Phylogeny of Palaearctic Pharyngodonidae parasite

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cult by the large numbers of species and individuals found in each host .... gonetria and Mehdiella are two distinct genera that can be distinguished by ..... Five states: 000, dodecagon; 100, triangular with sides equal; 110, triangular with sides ...
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Phylogeny of Palaearctic Pharyngodonidae parasite species of Testudinidae: a morphological approach Salah Bouamer and Serge Morand

Abstract: The phylogenetic relationships of 23 oxyurid species from five genera (21 parasite species of the Palaearctic Testudinidae, 1 parasite species of Uromastix acanthinurus Bell, 1825 from Algeria, and 1 parasite species of Ctenosaura pectinata (Wiegmann, 1834) from Mexico) were investigated using 30 morphological characters obtained from species descriptions. The nonweighted analysis produced one shortest tree. All species of the ingroup form a monophyletic group and the oxyurid species of Testudinidae form a monophyletic group. The type species of the genus Alaeuris Thapar, 1925 is the basal member of the species parasitizing Testudinidae. The analysis confirms the monophyly of the genus Thaparia Ortlepp, 1933, whereas the genera Mehdiella Seurat, 1918 and Tachygonetria Wedl, 1862 are considered paraphyletic groups. The large diversification in the genus Tachygonetria is linked to their position in the host caecum. The ancestral state is in the paramucous and the derived state is in the centre of the caecum. This suggests that recent speciation in the group occurs in the centre of the caecum. Résumé : L’utilisation de 30 caractères morphologiques tirés des descriptions des espèces nous a permis d’étudier les relations phylogénétiques de 23 espèces d’oxyures appartenant à cinq genres, soit 21 espèces parasites de Testudinidae paléarctiques, 1 espèce parasite d’Uromastix acanthinurus Bell, 1825 d’Algérie et 1 espèce parasite de Ctenosaura pectinata (Wiegmann, 1834) du Mexique. Une analyse non pondérée a généré un seul arbre de longueur minimale. Toutes les espèces du groupe interne forment un ensemble monophylétique et les oxyures parasites de Testudinidae sont aussi monophylétiques. L’espèce type du genre Alaeuris Thapar, 1925 est le taxon basal aux autres Pharyngodonidae parasites de Testudinidae. L’analyse confirme la monophylie du genre Thaparia Ortlepp, 1933; en revanche, elle indique que les genres Mehdiella Seurat, 1918 et Tachygonetria Wedl, 1862 sont paraphylétiques. La grande diversification du genre Tachygonetria est associée à la position des espèces dans le caecum. L’habitat ancestral est la paramuqueuse et l’habitat dérivé est le centre du caecum. Il semble donc que la spéciation récente de ce groupe se produise dans le centre du caecum. [Traduit par la Rédaction]

Bouamer and Morand

Introduction Several studies were devoted to the systematics of oxyuroid nematodes of the Palaearctic Testudinidae (Seurat 1918; Thapar 1925; Dubinina 1949; Petter 1961, 1963). Recently, four genera of Pharyngodonidae (13 species of the genus Tachygonetria Wedl, 1862; 4 species of the genus Mehdiella Seurat, 1918; 3 species of the genus Thaparia Ortlepp, 1933; and 1 species of the genus Alaeuris Thapar, 1925) 2004a, were cited (Bouamer and Morand 2000, 2002, 2003,2004b; 2004; Bouamer et al. 2001a, 2001b, 2003). The classification of these Pharyngodonidae is made difficult by the large numbers of species and individuals found in each host (more than 30 000 individuals can be observed in only one host caecum). The species often resemble each

