequencing was performed by Macrogen Inc. (Seoul, Korea) using primers 210 and 211.

The amplification of the small subunit of the mitochondrial ribosomal gene, rrnS, was performed according to the procedures reported in D'Amelio et al. (2007) with the primers MH3 (forward; 5′-TTGTTCCAGAATAATCGGCTAGACTT-3′) and MH4.5 (reverse; 5′-TCTACTTTACTACAACTTACTCC-3′). The PCR conditions were as follows: 10 min at 95 C (initial denaturation), 35 cycles of 30 sec at 95 C (denaturation), 30 sec at 55 C (annealing), 30 sec at 72 C (extension), and a final elongation step of 7 min at 72 C.

The amplification of the ITS-1 region was carried out according to the procedure reported in Shamsi et al. (2009a, 2009b) with the primers sets SS1 (forward; 5′-GTTTCCGTAGGTGAACCTGCG-3′) and NC13R (reverse; 5′-GCTGCGTTCTTCATCGAT-3′) and the ITS-2 region with the primers sets SS2 (forward; 5′-TTGCAGACACATTGAGCACT-3′) and NC2 (reverse; 5′-TTAGTTTCTTTTCCTCCGCT-3′). The PCR was performed in 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 3 mM MgCl2, 250 µM each of dNTP, 50 pmol of each primer, and 1.5 U Taq polymerase (Promega) in a thermocycler using the following conditions: 4 C for 5 min (initial denaturation), followed by 30 cycles at 94 C for 30 sec (denaturation), at 55 C for 30 sec (annealing), at 72 C for 30 sec (extension), and a final extension at 72 C for 5 min. The PCR products were examined on a 1% agarose gel, stained with 1.5 µl of Gel-Red (Biotium Inc., Hayward, California), and analyzed using a gel documentation system. Reference specimens and isolated DNA samples are stored at the Department of Public Health and Infectious Diseases, of the Sapienza – University of Rome, Rome, Italy.

The sequences of the specimens of Contracaecum spp. from Ph. brasilianus and C. chubutensis in Phalacrocorax atriceps were compared to those already obtained in our previous studies on mtDNA cox2 deposited in GenBank. Sequences examined were from the 2 sibling species of C. rudolphii (s. l.) complex, i.e., C. rudolphii A and C. rudolphii B of Bullini et al. (1986) from the Eurasian subspecies of the great cormorant Phalacrocorax carbo sinensis (Blumenbach) from Italian coastal lagoons; C. bioccai Mattiucci, Paoletti, Olivero-Verbel, Baldiris, Arroyo-Salgado, Garbin, Navone, and Nascetti, 2008, from the brown pelican P. occidentalis (L.) in Colombia; Contracaecum septentrionale Kreis, 1955 from Phalacrocorax aristotelis (L.) off Norway; Contracaecum microcephalum (Rudolphi, 1809) from Phalacrocorax pygmaeus (L.) off Montenegro; Contracaecum micropapillatum (Stossich, 1890) sampled in the white pelican Pelecanus onocrotalus (L.) in Egyptian waters; C. pelagicum Johnston and Mawson, 1942 from S. magellanicus (Forster) off Argentina; and, finally, C. gibsoni Mattiucci, Paoletti, Consuegra-Solorzano, and Nascetti, 2010, and C. overstreeti Mattiucci, Paoletti, Consuegra-Solorzano, and Nascetti, 2010 from the Dalmatian pelican, P. crispus, off the coast of Greece.

The sequences of the mitochondrial rrnS region of the ribosomal DNA obtained for the specimens of Contracaecum from Ph. brasilianus and C. chubutensis from Ph. atriceps were compared to those already obtained for C. rudolphii C from Ph. auritus and deposited in GenBank. Sequences obtained in the present study for Contracaecum spp. included, for comparative purposes: C. rudolphii A, C. rudolphii B, C. bioccai, C. septentrionale, C. microcephalum, C. micropapillatum, C. pelagicum, C. gibsoni, and C. overstreeti.

Finally, the sequences of ITS-1 and ITS-2 regions of the rDNA obtained for Contracaecum from Ph. brasilianus and C. chubutensis were also compared with those previously obtained for the same gene from C. rudolphii D and C. rudolphii E isolated from Ph. carbo and Ph. various, respectively.

