Systematic Parasitology 45: 185–197, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 185 Pseudoterranova decipiens species A and B (Nematoda, Ascaridoidea): nomenclatural designation, morphological diagnostic characters and genetic markers Lia Paggi1 , Simonetta Mattiucci1 , David I. Gibson2 , Bjorn Berland3 , Giuseppe Nascetti4 , Rossella Cianchi5 & Luciano Bullini5 1 Institute of Parasitology, University of Rome "La Sapienza", P. le Aldo Moro 5, I-00185 Rome, Italy Worms Division, Department of Zoology, The Natural History Museum, London SW7 5BD, UK 3 Zoological Laboratory, University of Bergen, Allegt. 41 N-5007, Bergen, Norway 4 Department of Environmental Sciences, Tuscia University, Via S. Camillo de Lellis, I-01100 Viterbo, Italy 5 Department of Genetics and Molecular Biology, University of Rome "La Sapienza", Via Lancisi 29, I-00161 Rome, Italy 2 Parasitic Accepted for publication 21st April, 1999 Abstract Five genetically distinct and reproductively isolated species have been detected previously within the morphospecies Pseudoterranova decipiens from the Arctic-Boreal, Boreal and Antarctic. Morphological analysis was carried out on male specimens identified by genetic (allozyme) markers, allowing the detection of significant differences at a number of characters between two members of the P. decipiens complex, namely P. decipiens A and B. On the basis of such differences, the nomenclatural designation for the two species is discussed. The names Pseudoterranova krabbei n. sp. and P. decipiens (sensu stricto) are proposed for species A and B, respectively. Morphological and genetic differentiation between the two species is shown using multivariate analysis. Allozyme diagnostic keys for routine identification of the four members of the P. decipiens complex, namely P. decipiens (s.s.), P. krabbei, P. bulbosa and P. azarasi, irrespective of sex and life-history stage, are provided. Introduction The systematics and nomenclature of anisakid nematodes which occur in the stomach of seals has long been enigmatic, the situation varying from complex and confused, to apparently stable and widely accepted, and back to semi-understood confusion. Three groups are involved: (i) those species lacking intestinal or ventricular appendages, i.e. members of the genus Anisakis Dujardin, 1845 – as far as seals are concerned, this is the least important group, as they are usually parasites of cetaceans; (ii) those species with an intestinal caecum only, i.e. members of the genus Pseudoterranova Mozgovoi, 1951; and (iii) those species with both an intestinal caecum and a ventricular appendix, i.e. members of the genus Contracaecum Railliet & Henry, 1912 originally comprising species from seals and fish-eating birds; preliminary genetic studies (Orecchia et al., 1986; Nascetti et al., 1990; Mattiucci et al., 1990) strongly support Berland’s (1964) suggestion that Contracaecum species from seals be moved to the genus Phocascaris Høst (1932). All three genera, and especially Pseudoterranova and Contracaecum, have been confused with other genera, even with genera in different families or subfamilies. They have also all had periods of stability as a result of taxonomic revision during the past 40 years, the foundations of which have been shattered by recent studies using molecular markers (Nascetti et al., 1986, 1993; Paggi et al., 1991; Mattiucci et al., 1997; inter alia). These investigations have shown that in these three genera many of the recognised morphospecies, such as Anisakis simplex (Rudolphi, 1809), Pseudoterranova decipiens (Krabbe, 1878) and Contracaecum osculatum (Rudolphi, 1802), considered to be cosmopolitan and parasites of a wide array of hosts, 186 Figure 1. Diagrammatic pattern of caudal papillae. Abbreviations: m, median papilla; pl1-pl3, caudal plates; p, posterior-most proximal papillae; pc, paracloacal papillae; d1-d4, distal papillae 1-4; ph, phasmids. are composites of several cryptic or “sibling” species. Such species have distinct gene pools characterised by diagnostic molecular markers; they are reproductively isolated by pre- and/or post-mating barriers, as in sympatric conditions no F1 fertile hybrids were detected (only a few sterile F1 hybrids were occasionally found between some species pairs). They are, therefore, true biological species sensu Mayr (1970). Once characterised genetically, the taxa previously included within each morphospecies revealed clearcut differences in their geographical range and definitive hosts. Moreover, analysis of specimens previously assigned