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other and live in the same niche, which renders the identification difficult even at the generic level (see for example the diagnoses of the genera Tachygonetria Wedl, 1862 and Mehdiella Seurat, 1918 in Bouamer et al. 2001b, 2002). Moreover, the females in this group of nematodes are morphologically very similar, which can make the pairing of males and females in some species difficult. Bouamer and Morand (2003) showed that the species in the genus Tachygonetria vary greatly in terms of their morphology, which may question the monophyly of this genus. A similar comment may apply to the genus Mehdiella, whose diagnosis is based only on the presence or absence of a caudal point (Bouamer et al. 2003). It is difficult to find homology in the evolution of various morphological structures (such as in the cephalic and caudal

Received 5 March 2003. Accepted 8 September 2003. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 9 January 2004. S. Bouamer.1 Centre de Biologie et d’Ecologie tropicale et méditerranéenne, Laboratoire de Biologie Animale (Unité Mixte de Recherche du Centre National de la Recherche Scientifique 5555), Université de Perpignan, Avenue de Villeneuve, 66 860 Perpignan, France. S. Morand. Centre de Biologie et de Gestion des Populations (CBGP), Campus International de Baillarguet CS 30 016, 34 988 Montferrier sur Lez CEDEX, France. 1

Corresponding author (e-mail: [email protected]).

Can. J. Zool. 81: 1885–1893 (2003)

doi: 10.1139/Z03-166

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structures). This is particularly true in the cephalic structure. For example, the apical structures of the two sexes are seldom identical (i.e., the males have six lips, whereas the females have three lips). In this study, we provide a phylogenetic hypothesis of the relationships among the species of the four genera of Pharyngodonidae nematodes found in the Palaearctic Testudinidae. This study is based on a comparative analysis of morphological characters obtained from recent descriptions and redescriptions of the genera and species (Bouamer and 2004a, Morand 2000, 2002, 2003, 2004;2004b; Bouamer et al. 2001a, 2001b, 2003). The parsimonious tree obtained is used to discuss the taxonomic positions of the species, the monophyly of the genera, and the evolution of this group of nematodes. The evolution of the group relative to their localization in the host caecum is discussed on the basis of a mapping of the characters (paramucous, centre of the caecum).

Materials and methods We included 23 species of five genera: Alaeuris Thapar, 1925; Mehdiella Seurat, 1918d; Tachygonetria Wedl, 1862; Thaparia Ortlepp, 1933; and Ozailamus Dujardin, 1845. The list of species, host localities, and localization in the host caecum are given in Table 1. A data matrix was compiled using characters derived from the comparative morphology of 23 oxyurid species and 2 outgroup species based on 30 morphological characters (Table 2). The choice of characters is based on morphological descriptions and redescriptions2004a, of the four genera (Bouamer 2004b; and Morand 2000, 2002, 2003, 2004; Bouamer et al. 2001a, 2001b, 2003). Character states were coded using outgroup comparison (Watrous and Weheeler 1980; Maddison et al. 1984) with the following two reference species: Angusticaecum holopterum (Rudolphi, 1819) (Ascaridida), which is a parasite of Testudo graeca from Morocco, and Raillietnema bainae Petter, 1966 (Cosmocercidae), which is a parasite of Kinixys erosa (Schweigger, 1812) from Central Africa. Characters were treated as unordered and were unweighted, and coded as either binary or multistate (Maddison 1993). Character states found to be unclear (or missing) were scored as ambiguous (?). All analyses were run using PAUP* 4.0b10 (Swofford 2002) and the character state distributions were investigated using MacClade version 4 (Maddison and Maddison 2000). Heuristic searches in PAUP* 4.0b10 (Swofford 2002) were performed using the following options: branch swapping = tree bisection and reconnection (TBR) with MULPARS on. Associated statistics included the consistency index (CI), rescaled consistency index (RC), retention index (RI), and homoplasy index (HI). To examine character evolution with respect to each branch in the cladogram, characters were mapped onto the resulting consensus tree. The evolution of the group relative to their localization in the host caecum was discussed on the basis of a mapping of the characters (paramucous, centre of the caecum) using MacClade.