Sequence analysis

The cox-2 and rrnS sequences obtained were aligned using Clustal X (Larkin et al., 2007). Phylogenetic scrutiny was performed using maximum parsimony (MP) and neighbor-joining (NJ) analyses, based on p-distance values, by PAUP* (Swofford, 2003) for mtDNA cox-2 and rrnS datasets. The optimal evolution schematic for the datasets was the GTR+I+G model, as determined by using Akaike Information Criterion (AIC) (Posada and Buckley, 2004), and implemented in the software Modeltest 3.6 (Posada and Crandall, 1998) among 56 possible alternative models. The parameters for the model inferred from the mtDNA cox2 sequences data were the proportion of invariable sites (I)  =  0.6020, distribution shape parameter (α)  =  0.8138, and nucleotide frequencies A  =  0.19, C  =  0.07, G  =  0.27, T  =  0.47. The parameters for the model inferred from rrnS rDNA were the proportion of invariable sites (I)  =  0.5937, distribution shape parameter (α)  =  0.7905, and nucleotide frequencies A  =  0.20, C  =  0.06, G  =  0.27, T  =  0.47. The reliabilities of the phylogenetic relationships were evaluated using nonparametric bootstrap analysis (Felsenstein, 1985) for the MP and NJ trees. Bootstrap values ≥70 were considered well supported (Hillis and Bull, 1993; Morrison, 2006).

Bayesian inference (BI) analysis (Larget and Simon, 1999) was performed using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001) on full consensus sequences. The optimal evolution model of our dataset for the Bayesian analysis was determined using Akaike Information Criterion (AIC) (Posada and Buckley, 2004), as implemented in the software Model test 3.7 (Posada and Crandall, 1998) associated with PAUP* (Swofford, 2003). This analysis supported GTR+I+G as the best-fit substitution model for the data. The parameters for the model inferred were the proportion of invariable sites (I)  =  0.6020, distribution shape parameter (α)  =  0.8524, and nucleotide frequencies A  =  0.19, C  =  0.07, G  =  0.27, T  =  0.45. For Bayesian analysis, 4 incrementally heated Markov chains (using default heating values) were run for 1,000,000 generations, sampling the Markov chains at intervals of 100 generations. Of 20,002 samples summarized from 2 runs, 19,502 were included in the analysis. Posterior probabilities were estimated and used to assess support for each branch in inferred phylogenies; probabilities where P < 0.05 were indicative of significant support (Reeder, 2003).

The sequences of Contracaecum from Ph. brasilianus and C. chubutensis from Ph. atriceps from Argentina were compared to those already obtained for mtDNA cox-2 for Contracaecum spp. from waterbirds in our previous studies (Mattiucci et al., 2008, 2010) and deposited in GenBank with the following accession numbers: EF513501, EF513502, EF513503, EF513505, EF558891, EF122202, EF535570 (C. rudolphii sp. A); EF558894, EF558896, EF513506, EF513507, EF513509, EU852349 (C. rudolphii sp. B); EF122205, EF513512, EF513513 (C. septentrionale); EF122208, EF5135017, EF5135018, EF513519 (C. microcephalum); EF122206, EF122207, EF513514, EF513515, EF513516, EU852350 (C. micropapillatum); EF513494, EF513495, EF558899, EF513497, EF513498, EF513499 (C. bioccai); EF122210, EF535568, EF535569 (C. pelagicum); EU852337–EU852342 (C. gibsoni); and EU852343–EU852348 (C. overstreeti).

Further, for a genetic comparison with other Contracaecum spp. so far described from other fish-eating species of Phalacrocorax, the sequences obtained for the mitochondrial gene rrnS in the present study were compared with those available for 1 specimen of C. rudolphii C (EF014283) deposited in GenBank. Finally, the sequences obtained for the ITS-1 and ITS-2 regions of the rDNA were compared with those of C. rudolphii D and C. rudolphii E, retrievable from GenBank under the accession numbers: FM210251, FM210252, FM210253, FM210262, FM210263, FM210264, FM210258, E-FM210259, FM210260, FM210270, FM210271, and FM210272. Sulcascaris sulcata from Caretta caretta of the Mediterranean Sea was included as outgroup to root the phylogenetic trees (GenBank HQ328505).