by molecular markers to their respective biological species may provide sets of morphological characters, whose combined use allows species recognition. As part of the clarification of this situation, it is necessary to assess the nomenclatural designation of the two sibling species, Pseudoterranova decipiens A and B, of the P. decipiens complex occurring in seals from the North Atlantic Boreal and Arctic-Boreal regions. P. decipiens was erected as a species of Ascaris L., 1758 by Krabbe (1878). Krabbe recognised that there were two common nematodes, A. decipiens and A. osculata Rudolphi, 1802, in the stomach of seals in a collection of material, mainly from Greenland waters, in Copenhagen Museum. Despite the complexities of 100 years of systematics, by the 1960s the same situation, i.e. that there were essentially only two common species, had again become accepted, although A. osculata had been transferred to Contracaecum and A. decipiens attributed to several other genera. A. decipiens was initially linked with Terranova Leiper & Atkinson, 1914 (a genus erected for parasites of elasmobranchs) by Baylis (1916), because of the presence of an intestinal caecum, but he did not make the combination1 . Subsequently, Baylis (1920) placed this species in Porrocaceum Railliet & Henry, 1912 (an ascaridid genus now restricted to terrestrial birds), which he considered to be a senior synonym of Terranova. Terranova was resurrected at full generic level by Johnston & Mawson (1945) and as a subgenus of Porrocaecum by Karokhin (1946), the latter apparently being the first to use the combination T. decipiens. T. decipiens was accepted by Mozgovoi (1951, 1953), Hartwich (1957) and Yamaguti (1961). Hartwich pointed out, however, that Porrocaecum spp. (sensu stricto) had a quite different excretory system and were not anisakids. In 1959 Myers erected the genus Phocanema, with decipiens as the type and only species, on the basis that it was a parasite of marine mammals and on unspecified differences in the cephalic and tail region. Phocanema was subsequently suppressed by Gibson (1983) in favour of Pseudoterranova, which had been erected by Mozgovoi (1951; listed by Mozgovoi, 1950, without indication) for parasites from sperm whales, and decipiens was transferred to this genus (combination first used in Gibson & Colin, 1982). This is the status quo which had been generally accepted in recent years. Although several other nominal species related to P. decipiens (i.e. occurring in seals and possessing only an intestinal caecum) have been described, these species have tended to be considered as synonyms of P. decipiens (e.g. Myers, 1959). The situation has been transformed by the use of allozyme markers, which provided evidence of five genetically distinct, reproductively isolated species, within the morphospecies P. decipiens from the Arctic-Boreal, Boreal and Antarctic regions (Bullini et al., 1997; Paggi et al., 1991, 1998; Mattiucci et al., 1998). 1 Although the combination Terranova decipiens has been attributed to Baylis (1916) by many authors, he actually stated: “it would seem likely that Ascaris decipiens will have to be included in Terranova. . . . For the present, at all events, I prefer not to press the point, but rather to retain the older generic name for Ascaris decipiens”. 187 Table 1. Collection data for Pseudoterranova decipiens (sensu lato) samples from North Atlantic Ocean studied morphologically and identified genetically. Locality N Life-history stage Matre Masfjorden 16 Adult (Hordland, Norway) Faxafloi Bay 51 Adult (Iceland) Point May 24 Adult (Newfoundland, Canada) St. Bride’s 4 Adult (Placentia Bay, Newfoundland, Canada) Host species Nh Date of collection Phoca vitulina 1 January, 1986 Halichoerus grypus 1 April, 1986 Phoca vitulina 2 July, 1988 Phoca vitulina 2 April, 1990 N, number of specimens studied; Nh, number of hosts. Morphological study of specimens from the North Atlantic assigned genetically to three species, provisionally indicated as P. decipiens species A, B and C, permitted the detection of differences in the arrangement of caudal papillae on the male tail (Paggi et al., 1991), and the elucidation of a discriminant function for their separation, based on multivariate analysis of morphometric characters (Di Deco et al., 1994). The same approach, combining genetic and morphological analysis, carried out on Pacific material showed that P. decipiens species C corresponded to P. bulbosa (Cobb, 1888), whereas the fourth member of the complex, provisionally indicated as species