Morphological support for the phylogenetic positioning of Pentastomida and related fossils

Pentastomida is a group of parasites that infects the respiratory tracts of vertebrates. They have a mixture of annelid and arthropod characteristics. For that reason, the phylogenetic relationships of the pentastomids have been controversial in proposals of metazoan phylogeny. Forty-seven characters were selected for the analyses of the taxa Annelida, Arthropoda, Kinorhyncha, Loricifera, Nematoda, Nematomorpha, Onychophora, Pentastomida, Priapulida and Tardigrada. The analyses with PAUP resulted in a single shortest cladogram (length 89, ci 0.78, ri 0.86). Our results indicate that Pentastomida is a transitional group between the Arthropoda and some of the Nemathelminth groups such as Nematoda and Nematomorpha.


Introduction
Pentastomida are parasites that infect the respiratory tracts of vertebrates.About 131 species are known, classifi ed into seven recent families and three Middle Cambrian fossil groups (# 450 million years) (Almeida and Christoffersen, 1999).These endoparasites have a mixture of annelid and arthropod characteristics.For this reason, the phylogenetic relationships of the pentastomids have been controversial in proposals of metazoan phylogeny.
Following the fi rst descriptions of pentastomids, the group was related to Platyhelminthes (Fröhlich, 1789;Humboldt, 1811) or Nematoda (Diesing, 1850).These hypotheses were soon abandoned, mainly because Dujardin (1845) described striated muscles in Pentastomida.This represented the fi rst step towards relating Pentastomida with Arthropoda.Among the fi rst supporters of this new proposal, controversies surfaced over which group of the arthropods would be closest to the pentastomids.Van Beneden (1849) proposed that the new parasites were crustaceans, while Leuckart (1860) suggested that pentastomids represented modifi ed mites.This latter idea was supported by Sambon (1922), who emphasized the analogous example of how lice became adapted to endoparasitism.Upon studying the embryonic development of Reighardia (Pentastomida), Osche (1959Osche ( , 1963) ) proposed that the Pentastomida were phylogenetically related to the Myriapoda.Heymons (1935) presented a series of anatomical observations to suggest that pentastomids had greater affi nity with the Annelida.
Nevertheless, for Cuénot (1949), Nicoli (1963) and Haffner (1977), pentastomids are neither annelid nor arthropod.These authors argued that pentastomids occupied a unique intermediary position between Annelida and Arthropoda.This latter hypothesis was also supported by Self (1969) and Haugerud (1989), who that argued that Pentastomida should be considered a phylum with indeterminate phylogenetic relationships.
The goal of this paper is to deduce the phylogenetic position of the Pentastomida in relation to the Arthropoda and Annelida, thus reconsidering the two main hypotheses that are widely debated today: Pentastomida are closely related to the Arthropoda or even belong as a subgroup (of crustaceans) in this taxon; Pentastomida represent a transitional group between annelids and arthropods, or are included in the Ecdysozoa.

Material and Methods
The primary source of data was the literature pertaining to all studied groups.We also examined specimens of pentastomids deposited in the collections of the Universidade Regional do Cariri (0021, 0022, 0023, 0043, 0044) and FIOCRUZ (CHIOC 20420, 20421, 20429, 20431, 20432, 20434, 20439, 20402, 30325, 30326, 30327, 20328, 30329a, 30330a-b, 29176a-m, 29185a-o), in addition to non-catalogued specimens of nematodes and onychophorans from the Laboratório de Invertebrados Marinhos and the Laboratório de Entomologia of the Universidade Federal da Paraíba.These examinations allowed the selection and confrontation of comparative data on morphology and anatomy described in the literature contrasting with biological specimens.
The taxa selected for analyses were based on the proposals of Abele et al. (1989)
Binary and multistate non-ordered characters were admitted in the analyses (see Wilkinson, 1992 and1995;Slowinski, 1993).Polarization of characters was obtained by the method of comparison of multiple outgroups (Nixon and Carpenter, 1993;Amorim, 2002).All studied groups were considered a priori as being monophyletic.The selected outgroups were Platyhelminthes, Sipuncula and Mollusca, following the results obtained by Almeida and Christoffersen (2001) and Almeida et al. (2003).
The phylogenetic analyses followed Hennigian principles (Hennig, 1966;Wiley, 1981;Amorim, 2002) and were carried out using the software PAUP (version 3.1.1;Swofford, 1999).For the construction of the matrix and the graphic visualization of cladograms, the program MacClade (Maddison and Maddison, 1992) was used.The commands used in the analyses with PAUP were according to the branch-and-bound option.
The fact that subgroups of arthropods were not used as terminal taxa obviously reduces the ability of the present analysis to test the hypothesis of relationships of the Pentastomida with certain groups of crustaceans.This paper explores the relationships between the topology obtained in the analysis and new paleontological evidence related to the group.

Results
Forty-seven characters were selected (see list below) for the construction of a data matrix for analyses (Table 1).The analyses with PAUP resulted in a single shortest tree, which is presented in fi gure 1.
List of analyzed characters with their respective states (0-4).