Taxonomic summary

Remarks

According to the morphological characters considered as diagnostic for species of Contracaecum, i.e., the length of the spicules, the morphology of the distal end of the spicule, and the bifurcation of the interlabial tip (sensu Hartwich, 1964), our specimens collected from the Ph. brasilianus would be assigned to C. rudolphii Hartwich 1964 sensu lato (see Hartwich, 1964). However, according to the morphological comparison of the new specimens, the material has been assigned to C. australe n. sp.

The new species possesses a distal tail constriction which seems to be absent in the original description of C. rudolphii (Hartwich, 1964), even though it is present in other descriptions, i.e., that of Abollo et al. (2001) (Fig. 1). The median plaque (Fig. 1g) observed in C. australe also seems to be absent in C. rudolphii (s. l.), or perhaps it was not observed by other authors (Hartwich, 1964; Abollo et al., 2001; Amato et al., 2006). Moreover, C. australe appears shorter and thicker, as is suggested by the body length to maximum body width ratio: 23.00–23.09 versus 29.4–98.1 (Table I). In addition, male spicules of C. australe are longer than those of C. rudolphii (9.60–15.88 mm vs. 4.05–9.98 mm), with a larger BL∶SL, 1.41–2.77 versus 2.06–5.69. However, according to Hartwich (1964) and Barus et al. (2000), C. rudolphii (s. l.) has a great variability in the size of spicules (Table I). It has been also demonstrated that C. rudolphii (sensu Hartwich, 1964) (s. l.) is a complex of sibling species. Contracaecum australe differs from both C. rudolphii sp. A and C. rudolphii sp. B (of Bullini et al., 1986). The former species has longer spicules compared to those observed for the 2 sibling species infecting the great cormorant Phalacrocorax carbo sinensis. Spicules from C. rudolphii sp. A are 6.8–7.2 mm and 8.6–9.5 mm in C. rudolphii B (Mattiucci et al., 2008). Moreover, the geographical distribution and host of the 2 siblings are different. Finally, the sequence analysis of the mtDNA cox-2, rrnS and of the ITS-1 and ITS-2 regions presented here demonstrates that C. australe is genetically distinct from C. rudolphii A, C. rudolphii B, C. rudolphii C, C. rudolphii D, and C. rudolphii E (Figs. 5–Figure67). Contracaecum rudolphii C (see D'Amelio et al., 2007) from Ph. auritus has not been morphologically described. As for C. rudolphii D and C. rudolphii E from Australian cormorants (see Shamsi et al., 2009b), the new species differs from C. rudolphii D for the spicule length (3.90–6.60 mm in C. rudolphii D vs. 9.60–15.88 mm in C. australe and from C. rudolphii E, in which the spicule lengths range from 5.53–6.13 mm). Moreover, the host and geographical distributions are different.

Among those Contracaecum spp. reported from cormorants belonging to Phalacrocoracidae, C. caballeroi is also reported as a parasite of Ph. brasilianus; it possesses significantly shorter spicules (0.90–1.09 mm vs. 9.60–15.88 mm) and, therefore, has a much higher body to spicule length ratio: 24.74–26.98 versus 1.41–2.77 (Lent and Freitas, 1948) (Table I). Contracaecum australe also differs morphologically from C. chubutensis Garbin Diaz, Cremonte, and Navone, 2008; the new species has a well-marked constriction of the tail tip, an oblique position of the paracloacal papillae, lips with no notches, entire or barely bifurcated interlabia, longer spicules (9.60–15.88 mm vs. 5.34–12.60 mm), and a smaller body to spicule length ratio: 1.41–2.77 versus 2.18–3.14.

Contracaecum septentrionale Kreis, 1955 greatly resembles C. australe; however, the precloacal papillae number is smaller, the paracloacal papillae seem to have an inverse oblique disposition, the tail looks more curved, and the spicule end appears to be more blunt in C. septentrionale (Kreis, 1955). Further, the host and geographical distribution of C. septentrionale is different; all the genetic data presented here also support the distinction of C. australe from C. septentrionale.