D, corresponded to P. azarasi (Yamaguti & Arima, 1942). Both P. bulbosa and P. azarasi were accordingly resurrected from synonymy with P. decipiens (see Mattiucci et al., 1998). As to the Antarctic species recovered from the weddell seal Leptonychotes weddelli and provisionally named P. decipiens E (see Bullini et al., 1997), a direct comparison with a morphologically differentiated parasite taxon from the South American sea-lion Otaria byronia from the south-eastern Pacific Ocean (George-Nascimento & Llanos, 1995) is still needed. In addition, nomenclatural designations for species A and B has remained unresolved. The aims of the present paper, based on new material identified by genetic markers, are: to determine the best morphological features for recognising P. decipiens species A and B; to solve the nomenclatural designation of these two sibling species; and to provide diagnostic allozyme keys for routine identification of specimens of the four Boreal and Arctic-Boreal members of the P. decipiens complex irrespective of both sex and life-history stage. Materials and methods The collecting localities, host species and numbers of P. decipiens (sensu lato) specimens analysed are summarised in Table 1. Nematode samples were taken from the collection of frozen anisakid nematodes at the Institute of Parasitology of the University of Rome “La Sapienza”. From each adult specimen, the anterior and posterior parts of the body were preserved and cleared in lactic acid-phenol (1:1) for morphological studies, whereas the remaining part was used to identify the specimens at species level by allozyme markers. Morphological analysis was carried out with a microscope equipped with a camera lucida at a total magnification of 100–1,000×, except for over-all body length, which was measured directly, and spicule length, which was measured at 35×. All measurements are in millimetres. Several characters analysed have been considered of diagnostic use for anisakid nematodes (Fagerholm, 1989; Mattiucci et al., 1998), including body length, spicule length, size of caudal plates, and size and pattern of caudal papillae which were labelled according to the nomenclature proposed by Fagerholm (1989) (Figure 1). In order to consider allometric variation, caudal measurements of each specimen were related either to total body length or to tail length (cf. Fagerholm et al., 1998). Student’s t tests (pairwise) were performed to detect significant differences in absolute and relative morphometric variables between samples identified by allozyme markers. A multivariate factor analysis was carried out on relative measurements of those characters which were significantly differentiated between the two species by the former test using STATISTICA software (StatSoft Inc.). 188 Table 2. Characteristic alleles (in order of frequencies) at the loci found diagnostic above the 95% level between P. decipiens A and P. decipiens B (data from Paggi et al., 1991, and Mattiucci et al., 1998). Locus E.C. P. decipiens A P. decipiens B Iddh Mdh-1 6Pgdh Np cEst-2 Pgm 1.1.1.14 1.1.1.37 1.1.1.43 2.4.2.1 3.1.1 5.4.2.2 100 100 100, 90 100, 117 100, 95 100 70, 80 98, 110, 88 93, 105 125,133 85 107, 114, 95 E.C., International code number. For genetic identification, specimens were crushed individually in distilled water. Standard horizontal starch gel electrophoresis was performed at 5 ◦ C and 7–9 V/cm for 3–6 h, depending on the various geneenzyme systems. The following enzymes, previously found to be diagnostic for the Boreal and Artic-Boreal members of the P. decipiens complex (see Paggi et al., 1991, 1998; Mattiucci et al., 1998; and unpublished data) were routinely studied: idditol dehydrogenase (IDDH), malate dehydrogenase (MDH), 6phosphogluconate dehydrogenase (6PGDH), superoxide dismutase (SOD), nucleoside phosphorylase (NP), adenylate kinase (ADK), colorimetric esterase (cEST), mannose phosphate isomerase (MPI) and phosphoglucomutase (PGM). The specimens were also compared genetically to reference populations of the four members of the P. decipiens complex. Isozyme and allozyme nomenclature follows that used by Paggi et al. (1991) and Mattiucci et al. (1998), with P. decipiens A from the Norwegian Sea as the reference. The diagnostic power of each locus was defined using the criteria of 99% (probability of misidentification of one in 100 specimens) and 95% (probability of misidentification of 5 in 100 specimens). The genetic divergence of populations and species was estimated using the formulae proposed by Nei (1972; standard genetic