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Results Phylogenetic analysis The analysis gave one parsimonious tree with the following indices: tree length (L) = 87, CI = 0.71, HI = 0.29, CI excluding uninformative characters = 0.69, HI excluding uninformative characters = 0.30, RI = 0.82, and RC = 0.58. The trees were rooted using the species A. holopterum and R. bainae as the outgroups. One parsimonious tree is given in Fig. 1 and the character diagnostics are given in Table 3. The parsimonious tree is fully resolved except the trichotomies, which include Tachygonetria longicollis, Tachygonetria setosa and the adjacent clades; Tachygonetria poulini and Tachygonetria numidica and the adjacent clades; and the species of the genus Thaparia (Fig. 1). All species of the ingroup form a monophyletic group, with Ozolaimus sp. being the basal member of the ingroup. The oxyurid species of Testudinidae form a monophyletic group, with Alaeuris numidica being the basal member of the species parasitizing Testudinidae and the sister group of all other species of Pharyngodonidae. The position of Ozolaimus sp. and A. numidica can be explained by the shape of the caudal alae (character 23) and the pedunculate shape of the postcloacal papillae (character 17). The monophyly of the genus Mehdiella is not supported. The species of this genus form two paraphyletic groups in spite of a high CI (CI = 1) of the character 21 (absence or presence of a robust caudal point in males). The monophyly of the genus Thaparia is supported by two synapomorphies: characters 4.3 (shape of female lips; Fig. 3) and 10 (shape of oesophagus corpus). The group formed by the species of the genera Thaparia and Tachygonetria is not supported by any synapomorphy. In our analysis, the genus Tachygonetria is recognized as a paraphyletic group. Tachygonetria robusta is defined as a sister species of the genera Thaparia and Mehdiella. The monophyly of the other species of the genus Tachygonetria is not supported by any synapomorphy. Tachygonetria numidica and T. poulini form a paraphyletic group. Tachygonetria palearcticus and Tachygonetria conica form a monophyletic group that is supported by two synapomorphies (characters 2 and 4.1). This group is defined as a sister group of the monophyletic group formed by T. longicollis and T. setosa. Tachygonetria viviparas is defined as sister to the group formed by (Tachygonetria marocana and Tachygonetria khallaayounei, (Tachygonetria settatensis (Tachygonetria pusilla (Tachygonetria longicollis and Tachygonetria setosa)))) and the monophyletic group formed by Tachygonetria combesi and Tachygonetria dentata. Mapping of the localization in the host caecum Optimization of the localization of each species was obtained using MacClade (Maddison and Maddison 2000). The ancestral state is in the paramucous and the derived state is in the centre of the caecum (Fig. 2). This suggests that recent speciation in the group occurs in the centre of the caecum. © 2003 NRC Canada

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Table 1. List of Pharyngodonidae species and the outgroup species cited in this study. Nematode

Host

Localities

Alaeuris numidica (Seurat, 1918)

Testudo graeca Testudo hermanni Ctenosaura pectanita Testudo hermanni Testudo graeca Testudo hermanni Testudo horsfieldii Testudo graeca Testudo hermanni Testudo graeca Testudo graeca Testudo graeca Testudo graeca Testudo graeca Testudo hermanni Testudo graeca Testudo graeca Testudo graeca Testudo hermanni Testudo horsfieldii Testudo graeca Testudo graeca Uromastix acanthinurus Testudo graeca Testudo hermanni Testudo hermanni Testudo graeca Testudo horsfieldii Testudo hermanni Testudo hermanni Testudo graeca Testudo hermanni Testudo graeca Testudo hermanni Testudo horsfieldii Testudo graeca Kinixys erosa

Morocco Spain Mexico Spain Morocco Spain Iran Morocco Spain Morocco Morocco Morocco Morocco Morocco Spain Morocco Morocco Morocco France Iran Morocco Morocco Algeria Morocco Spain Spain Morocco Iran France Spain Morocco Spain Morocco Spain, France Iran Morocco Benin

Ozolaimus sp. Tachygonetria combesi Bouamer and Morand, 2002 T. conica (Drasche, 1884)