Contracaecum travassosi is also similar to C. australe, although spicules in the former species are shorter (7.70–11.10 vs. 9.60–15.88). Therefore, the BL∶SL ratio is less variable (2.09–2.28 vs. 1.41–2.77), the bifurcation of interlabia is more marked, the body width is greater and, consequently, so is the body to maximum body width ratio: 23.00–23.09 versus 28.31–39.12. Further, according to the original description given by Gutierrez (1943), the paracloacal papillae are double, even if in the original figure they appear to be as 2 separate papillae, but very close to each other. Specimens of C. travassosi described from osprey, Pandion haliaetus (L.) (Accipitridae), in North America (Morgan et al., 1949) are significantly thinner (Table I) and the tail length is longer based on the body to tail length ratio: 84.74–97.69 versus 97.92–138.89. A morphological re-examination of a male paratype of C. travassosi provided clear evidence that the paracloacal papillae are double.

Contracaecum australe is also morphologically distinct from other Contracaecum species that parasitize waterbirds, i.e., it differs from Contracaecum variegatum Rudolphi, 1809 from red-throated loon, Gavia stellata (Pontoppidan) (Gaviidae), because the latter 2 species possess almost double the number of precloacal papillae and shorter spicules (4.40–4.86 mm vs. 9.60–15.88 mm), and, therefore, a larger body to spicule length ratio (BL∶SL 4.00–6.50 vs. 1.41–2.77) (Fagerholm et al., 1996). Moreover, C. variegatum is genetically distinct from the new species based on mtDNA cox-2 analysis (data not shown).

Contracaecum magnipapillatum ( =  Contracaecum magnicollare) Johnston and Mawson, 1941 differs from C. australe of black noddy Anous minutus Chapin (Laridae) because it lacks bifurcated interlabia (Fagerholm et al., 1996) and its spicules are smaller (2.62–3.70 vs. 9.60–15.88 mm) and, therefore, has a higher BL∶SL ratio (4.20–6.00 vs. 1.41–2.77). Contracaecum plagiaticum has 8 postcloacal papillae pairs (1 more subventral papillae pair) and shorter spicules (2.32–3.49 mm vs. 9.60–15.88 mm), BL∶SL ratio 4.80–5.40 versus 1.41–2.77 (Lent and Freitas, 1948). Contracaecum pelagicum Johnston and Mawson, 1942 from several hosts (Portes-Santos, 1984; Silva et al., 2005; Garbin et al., 2007; Garbin, 2009) can be differentiated from C. australe, mainly for its bifurcation on interlabia and shorter spicules (3.07–5.07 mm vs. 9.60–15.88 mm); moreover, C. pelagicum is also genetically distinct from C. australe (Figs. 3, 4). Contracaecum multipapillatum (von Drasche, 1882) from great egret Ardea alba greatly differentiates from C. australe in terms of the number of papillae and the pattern of postcloacal papillae; further, C. multipapillatum s. l. has no bifurcated interlabia (Navone et al., 2000). Contracaecum australe differs also from C. gibsoni and C. overstreeti described from P. crispus; the latter species does not have a bifurcated interlabia, and they also have shorter spicules and a different pattern of distribution of proximal papillae (see Mattiucci et al., 2010). Contracaecum bioccai Mattiucci et al., 2008 from P. occidentalis has shorter and subequal spicules (right 5.80–6.20 mm, left 6.00–6.50 mm vs. 9.60–15.88 mm), conspicuous bifurcated interlabia, and the a4 sublateral papillae pair unites with the a2 subventral pair, forming a double, 3-paired subventral row. Contracaecum microcephalum (Rudolphii, 1809) has very short spicules (1.40–3.65 mm vs. 9.60–15.88 mm), different in the BL∶SL ratio (5.06–15.05 vs. 1.41–2.77).

Genetic differentiation between C. australe n. sp. and C. chubutensis with respect to other congeners from waterbirds

To provide support for the existence and the validity of C. australe as a new species and of C. chubutensis, morphologically described in our previous studies, the same 13 specimens of the first taxon and 3 of the second were sequenced at the mtDNA cox-2 locus. Further, some specimens among those sequenced at the mtDNA cox-2 locus were also sequenced at the rrnS locus and at the ITS-1 and ITS-2 regions of the nuclear rDNA (Table III). The sequences obtained for the specimens of C. australe n. sp. and C. chubutensis are deposited in GenBank under the accession numbers indicated in Table III.