identity, I, and distance, D) and by Rogers (1972, modified by Wright, 1978). The genetic relationships between species were also analysed by factor analysis performed using allele frequencies at differentiated loci as variables using STATISTICA software (StatSoft Inc.). Results and Discussion Morphological differentiation of P. decipiens A and B Adult male specimens were genetically identified as P. decipiens species A or B on the basis of six allozyme markers, as shown in Table 2 (the locus Iddh was not reported by Paggi et al., 1991). Each specimen was then analysed for various morphological characters (Table 3), including most of those considered for morphometric analysis by Di Deco et al. (1994). Moreover, numerous ratios between the variables were calculated to account for allometric variation. The resulting data are shown in Table 3. Females were not included, as no discriminant morphological features have been elucidated so far between the sibling species detected. Highly significant differences between averages were found for several characters, both as absolute measurements and when related to body/tail length, between species A and B (Table 3): mean spicule length (spi: 2.15 ± 0.23 versus 2.34 ± 0.17; spi/len: 0.06 ± 0.01 versus 0.05 ± 0.01); diameter of proximal papilla (dp: 0.016 ± 0.003 versus 0.025 ± 0.002; dp/tail: 0.07 ± 0.01 versus 0.08 ± 0.01); relative sizes of proximal papilla and distal papilla 1 (dp/dd1: 0.63±0.10 versus 0.94±0.05); distance between distal papillae 1–2, 3–4 and 4–2, (d1d2: 0.083 ± 0.011 versus 0.128 ± 0.018; d1d2/tail: 0.34 ± 0.04 versus 0.40 ± 0.05; d3d4: 0.031 ± 0.009 versus 0.058 ± 0.015; d3d4/tail: 0.13 ± 0.04 versus 0.18 ± 0.04; d4d2: 0.004 ± 0.003 versus 0.008 ± 0.004; d4d2/tail: 0.01 ± 0.01 versus 0.02 ± 0.01); absolute width of caudal plates 1 and 3 (wpl1 0.070 ± 0.009 versus 0.090 ± 0.013; wpl3: 0.068 ± 0.007 versus 0.100 ± 0.012; wpl3/tail: 0.28 ± 0.04 versus 0.32 ± 0.04); and relative width of plates 1–3 and 2–3 higher in species A (wpl1/wpl3: 1.04 ± 0.15 versus 0.90 ± 0.15; wpl2/wpl3: 1.02 ± 0.13 versus 0.90 ± 0.09). Accordingly, species A exhibits, in comparison with species B, the following morphological diagnostic characters: shorter spicules; proximal papilla (p) smaller than d1 versus the same size in species B; distal papillae 1, 2 and 4 closer to each other; and caudal plates of similar width and narrower than in species B where wpl1 and wpl2 are of similar width but wpl3 is narrower. Although no character, taken in isolation, allows the discrimination of 100% of specimens, as the variation observed exhibits some overlap, a reliable iden- 189 Table 3. Univariate statistics of 16 morphometric variables in samples of adult males classified as P. decipiens A and P. decipiens B, on the basis of allozyme diagnostic loci (see Table 2). All measures in millimetres. P. decipiens B (n = 36) AVG SD Student’s t Character P. decipiens A (n = 42) AVG SD t P len (mm) spi (mm) spi/len tail tail/len dp dp/tail dpc dpc/tail dd1 dd1/tail dp/dpc dpc/dd1 dp/dd1 ped1 ped1/tail d1d2 d1d2/tail d3d4 d3d4/tail d4d2 d4d2/tail wpl1 wpl1/tail wpl2 wpl2/tail wpl3 wpl3/tail wpl1/wpl3 wpl2/wpl3 bwcl bwpc bwd2 bwcl/tail bwpc/tail bwd2/tail 36.12 2.15 0.06 0.243 6.77 0.016 0.07 0.037 0.15 0.026 0.11 0.45 1.41 0.63 0.171 0.70 0.083 0.34 0.031 0.13 0.004 0.01 0.070 0.29 0.068 0.28 0.068 0.28 1.04 1.02 0.251 0.207 0.064 1.04 0.86 0.26 44.78 2.34 0.05 0.318 7.10 0.025 0.08 0.037 0.12 0.027 0.08 0.67 1.39 0.94 0.230 0.73 0.128 0.40 0.058 0.18 0.008 0.02 0.090 0.29 0.090 0.28 0.100 0.32 0.90 0.90 0.310 0.251 0.078 0.98 0.80 0.25 −7.46 −4.06 3.96 −11.41 −1.55 −13.02 −3.87 −0.17 8.77 −0.59 7.99 −15.07 0.86 −16.08 −11.86 −1.47 −12.71 −5.39 −9.78 −6.10 −4.94 −2.86 −8.01 0.24 −7.78 −0.08 −13.81 −3.41 4.21 4.28 −7.61 −5.39 −4.29 1.72 1.80 1.08 ∗∗∗ 3.47 0.23 0.01 0.025 0.82 0.003 0.01 0.005 0.02 0.004 0.01 0.07 0.13 0.10 0.020 0.07 0.011 0.04 0.009 0.04 0.003 0.01 0.009 0.04 0.008 0.04 0.007 0.04 0.15 0.13 0.030 0.030 0.015 0.12 0.12 0.06 6.45 0.17 0.01 0.031 1.02 0.002 0.01 0.004 0.02 0.003 0.01 0.07 0.12 0.05 0.022 0.06 0.018 0.05 0.015 0.04 0.004 0.01 0.013 0.04 0.014 0.05 0.012 0.04 0.15 0.09 0.035 0.037 0.012 0.16 0.17 0.05 ∗∗∗ ∗∗ ∗∗∗ NS ∗∗∗ ∗∗∗ NS ∗∗∗ NS ∗∗∗ ∗∗∗ NS ∗∗∗ ∗∗∗ NS ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗ ∗∗∗ NS ∗∗∗ NS ∗∗∗ ∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ NS NS NS NS = not significant; ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. n, number of specimens; AV, average; SD, standard deviation. Character codes: len, total body length; spi, mean spicule length; tail, distance between posterior end and cloaca; dp, diameter of proximal papilla; dpc, diameter of paracloacal papilla; dd1, diameter distal papilla 1; ped1, distance between posterior end and papilla d1; d1d2, d3d4, d4d2, distance between distal papillae d1 and d2, d3 and d4, d4 and d2, respectively; wpl1, wpl2, wpl3, width of caudal plates 1, 2 and 3, respectively; bwcl, body width at level of cloaca; bwpc, body width at level of proximal papilla; bwd2, body width at level of distal papilla 2. 