T. dentata Wedl, 1862 T. T. T. T. T.

khallaayounei Bouamer and Morand, 2004 longicollis (Schneider, 1866) numidica Seurat, 1918 marocana Bouamer and Morand, 2003 palearcticus (Petter, 1966)

T. poulini Bouamer and Morand, 2003 T. pusilla Petter, 1966 T. robusta (Drasche, 1884)

T. settatensis Bouamer and Morand, 2004 T. setosa Petter, 1966 T. vivipara Wedl, 1862 Thaparia thapari (Dubinina, 1949) Thap. bourgati Bouamer and Morand, 2000 Thap. carlosfeliui Bouamer and Morand, 2000 Mehdiella microstoma (Drasche, 1884)

M. petterae Bouamer, Morand, and Bourgat, 2001 M. stylosa (Thapar, 1925) M. uncinata (Drasche, 1884)

Angusticaecum holopterum (Rudolphi, 1819) Raillietnema bainae Petter, 1966

Discussion Taxonomic implication The genera Ozolaimus and Alaeuris are the basal members and the sister group of the genera Mehdiella, Thaparia, and Tachygonetria. The presence of caudal alae and the elongate or pedunculate shape of papillae are diagnostic characters of the genera Ozolaimus and Alaeuris. Species of the genera Mehdiella, Thaparia, and Tachygonetria have tiny caudal alae. The reduction of the caudal ala can be interpreted as a derived character. The genus Thaparia forms a monophyletic group and is the sister group of the genus Tachygonetria. These two genera share some characters in the cephalic and caudal ends: the presence of cut cephalic lips, pedunculate amphids, and a divided corpus in the genus Thaparia.

Localization in the caecum 2 ? 0 0

0 0 0 0 0 0 0 0 2

0 0 ? 1 1 1 1

2 2 2

? ?

Seurat (1918) revised the genus Tachygonetria and transferred two species (Oxyuris microstoma Drasche, 1884 and O. uncinata Drasche, 1884) to a new genus Mehdiella. Seurat (1918) based the diagnosis on the presence of a truncate tail in the genus Tachygonetria and the presence of a robust caudal point in the males of the genus Mehdiella. But Thapar (1925) synonymized the genus Mehdiella with the genus Tachygonetria in view of fact that the presence of a caudal point cannot be regarded as having generic value. Petter (1961, 1966) agreed with Seurat (1918) that Tachygonetria and Mehdiella are two distinct genera that can be distinguished by the presence of a truncate caudal end in the male of the genus Tachygonetria and the presence of a pointed caudal end in the male of the genus Mehdiella. In our analysis, the genus Mehdiella does not form a monophyletic group and is formed with two groups. The © 2003 NRC Canada

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Table 2. Matrix of character coding Character Taxon

1

2

3

4

4

4

5

5

5

6

7

8

9

9

10

11

12

Alaeuris numidica Tachygonetria combesi T. conica T. dentata T. khallaayounei T. longicollis T. marocana T. numidica T. palearcticus T. poulini T. pusilla T. robusta T. settatensis T. setosa T. vivipara Thaparia thapari Thap. bourgati Thap. carlosfeliui Mehdiella microstoma M. petterae M. stylosa M. uncinata Ozolaimus sp. Angusticaecum holopterum Raillietnema bainae

0 2 2 2 2 2 2 2 2 2 2 1 2 2 1 1 1 1 1 1 1 1 0 0 0

1 1 2 1 1 1 1 1 2 1 1 0 1 1 1 0 0 0 1 1 1 1 1 1 1

1 1 0 1 1 1 1 1 0 1 1 1 1 1 1 0 0 0 0 0 0 1 1 0 0

0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 0

0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 ? 0

0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 1 1 1 0 0 0 0 ? 0

1 ? 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 0 1 1 ? 1

0 ? 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 ? 0 0 0 ? 0

0 ? 0 1 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 ? 0 2 0 ? 0

1 2 1 2 2 1 1 1 1 1 1 0 0 1 1 1 1 1 1 3 3 3 1 ? 1

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 ? 0 1 1 1 1

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 1 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0

2 1 0 1 1 0 1 1 1 1 0 0 1 0 1 3 3 3 2 0 0 2 2 2 2

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ? ? ?