Table III

GenBank accession numbers of the specimens of Contracaecum australe n. sp. and Contracaecum chubutensis sequenced at cox-2, rrnS, ITS-1, and ITS-2 loci. They are reported with their codes appearing in the text and figures.

The specimens of Contracaecum from Ph. brasilianus indicated that C. australe did not match any of the previously reported sequences for the 3 genes examined here or any of those previously deposited in GenBank. Similarly, the specimens of C. chubutensis did not match any of the congener species previously scrutinized or deposited in GenBank. The sequence alignments of C. australe n. sp. and C. chubutensis, in comparison with others that have been investigated, are shown in Figures 2, 3, 4a, b.

Figure2

Alignment of mtDNA cox-2 (519 bp) sequences of C. australe n. sp. and C. chubutensis with other Contracaecum spp. previously sequenced (Mattiucci et al., 2010) and deposited in GenBank. The alignment was performed using BioEdit (Hall, 1999). One representative of each unique sequence was included for the comparison. Dot indicates identity.

Figure3

Alignment of rrnS (470 bp) sequences of C. australe n. sp. and C. chubutensis with other Contracaecum spp. previously deposited in GenBank under the accession numbers as published in D'Amelio et al. (2007). The alignment was performed using BioEdit (Hall, 1999). One representative of each unique sequence was included for the comparison. Dot indicates identity and dash indicates gap.

Figure4

Alignment of sequences of nuclear ribosomal DNA ITS-1 (a) (451 bp) and ITS-2 (b) (284 bp) regions of C. australe n. sp. and C. chubutensis with respect to the siblings of the C. rudolphii complex sequenced so far at those loci and deposited in GenBank under the accession numbers as given in 49Shamsi et al. (2009). The alignment was performed using BioEdit (Hall, 1999). Three representative sequences for each C. rudolphii A, C. rudolphii B, C. rudolphii D, and C. rudolphii E were included for the comparison. Dot indicates identity and dash indicates gap.

Continued.

The individuals corresponding to C. australe n. sp. all clustered in the same clade, well supported in the MP tree (Fig. 5) as well as in the BI (Fig. 6) inferred from the mtDNA cox-2 sequence analysis. In these trees, the clade formed by the specimens of C. australe was quite distinct from all the previously genetically characterized species of Contracaecum. Similarly, C. chubutensis forms, at both MP and BI (Figs. 5, 6) inferred from the same mtDNA cox2 sequences analysis, a distinct clade from all the Contracaecum spp. Moreover, both C. australe n. sp. and C. chubutensis form 2 quite-distinct clades (Fig. 6). Nonetheless, C. australe and C. chubutensis are closely related to the other Contracaecum parasites of cormorants, i.e., C. rudolphii A, C. rudolphii B, C. rudolphii C, and C. septentrionale. Indeed, a congruent tree topology inferred from mtDNA cox-2 and rrnS sequence analyses (Figs. 5–Figure67) was generated. The same sub-clade was produced with the tree topology inferred from MP and BI from mtDNA cox-2 (Figs. 5, 6), as well as from MP and NJ of the rrnS (Fig. 7), including C. australe, the species in the C. rudolphii complex (C. rudolphii sp. A, C. rudolphii sp. B, and C. rudolphii C) plus C. septentrionale and C. chubutensis. Moreover, this sub-clade (Fig. 5, 7) was distinct from all other Contracaecum species considered in the comparison, although it did not receive a high bootstrap value (<70) in all the analyses inferred from different genes.

Figure5

Maximum parsimony (MP) bootstrap consensus tree performed by PAUP* (Swofford, 2003) on 1,000 replicates, using the GTR+I+G evolution model (by Modeltest 3.1; Posada and Crandall, 1998) showing the genetic relationship of C. australe n. sp. and C. chubutensis with respect to Contracaecum spp. previously sequenced at the mtDNA cox-2. Bootstrap values are reported at the nodes (MP values: above; neighbor-joining values: below). Sulcascaris sulcata was used as outgroup.