Discussion
Analyses using the molecular marker 18S rRNA led to the proposal of the taxon Ecdysozoa, which includes all animals presenting ecdysis (periodical moulting during development): Pentastomida, Onychophora, Tardigrada, Arthropoda, Nematoda, Nematomorpha, Priapulida and Loricifera (see Aguinaldo et al., 1997;Giribet and Ribera, 1998;Giribet, 2003).The congruence between molecular and morphological data was also demonstrated by Schmidt-Rhaesa et al. (1998).Our analysis corroborates the monophyly of the Ecdysozoa.However, data derived from the DNA analysis diverge in different degrees from morphological studies.
The problem of the incongruence between morphological and molecular data has already been discussed by Wägele (1996) and Nielsen (2001).These authors demonstrated that the results of molecular analyses vary according to the selected species, sample size, and particular methods of analysis used.Although still controversial, there is additional evidence that at least the sequences of 18S rRNA may not contain consistent phylogenetic information for cladogenetic events as old as the Median Cambrian (Philippe et al., 1994).Regarding the phylogenetic positioning of the Pentastomida, our results do not corroborate conclusions obtained based on spermatological (Wingstrand, 1972;Riley et al., 1978;Storch and Jamieson, 1992) or molecular data (Abele et al., 1989;Lavrov et al., 2004).Our results agree with the proposals of Waloszek and Müller (1994), Maas and Waloszek (2001) and Waloszek et al. (2006), in which neither the Cambrian fossils nor the recent forms of pentastomids present any morphological evidence for their inclusion within any specifi c group of euarthropods.
Among the characters studied we stress the implications of metamery (#15) and the number of metameres (#16) for the phylogenetic position of the Pentastomida and for the history of the fossils related to them.In the fi rst case (#15) we agree with Aguinaldo et al. (1997) and Lavrov et al. (2004), whose results indicate that the absence of metamery in ecdysozoan groups (e.g., Nematoda and Nematomorpha) must be secondary.
In the second case (#16), the reduction in the number of metameres is a character related to the reduction in body size of the animals and to adaptations to other modes of life (for example, parasitism).In this evolutionary scenario the position of the Pentastomida in our results would indicate that, before the loss of metamery, some groups reduced their number of somites.According to Moura and Christoffersen (1996), depending on some premises of homology of the cephalic structures, there are 22 metameres present in the groundplan of the Arthropoda, and this number becomes reduced to 13 in higher arthropods.Tardigrada has only eight nervous ganglia and Pentastomida has seven.This reduction is also related to the reduction in the size and number of the locomotory appendages.In Tardigrada there are three pairs of legs for locomotion.The fourth pair, located in the posterior region of the body, similar to those of †Aysheaia and Onychophora, has the function of embracing the substrate, only occasionally being used for walking.However, in the basic plan of the Tardigrada there are seven pairs of appendages, the fi rst three being reduced on the cephalic tagma.In Pentastomida, there are two distinct pairs of appendages, as well as two pairs of vestigial anterior appendages in the fossil †Bockelericambria (Waloszek and Müller, 1994) and in the groundplan of the Pentastomida.Almeida and Christoffersen (1999) considered the basic number of segments in Pentastomida to be seven.
The evolutionary scenario presented herein has profound implications for the positioning of several enigmatic Cambrian fossils.Integrating our results with those obtained by Eloy (2001), fossils of some lobopodian animals must be positioned at the stemgroup of Ecdysozoa.Others, such as †Anomalocaris and related forms, must belong to the stem-group of Arthropoda (Figure 2).Hou and Chen (1989) described fi ve pairs of appendages for †Facivermis, but Hou and Bergström (1995) considered the differences between †Facivermis and the lobopodians to be profound.Hou and Bergström (1995) argued that the appendages of †Facivermis were restricted to the anterior region of the body and were arranged along the midventral line of the body, not ventrolaterally as in lobopodians (Figure 2).The appendages become vestigial in the heads of some of the nemathelminths (Chang et al., 1998a and1998b).However, we understand that these differences are not signifi cant, because the appendages of †Facivermes are similar in shape to those of the remaining lobopodians.Furthermore, other characters (e.g., the presence of cutaneous papillae) indicate that †Facivermes must be interpreted as a lobopodian.Its proximity to Pentastomida and other nemathelminths is also indicated by the fact that its long and delicate spines at the tips of the lobopodes were probably not used for walking, but rather for anchorage to the substrate or to a host animal.
The Pentastomida must, hence, have evolved from an ancestor similar to †Facivermes and seem to be related to Tardigrada and other groups of nemathelminths.Today, even though very modifi ed towards the parasitism of terrestrial vertebrates, pentastomids still retain vestiges of the early stages of the evolution of a group that originated more than 450 million years ago and that resulted in a great diversity of ecdysozoans, containing groups as different as nematodes and priapulids.The presence of the onychophorans as the sister clade of the Arthropoda differs from other recent phylogenetic studies of the group that point to the Panarthropoda (including Tardigrada and Arthropoda) as monophyletic, implying that the presence of the tritocerebral ganglia would be a synapomorphy.We agree with this later hypothesis, and the connection of Onychophora and Arthropoda in a clade may result from the undersampling of features involving onychophorans and secondary losses in the evolution of the group.et al., 1995), †Hallucigenia (modifi ed from Ramsköld and Hou, 1991), †Aysheaia (modifi ed from Whittington, 1978), †Anomalocaris (modifi ed from Collins, 1996), and †Facivermis (modifi ed from Delle Cave and Simonetta, 1991).

TABLE 1 :
Character matrix of data for the investigation of interrelationships of Pentastomida.0-4,characterstates arranged into transformation series; "?", uncertainty or absence of information on the state of a character.W. O.Almeida et al.