0 0 0 0 0 1 0 0 0 0 1 0 1 1 0 0 0 0 0 0 0 ? ? ? ?

Note: Refer to Appendix A for an explanation of the character states.

Fig. 1. Results of the cladistic analysis (one parsimonious tree) of the Palaearctic parasite species of Testudinidae.

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13

14

14

15

16

17

17

18

19

20

21

22

23

24

25

26

27

28

29

30

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 ? ?

0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 ? ?

5 0 2 0 0 3 0 1 2 1 3 2 0 3 0 1 1 1 4 4 4 4 5 ? ?

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1

0 0 1 0 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 3 ? ?

4 0 0 0 1 0 1 2 0 2 1 1 0 0 0 1 1 1 3 3 3 3 2 ? ?

2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 2 ? ?

2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 ? ?

0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 0 1 0 0 1 ? ? ?

? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 2 2

1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 0 0 2 1 1

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? 1 1

2 1 3 1 2 2 2 1 2 1 2 1 2 2 1 1 1 1 3 1 1 1 0 ? ?

0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 ? ?

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 ? ?

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1

Palaearctic and Ethiopian species of this genus were divided into three groups (Bouamer et al. 2003). The only character that maintains its cohesion is the presence of the robust caudal point of the tail in males. The basal position of Mehdiella microstoma can be explained by the shape of the mouth and the number of lips in the female and by the anterior end without vesicular swellings in both sexes. Mehdiella stylosa and Mehdiella petterae present a tail with a caudal point less robust than that of M. microstoma and Mehdiella uncinata. In certain species of the genera Tachygonetria (T. dentata, T. numidica, T. poulini) and Thaparia (Thaparia thapari), a hyaline point is observed that can be compared with the caudal point in the genus Mehdiella. The numerous morphological differences in the species of the genus Mehdiella (Bouamer et al. 2003) and the results of this study allow us to divide this genus in two groups: (M. stylosa, M. petterae) and (M. microstoma, M. uncinata). The basal position of T. robusta in the genus Tachygonetria can be explained (i) by the shape of the female oral opening (cephalic left lip, stretching from the ventral side to the dorsal side, and two right lips of subequal size), which is similar to those from females of M. microstoma, and (ii) by the shape of the body (character 1) in the genus Thaparia and the structure of the cephalic end of M. stylosa and M. petterae. Moreover, the genus Tachygonetria is defined as a paraphylelic group with T. robusta, which is a sister

species of the genus Thaparia. This observation questions the positions of T. robusta among the genus Tachygonetria. In spite of the similarity in morphology of the body, the structure of the genital cone, the cloacal papillae, and the tail shape, the position in our analysis of T. poulini and T. numidica can be explained by the numerous morphological differences in the shapes of the cephalic lips and cephalic end. Tachygonetria numidica shows two rows of cephalic lips, the anterior opaque 2004a). lips and the posterior transparent lips (Bouamer and Morand 2003, 2004). This single structure of the cephalic end in the genus Tachygonetria and the basal position in the phylogeny question the position of the T. numidica in the genus. Bouamer and Morand (2003) considered the genus Tachygonetria a complex group with at least four subgroups. The only character that maintains the cohesion of the genus is the presence of a truncated tail in the male (character 21), but this character cannot be considered a synapomorphy. The numerous morphological differences between the species of the genus Tachgonetria and the results of this study question the current status of the genus. Evolution and implication According to Schad (1963a, 1963b), Petter (1966), and personal observations, each species is selectively distributed along the caecum of Testudo graeca. Schad (1963a, 1963b), by freezing and sectioning the host caecum, established the fact that the parasites are selectively distributed radially as © 2003 NRC Canada

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Fig. 2. Mapping of the localization of the Palaearctic parasite species of Testudinidae in the host caecum on a cladogram.