Figure6

Consensus tree inferred by Bayesian analysis (BI) carried out using MrBayes3.1 software program (Ronquist and Huelsenbeck, 2003), inferred from mtDNA cox-2 sequences analysis of Contracaecum spp. studied, on 1,000,000 generations. The BI was performed using the GTR+I+G model as the best-fit substitution model for the data. The parameters for the model inferred were the proportion of invariable sites (I)  =  0.6020, distribution shape parameter (α)  =  0.8524, and nucleotide frequencies A  =  0.19, C  =  0.07, G  =  0.27, T  =  0.45. Numbers at the nodes are posterior probabilities recovered by the Bayesian analysis with a significant support of P ≥ 95%. Sulcascaris sulcata was used as outgroup.

Figure7

Condensed MP and neighbor-joining (NJ) bootstrap consensus trees by PAUP* (Swofford, 2003) on 1,000 replicates, based on rrnS sequences analysis of specimens of Contracaecum spp. sequenced in the present study in comparison with those sequenced by D'Amelio et al. (2007) and reported in the analysis with the following GenBank accession numbers: EF014281 (A-cor31), EF014279 (B-cor41), EF014283 (C-cor52), EF030716 (Pel3), EF014282 (Php1), EF014280 (Pc1). MP tree was obtained by bootstrap method on 1,000 replicates. The MP was performed using the GTR+I+G evolution model (by Modeltest 3.1; Posada and Crandall, 1998). Proportion of invariable sites (I)  =  0.5937; gamma distribution shape parameter (G)  =  0.7905; 166 polymorphic sites; 154 parsimony-informative sites; base frequencies A  =  19%, C  =  7%, G  =  27%, T  =  47%. Bootstrap values are shown at the nodes: MP and NJ values are shown above and below the nodes, respectively. Sulcascaris sulcata was used as outgroup.

Pairwise comparisons of the p-distance values inferred from mtDNA cox-2 (Table IV) sequences range from 0.08 to 0.10 between C. australe and C. chubutensis with respect to C. rudolphii A and C. rudolphii B. On the other hand, C. australe versus C. chubutensis shows a value of p-distance  =  0.09. Moreover, C. chubutensis was found to be genetically more related to C. septentrionale, from which it has been demonstrated to show, however, a p-distance value of 0.06. With respect to C. septentrionale, C. australe exhibits a p-distance value of 0.10, whereas C. australe shows much larger values, i.e., 0.13 to 0.14 with respect to other morphologically distinct species such as C. micropapillatum or C. gibsoni (Table III).

Table IV

Pairwise p-distance values inferred from mtDNA cox-2 (above the diagonal) and rrnS (below the diagonal) sequences analysis between Contracaecum australe n. sp. and Contracaecum chubutensis and versus other Contracaecum spp. so far sequenced at the same loci. Accession number for rrnS of Contracaecum rudolphii C deposited in GenBank is reported.

Similarly, the p-distance estimated at the rrnS DNA (Table IV) exhibits a value of 0.03 between C. australe and C. chubutensis, whereas a value of p-distance  =  0.04 was observed between C. australe and C. rudolphii C. Similar numbers were observed between C. australe and C. chubutensis with respect to C. rudolphii A, C. rudolphii B, and C. septentrionale as well (Table IV).

Finally, at the ITS-1 and ITS-2 regions of the rDNA, C. australe exhibits p-distance values (Table V) ranging from 0.02 to 0.03 with respect to C. chubutensis and other species of the C. rudolphii complex, i.e., C. rudolphii D and C. rudolphii E. Sequence polymorphisms at the ITS-1 and ITS-2 were detected in C. australe at alignment positions 303, 333, and 418, and 74, 83, 88, 159, 160, 273, and 280, respectively (Fig. 4a, b). Sequence polymorphism was detected at alignment position 116 of the ITS-1 and at position 31 of the ITS-2 in C. chubutensis (Fig. 4a, b).

Table V

Pairwise p-distance values inferred from the ITS-1 (above the diagonal) and ITS-2 (below the diagonal) sequences analysis between Contracaecum australe n. sp. and Contracaecum chubutensis and versus members of the Contracaecum rudolphii complex. Sequences and their accession numbers of the species C. rudolphii D and C. rudolphii E are those reported in Table III.