190 Figure 2. Plot of the first two factors extracted by a multivariate factor analysis carried out using nine morphological variables (spi/len, dp/dpc, d1d2/tail, d3d4/tail, d4d2/tail, dd1/tail, wpl1/wpl3, wpl2/wpl3, wpl3/tail) on specimens previously assigned to Pseudoterranova decipiens species A or B on the basis of diagnostic allozyme markers. The percentage variance explained by the two factors are 44.1% and 16.2%. Symbols: # P. decipiens A;  P. decipiens B. Table 4. Average values and ranges (in parentheses) of genetic distance between the members of the Pseudoterranova decipiens complex, calculated using the indices of Nei (1972, below the diagonal) and Rogers (1972, modified by Wright, 1978, above the diagonal). Intraspecific DNei values are given along the diagonal. P. krabbei n. sp. P. decipiens (s.s.) P. bulbosa P. azarasi P. krabbei n. sp. P. decipiens (s.s.) P. bulbosa P. azarasi 0.005 (0.001–0.011) 0.425 (0.387–0.446) 0.934 (0.880–0.977) 0.562 (0.537–0.584) 0.569 (0.535–0.585) 0.010 (0.001–0.023) 0.637 (0.613–0.677) 0.385 (0.365–0.404) 0.756 (0.736–0.767) 0.665 (0.654–0.674) 0.004 (0.001–0.009) 0.634 (0.595–0.661) 0.616 (0.602–0.632) 0.531 (0.504–0.546) 0.646 (0.624–0.666) 0.018 (0.004–0.027) tification can be provided by using a combined set of characters, as shown by factor analysis (Figure 2). Nomenclature P. decipiens A This species appears to occur only in the North-East Atlantic, where it is a parasite at the adult stage mainly of the grey seal Halichoerus grypus, although rare specimens have been also recovered from the common 191 Figure 3. a–c. Pseudoterranova krabbei n. sp.: (a) anterior body; (b) head, dorsal view; (c) tail of male; (d) P. decipiens (s.s.): tail of male. Scale-bars: a, 0.5 mm; b, 0.1 mm; c,d, 0.05 mm. 192 seal Phoca vitulina. A search of the possible names using the Host-Parasite Data-base and Catalogue at The Natural History Museum (NHM), London, and using synonymy lists, such as that given by Myers (1959), has indicated that there are no available names which can be reasonably used for this taxon based on the host and distribution. We consider it appropriate, therefore, to erect a new specific name. Consequently, P. decipiens A becomes Pseudoterranova krabbei n. sp., named for Dr H. Krabbe, who originally described Ascaris decipiens. P. decipiens B In the North-East Atlantic, where it occurs sympatrically and syntopically with P. decipiens A, this species at the adult stage is primarily a parasite of the common seal Phoca vitulina, although it also occurs rarely in the grey seal Halichoerus grypus. In geographical areas where P. decipiens A is not present, P. decipiens B is a parasite both of P. vitulina and H. grypus: in the northern North Atlantic Ocean it has been reported from both of these seals (see Paggi et al., 1991) and, in the North Pacific Ocean, as a parasite of P. vitulina richardsii (see Mattiucci et al., 1998). Thus, this species appears to have a circumpolar distribution, although its range in the North Pacific Ocean tends to be south of that of P. bulbosa (see Mattiucci et al., 1998). Judging from the hosts and distribution of the material listed in Krabbe’s original (1878) description of Ascaris decipiens, it is likely that he had material of more than one species; however, some of the material was collected from various species of seal including Phoca vitulina from off the coast of Denmark. The type-material from the Copenhagen Museum is also likely a mixture of more than one species; however, the majority of the material which we have seen came from P. vitulina in Greenlandic and Danish waters. Although the latter material is not in a suitable condition (the specimens in spirit, lacking intact males, are very discoloured, as are the mounted tails) for us to be able to check its identity, it seems reasonable to suppose that, in view of its host and locality, it is conspecific with Pseudoterranova decipiens B. We propose, therefore, to consider P. decipiens B to be P. decipiens (sensu stricto). However, due to the conditions of Krabbe’s material we are not in a position to select a lectotype. Figure 4. Plot of the first three factors extracted by a multivariate factor analysis showing the genetic relationships among populations of P. krabbei n. sp. (= P. decipiens A), P. decipiens (s.s.) (= P. decipiens B), P. bulbosa (= P. decipiens C) and P. azarasi (= P. decipiens D). 