Fig. 3. Shape of the female lips of the Palaearctic parasite species of Testudinidae.

Fig. 4. Shape of the anterior cloacal lips of males of the Palaearctic parasite species of Testudinidae.

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Table 3. List of each character and the corresponding consistency index (CI) of characters from the original-set one parsimonious tree.

Car1 Car2 Car3 Car4.1 Car4.2 Car4.3 Car5.1 Car5.2 Car5.3 Car6 Car7 Car8 Car9.1 Car9.2 Car10 Car11 Car12.1 Car12.2 Car13 Car14.1 Car14.2 Car15 Car16 Car17.1 Car17.2 Car18 Car19 Car20 Car21 Car22 Car23 Car24 Car25 Car26 Car27 Car28 Car29 Car30

Character

CI

Shape of body Shape of cephalic end Shape of male lips Shape of female lips Shape of female lips Shape of female lips Shape of female oral opening Shape of female oral opening Shape of female oral opening Shape of male oral opening Shape and position of female cephalic papillae Shape and position of male cephalic papillae Shape and position of amphids Shape and position of amphids Shape of oesophagus corpus Shape of precloacal membrane Shape of anterior cloacal lips Shape of anterior cloacal lips Shape of posterior cloacal lips Shape of postcloacal papillae Shape of postcloacal papillae Number of cloacal papillae Ad-anal papillae Shape of precloacal papillae Shape of precloacal papillae Position of caudal papillae Shape of caudal papillae Shape of tail Tail truncate or non-truncate Vesicular swellings Caudal alae Oesophagus bulb Pharynx Precloacal membrane Shape of postcloacal lips Position of postcloacal lips Number of postcloacal papillae Number of spicules

1.000 0.667 0.250 1.000 0.500 1.000 0.500 0.500 0.667 0.500 1.000 1.000 1.000 0.600 0.333 1.000 0.500 1.000 1.000 0.500 0.833 1.000 1.000 1.000 0.500 1.000 1.000 0.200 1.000 0.500 1.000 1.000 1.000 1.000 0.500 1.000 0.500 1.000

Note: Car, character. Shapes of the female lips and anterior cloacal lips of males are illustrated in Figs. 3 and 4.

well as longitudinally in the host caecum; hence, some of the parasites are closely associated with the mucosa. There are two groups. One group of species prefers the centre of the caecum (central portion of the anterior portion of the caecum) and the second group prefers the paramucus. Petter (1966) reported that two groups of species can be identified according to the preference of the ecological niche and the cephalic structure. The first group contains all Tachygonetria species except T. robusta and all Thaparia spp. The second group contains T. robusta, A. numidica, and all Mehdiella spp. The first group is more diversified with 13 species and the second group is relatively poor with 6 species (Bouamer and Morand 2000, 2002, 2003, 2004; Bouamer et al. 2001a, 2001b, 2003).

2004a, 2004b;

According to this study, the species of the genus Tachygonetria occupy the crown of the cladogram. The speciation gives rise to a highly diversified group with similar species difficult to distinguish (Bouamer and Morand 2003). According to Petter (1966), a similar diversification of the genus Tachygonetria occurs in the Ethiopian region (South Africa and Madagascar). The phylogenetic position and the ecological data of the genus Tachygonetria can be explained by the large number of species in the genus. The low number of species in the genera Alaeuris, Mehdiella, and Thaparia is linked with a low morphological variation compared with species of the genus Tachygonetria. According to the results of this phylogeny, the ancestral state is in the paramucous and the derived state is in the centre of the caecum (Fig. 2). This suggests that recent speciation in the group occurs in the centre of the caecum. In spite of the relative richness of the morphological characters (based primarily on our previous studies) of this group, the extent of homoplasy indicates that robust phylogenetic analyses depending only on morphology will be difficult. The study of other Pharyngodonidae members from the Ethiopian, Neotropical, and Nearctic regions will most certainly provide new information (including morphological and molecular data on both hosts and parasites), which should improve our resolution, and will allow future studies to develop more precise explanations regarding (i) the processes driving the patterns of diversification in this group and (ii) the cladistic biogeographical analysis of the distribution of these nematodes and their Testudinidae hosts.

Acknowledgements We thank the anonymous reviewers for their helpful comments and suggestions that improved the manuscript.

References Bouamer, S., and Morand, S. 2000. Oxyuroids of Palearctic Testudinidae: new definition of the genus Thaparia Ortlepp, 1933 (Nematoda: Pharyngodonidae), redescription of T. thapari thapari, and descriptions of two new species. Comp. Parasitol. 67: 169–180. Bouamer, S., and Morand, S. 2002. Description of T. combesi n. sp., and redescriptions of four species of the genus Tachygonetria Wedl, 1862 (Nematoda: Pharyngodonidae), with a new diagnosis of the genus. Syst. Parasitol. 53(2): 121–139. Bouamer, S., and Morand, S. 2003. Descriptions of two new species of the genus Tachygonetria Weld, 1862 (Nematoda — Pharyngodonidae) and discussion of the relationships among the species of the genus. Parasitol. Res. 91(1): 68–73. Bouamer, S., and Morand, S. 2004. Descriptions of Tachygonetria africana n. sp. and T. pretoriensis n. sp., and redescriptions of two species of Tachygonetria Wedl, 1862 (Nematoda: Pharyngodonidae) parasite of Geochelone pardalis (Testudinidae) from South Africa. Syst. Parasitol. In press. Bouamer, S., Morand, S., and Bourgat, R. 2001a. Oxyuroids of Palaearctic Testudinidae — new definition of the genus Alaeuris Seurat, 1918 (Nematoda: Pharyngodonidae), redescription of Alaeuris numidica (Seurat, 1918). J. Parasitol. 87(1): 128–133. Bouamer, S., Morand, S., and Bourgat, R. 2001b. Redescription of Mehdiella microstoma and description of Mehdiella petterae sp. n., with a new definition of the genus Mehdiella Seurat, 1918 © 2003 NRC Canada

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(Nematoda: Pharyngodonidae). Folia Parasitol. (Prague), 48: 132–138. Bouamer, S., Morand, S., and Karat, M. 2003. Redescription of four species of Mehdiella from Testudinidae with a key to the species of the genus. Parasite. In press. Maddison, D.R., and Maddison, W.P. 2000. MacClade 4: analysis of phylogeny and character evolution. Sinauer Associates, Inc., Sunderland, Mass. Maddison, W.P. 1993. Missing data versus missing characters in phylogenetic analysis. Syst. Biol. 42: 576–581. Maddison, W.P., Donoghue, M.J., and Maddison, D.A. 1984. Outgroup analysis and parsimony. Syst. Zool. 33: 83–103. Petter, A.J. 1961. Redescription et analyse critique de quelques espèces d’Oxyures de la tortue grecque (Testudo graeca L.) Diversité des structures céphaliques. Ann. Parasitol. Hum. Comp. 10: 648–671. Petter, A.J. 1963. Equilibre des espèces dans les populations de Nématodes parasites du côlon des tortues terrestres. C. R. Acad. Sci. Paris, 257: 2152–2154.