31 alleles at 14 differentiated loci were used as variables. Percentage variance explained by the three factors are: 34.25%, 30.26% and 18%. Pseudoterranova krabbei n. sp. (Figure 3a,b,c) Type-material Holotype: one male from the stomach of Halichoerus grypus, Faxafloi Bay, Iceland, North-East Atlantic. Anterior and posterior ends of the holotype deposited in the collection of the NHM, London, BMNH 1999.4.14.1. Allotype: one female from the stomach of H. grypus, Faxafloi Bay, Iceland, North-East Atlantic. Anterior and posterior ends deposited in the collection of the NHM, London, BMNH 1999.4.14.2. Paratypes: 19 males and 7 females collected from the same host and locality as the holotype. Anterior and posterior ends of the paratypes deposited in the collection of the Institute of Parasitology, University of Rome “La Sapienza”. Other material examined: 22 males from the same host and locality. Description General. Medium-sized, reddish nematodes with inconspicuous annulate cuticle; body reaches greatest width near mid-body. Anterior end with 3 lips approximately equal in size, widest at base; all wider than long and bearing bilobed projection on anterior extremity; projection with dentigerous border; dorsal lip with 193 Figure 5. a. Zymograms corresponding to the Idditol dehydrogenase (Iddh) genotypes observed in P. krabbei (1: 100/100), P. decipiens (s.s.) (2: 70/70), P. bulbosa (3: 70/70; 4: 70/85; 5: 85/85) and P. azarasi (6: 70/70; 7: 70/80; 8: 80/80). b. Zymograms corresponding to the Adenylate kinase-2 (Adk-2) genotypes observed in P. krabbei n. sp., P. decipiens (s.s.), P. azarasi (1: 100/100) and P. bulbosa (2: 107/107). c. Zymograms corresponding to the Phosphoglucomutase (Pgm) genotypes observed in Pseudoterranova krabbei (1: 100/100), P. decipiens (s.s.) (2: 107/107; 3: 107/114; 4: 114/114 ), P. bulbosa (5: 103/103; 6: 98/103; 7: 98/98) and P. azarasi (8: 105/105; 9: 105/117; 10: 117/117). 2 lateral double papillae; ventro-lateral lips each with single lateral papilla, amphid and medio-lateral double papilla. Nerve-ring encircles approximately middle of anterior half of oesophagus. Deirids slightly posterior to nerve-ring. Oesophagus long, slightly broader posteriorly than anteriorly. Ventriculus narrower than widest part of oesophagus, longer than broad. In- testinal caecum short, extending slightly anteriorly to anterior border of ventriculus. Excretory pore between bases of subventral lips. Male (based on 20 specimens from H. grypus from Faxafloi Bay, Iceland); (measurements in millimetres; holotype in parentheses). Total length 31.5–43.0 194 (35.0), maximum width 1.27–1.54 (1.40). Deirids (n = 8) 0.50–0.82 (0.76) and nerve-ring (n = 8) 0.50–0.82 (0.77) from anterior extremity. Oesophagus 2.38–3.05 (2.85) long. Ventriculus 0.76–1.25 (1.25) long. Intestinal caecum 0.99–1.75 (1.30) long. Spicules slender, subequal, 1.82–2.55 (2.05–2.06) long. Three denticulated caudal plates; width of pl1 0.055–0.090 (0.070), of pl2 0.058–0.080 (0.070), of pl3 0.060–0.080 (0.070). Caudal papillae (nomenclature according to Fagerholm, 1989): one medial precloacal papilla; one pair of proximal papillae (p) posterior to cloacal opening, diameter 0.012–0.018 (0.017); one pair conspicuously larger double paracloacal papillae (pc), diameter 0.030–0.042 (0.039); and 4 pairs of distal papillae (d1, d2, d3, d4): distal papillae (d1) diameter 0.021–0.033 (0.025), larger than proximal papillae, slightly posterior to paracloacal papillae; distance between d1 and d2 0.060–0.100 (0.070), between d3 and d4 0.020–0.040 (0.020), between d2 and d4 0.007–0.020 (0.010). One pair of very small papilla-like phasmids, situated more laterally, occur slightly posterior to last pair of distal papillae (d2) and almost at same level as distal papillae (d2). Tail conical 0.21–0.29 (0.23), terminates in small spined conical process. Under cover-slip pressure, lateral body gives impression of forming caudal alae. Female (based on 8 specimens from H. grypus, Faxafloi Bay, Iceland; (measurements in millimetres; allotype in parentheses). Total length 30.0–44.0 (44.0), maximum width 1.15–1.46 (1.33). Deirids (n = 4) 0.56–0.84 (0.78) and nerve-ring (n = 5) 0.66–0.88 (0.66) from anterior end. Oesophagus 2.50– 3.39 (3.30) long. Ventriculus 0.93–1.21 (1.21) long. Intestinal caecum 1.07–1.85 (1.84) long. Vulva approximately at 40% of body length from anterior extremity. Eggs spherical, thin-walled, smooth, not embryonated 0.038–0.041. Tail short 0.20–0.41 (0.30) long. Pseudoterranova decipiens (Krabbe, 1878) (sensu stricto) (Figure 2d) Deposited material Voucher male: from the stomach of Phoca vitulina from St. Bride’s, Placentia Bay (Newfoundland, Canada = NFLD). Voucher female from the stomach of P. vitulina, Point May (NFLD), Canada. Anterior and posterior ends deposited in the collection of the Table 5. Two examples of allozyme keys for the identification of members of the Pseudoterranova decipiens complex (data from Paggi et al., 1991, and Mattiucci et al., 1998). A B Locus Alleles 1 Adk-2 2 Pgm 3 Iddh 1 Np 2 Mpi 3 Sod-1 107 100 107, 114 100 100 70, 80 100, 117 125, 133 85 94, 100, 107 100, 120 80 Species → → → → → → → → → → → → P. bulbosa 2 P. decipiens (s.s.) 3 P. krabbei P. azarasi P. krabbei 2 P. bulbosa 3 P. decipiens (s.s.) P. azarasi NHM, London, BMNH 1999.4.14.3–4. Other vouchers: 3 males collected from P. vitulina from Matre Masfjorden (West Norway); 5 males and 8 females from P. vitulina from Point May (NFLD); 3 males from P. vitulina from St. Bride’s, Placentia Bay (NFLD); one male from Halichoerus grypus from Faxafloi Bay (Iceland), North East Atlantic Ocean. Anterior and posterior ends deposited in the collection of the Institute of Parasitology, University of Rome “La Sapienza”. Other material examined: 13 males from P. vitulina from Matre Masfjorden (Norway) and 10 males from Point May (NFLD). Description General. Medium-sized, reddish nematodes with inconspicuous annulate cuticle; body reaches greatest width near mid-body. Anterior end with 3 lips of approximately equal size, widest at base; all wider than long and bearing bi-lobed projection on anterior extremity; projections with dentigerous border; dorsal lip with 2 lateral double papillae; sub-ventral labia each with single lateral papilla, amphid and medio-lateral double papilla. Nerve-ring encircling approximately middle of anterior half of oesophagus. Deirids slightly posterior to nerve-ring. Oesophagus long, slightly broader posteriorly than anteriorly. Ventriculus narrower than widest region of oesophagus, longer than broad. Intestinal caecum short, extending slightly anteriorly to anterior border of ventriculus. Excretory pore between bases of subventral lips. 195 Male (based on 13 specimens: 3 from P. vitulina, Matre Masfjorden (Norway); 5 from P. vitulina Point May (NFLD); 4 from P. vitulina from St.Bride’s, Placentia Bay, (NFLD); one from H. grypus from Faxafloi Bay, Iceland); (all measurements in millimetres; voucher male (NHM) in parentheses). Total length 42.5–54.0 (48.0), maximum width 1.32–1.62 (1.62). Deirids (n = 4) 0.75–0.92 (0.90) and nervering (n = 5) 0.80–0.98 (0.92) from anterior end. Oesophagus 2.23–3.74 (2.48) long. Ventriculus 0.78– 1.53 (0.95) long. Intestinal caecum 0.92–1.73 (1.32) long. Spicules slender, subequal 1.84–2.55 (2.48– 2.55) long. Three denticulate caudal plates; width of pl1 0.070–0.110 (0.080), of pl2 0.060–0.107 (0.085), of pl3 0.085–0.110 (0.085). Caudal papillae (nomenclature according to Fagerholm, 1989) as follows: one medial precloacal papilla; one pair of proximal papillae (p), diameter 0.020–0.027 (0.026); one pair of conspicuously larger double paracloacal papillae (pc) diameter 0.027–0.043 (0.038) and 4 pairs of distal papillae (d1, d2, d3, d4): distal papillae (d4) diameter 0.020–0.030 (0.028), almost same size as proximal papillae, slightly posterior to paracloacal papillae; distal papillae (d2) slightly posterior to distal papillae (d4); distance between d1 and d2 0.110–0.198 (0.140), between d3 and d4 0.040–0.090 (0.080), between d2 and d4 0.007–0.030 (0.010). One pair of very small papilla-like phasmids situated more laterally, slightly posterior to last pair of distal papillae. Tail conical, 0.26–0.35 (0.35), terminates in small spined conical process. Under cover-slip pressure, lateral body gives impression of forming caudal alae. Female (based on 8 specimens from P. vitulina, Point May, NFLD; (all measurements in millimetres; voucher in parentheses). Body 45.0–90.0 (80.0) long, maximum width 1.54–2.20 (1.74). Deirids (n = 3) 0.74–1.80 and nerve-ring (n = 5) 0.70–1.05 (1.00) from anterior extremity. Oesophagus 3.23–4.46 (4.30) long. Ventriculus 1.12–1.80 (1.57) long. Intestinal caecum 1.22–2.49 (2.00) long. Vulva pre-equatorial