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Appendix A Table A1. Character traits for the character matrix used to distinguish the phylogeny of Palaearctic Pharyngodonidae parasite species of Testudinidae. Character number 1

Character trait

Character-state score

2

Ratio of maximum body width to body width at level of cloaca Shape of anterior end

3 4

Number of male lips Shape of female lips

5

Shape of female oral opening

6 7 8 9

Shape Shape Shape Shape

Three states chosen arbitrarily: 0, ratio between 0.65 and 0.70; 1, ratio between 0.77 and 0.82; 2, ratio greater than 0.86 Three states: 0, broad (large) and flattened; 1, slightly broad (blunt); 2, attenuated or pointed Two states: 0, three lips; 1, six lips Four states: 000, simple equal transparent; 001, equal transparent indented; 100, simple equal opaque, 011, unequal asymmetrical transparent (Fig. 3) Five states: 000, dodecagon; 100, triangular with sides equal; 110, triangular with sides unequal; 101, semi-triangular; 102, circular Three states: 0, hexagonal; 1, triangular; 2, semi-triangular; 3, circular Two states: 0, sessile; 1, projecting Three states: 0, sessile; 1, projecting Five states: 00, sessile and internal with a small slit; 01, sessile and external with a large slit; 10, slightly projecting and internal with a small slit; 02, slightly projecting and external; 03, pedunculate and external Two states: 0, corpus divided; 1, corpus not divided Two states: 0, thin; 1, thick Seven states: 00, tiny lobes; 01, slightly robust lobes; 02, lobes robust with a pointed extremity; 03, large and robust lobes; 13, curved lobes; 04, flat and robust lobes rectangular distally; 05, robust and conical shape lobes (Fig. 4) Two states: 0, postcloacal lips with narrow lateral alae; 1, postcloacal lips without narrow lateral alae Six states: 00, rosette-shaped and large with two tapered points; 10, kidney shape and transversely to the anterolateral symmetry of the body; 01, lengthened and oblique to the anterolateral symmetry of the body; 02, lengthened to the anterolateral symmetry of the body; 03, longitudinal to the anterolateral symmetry of the body, slightly pedunculate with a small nipple; 04, longitudinal to the anterolateral symmetry of the body, slightly pedunculate with a large nipple; 05, pedunculate shape (Fig. A1) Two states: 0, two pairs; 1, more than two pairs

of male oral opening of female cephalic papillae of male cephalic papillae and position of amphides

10 11 12

Shape of oesophagus corpus Precloacal membrane Shape of anterior cloacal lips

13

Shape of posterior cloacal lips of male

14

Shape of postcloacal papillae

15

Number of cloacal papillae

© 2003 NRC Canada

Bouamer and Morand

1893

Table A1 (concluded). Character number 16 17

Character trait Ad-anal papillae Shape of preanal papillae

18 19 20 21 22 23 24 25 26 27

Position of caudal papillae Shape of caudal papillae Shape of tail Robust caudal point in males Vesicular swellings Caudal alae Oesophagus bulb Pharynx Preanal membrane Shape of postanal lip

28 29 30

Position of postanal lip papillae Number of postanal lip papillae Number of spicules

Fig. A1. Shape of the postanal papillae of males of the Palaearctic parasite species of Testudinidae.

Character-state score Two states: 0, present; 1, absent Six states: 00, rosette-shaped; 10, rosette-shaped with a thin posterior expansion; 01, rosette-shaped with a robust posterior expansion; 02, lengthened; 03, slightly pedunculate; 04, pedunculate (Fig. A2) Three states: 0, lateral; 1, lateroventral; 2, ventral Two states: 0, sessile; 1, slightly projecting; 2, projecting Three states: 0, robust and straight; 1, robust and curved Two states: 0, absent; 1, present Two states: 0, present; 1, absent Two states: 0, absent; 1, slightly developed; 2, developed Two states: 0, absent; 1, present Two states: 0, absent; 1, present Two states: 0, absent; 1, present Four states: 0, distal end broader than the proximal end; 1, simple; 2, proximal end broader than stalk; 3, distal end with side expansion Three states: 0, lateral; 1, lateroventral; 2, ventral Two states: 0, one papilla; 1, two papillae Two states: 0, one spicule; 1, two spicules

Fig. A2. Shape of preanal papillae of males of the Palaearctic parasite species of Testudinidae.

© 2003 NRC Canada