approximately 38–40% of body length from anterior extremity. Eggs spherical, thin-walled, smooth, unembryonated, 0.040–0.048. Tail short: 0.20–0.41 (0.23). Genetic identification of the Boreal and Arctic-Boreal members of the P. decipiens complex Distinct allozyme markers were detected at six of the 19 loci tested, allowing us to distinguish between specimens of P. krabbei n. sp. (= P. decipiens A) and P. decipiens (s.s.) (= P. decipiens B); the different sets of alleles observed in the two species are listed in Table II. The probability of the correct identification of a specimen to one or other of the species is >95% for each locus; the compound probability of misidentification over the six markers is, therefore, virtually non-existent: P = 1.56 × 10−8. Only one F1 hybrid was detected between the two species in more than 300 specimens tested; it is recognised by the contemporary presence of both a krabbei and a decipiens (s.s.) allele at each of the six diagnostic loci. The rare F1 hybrids observed do not lead to any gene exchange between P. krabbei and P. decipiens (s.s.), as indicated by the lack of recombinant, backcross or introgressed genotypes (which would be recognised by having different combinations of alleles at the six diagnostic markers). Accordingly, P. krabbei and P. decipiens (s.s.) correspond to two distinct biological species which are reproductively isolated (Paggi et al., 1991). Detailed allele frequencies of the four Boreal and Arctic-Boreal members of the P. decipiens complex, P. krabbei n. sp., P. decipiens (s.s.), P. bulbosa and P. azarasi, at 19 enzyme loci are presented in our earlier papers (Paggi et al., 1991; Mattiucci et al., 1998). A matrix of Nei’s genetic distance values for each pair-wise comparison is given in Table 4. The genetic relationships between the four members of the P. decipiens complex are summarised in Figure 4; these were obtained by plotting the first three components of a PCA analysis carried out using the frequencies of 31 alleles at 14 differentiated loci. A closer relationship of P. decipiens (s.s.) to P. azarasi and P. krabbei than to P. bulbosa is apparent. On the basis of the loci showing fixed differences between the different members of the P. decipiens complex, molecular diagnostic keys can be set up using a minimum number of markers, enabling the routine identification of large numbers of specimens of different life-history stage and sex, and even from small portions of these worms. Two examples of diagnostic keys, for the identification of P. decipiens (s.s.), P. krabbei, P. bulbosa and P. azarasi are given in Table 5. They were assembled by selecting those diagnostic loci showing allozymes with a well-separated 196 electrophoretic mobility for the different species (e.g. Figure 5), thus allowing an easy and clear assignment of the specimens tested. For example, only the Pgm locus will discriminate each of the four species, i.e. using the following allozymes: 100 in P. krabbei, 107 and 114 in P. decipiens (s.s.), 98 and 103 in P. bulbosa, and 105 and 117 in P. azarasi (Figure 4); however, some of these allozymes exhibit a similar electrophoretic mobility and could be difficult to be distinguished from each other (e.g. 98 versus 100 and 103; 105 versus 107; etc.). Therefore, it is strongly recommended that a combination of at least two or more diagnostic loci are used and in two different keys, as shown in Table V, for a ready and accurate identification at the specific level of the four members of the P. decipiens complex so far detected. Acknowledgements We thank anonymous referees for their comments and suggestions. The research was supported by grants from the Italian Ministero per le Politiche Agricole (Direzione Generale della Pesca e dell’Acquacoltura) and from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica, Progetti di Rilevante Interesse Nazionale – Cofinanziamento 1997. References Baylis, H.A. (1916) Some ascarids in the British Museum (Nat. Hist.). Parasitology, 8, 360–378. Baylis, H.A. (1920) On the classification of the Ascarididae. I. The systematic value of certain characters of the alimentary canal. Parasitology, 12, 253–264. Berland, B. (1964) Phocascaris cystophorae sp. nov. (Nematoda) from the hooded seal, with an emendation of the genus. Arbok for Universitetet i Bergen, Mat.- Naturv. Ser., 17, 1–21. Bullini, L., Arduino, P., Cianchi, R., Nascetti, G., D’Amelio, S., Mattiucci, S., Paggi, L., Orecchia, P., Plotz, J., Berland, B., Smith, J.W. & Brattey, J. 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