Phylogenetic analysis of Petaloproctus ( Maldanidae : Polychaeta ) , with description of a new species from southeastern Brazil

We used morphological data for a phylogenetic analysis of the genus Petaloptoctus (Maldanidae: Polychaeta). We found three most parsimonious phylogenetic trees with length = 37, Ci = 0.89 and ri = 0.92. the genus Petaloproctus is monophyletic, and is supported by a strongly arched prostomium, pinnate capillary setae, and an anal cup with a reduced dorsal border. our proposal for the systematisation of the subgroups of Petaloproctus is ((P. tenuis (P. terriculus + P. neoborealis)) (P. ornatus (((P. cirratus + P. dentatus) (P. macrodentatus (P. borealis (P. vallejoi n. sp. + P. socialis)))))). We also regard nicomachinae as a monophyletic group with the following synapomorphies: prostomium short and arched; nuchal grooves short and curved; cephalic plate lost; anal pore opening on margin of anal plate. Monophyly of Micromaldane and Nicomache was also supported in the analysis. Petaloproctus vallejoi n. sp. has 19 setigerous and one asetigerous pre-anal segment. the prostomium is rounded, forming a keel with curved laterals. nuchal grooves are short and deep, strongly curved outwards. Each neuropodium has one acicular spine on setigers 1-3. the pygidium has a large ventral border, and a reduced dorsal border. the anus is terminal, and close to the margin of the anal plate, surrounded by divergent folds.

introDuCtion Maldanids are sedentary, tube-builder polychaetes, commonly known as bamboo worms because of the elongate median segments with globose tori at the extremities (fauchald and rouse, 1997). there are no head appendages, but some species may have a cephalic plate. the keel-shaped prostomium is completely fused to the peristomium. the pygidium may be cone-shaped and bear a truncate plate, or this plate may be within the anal cup (Day, 1967;hartmann-schröder, 1971;fauchald, 1977;imajima and shiraki, 1982).
Maldanids are found from the intertidal region to the deep sea (Arwidsson, 1907;Chamberlin, 1919). their tubes are constructed both horizontally, with sand and shell fragments under rocks, or vertically, in sandy bottoms with fine and hyaline sand (De Assis et al., 2007a, b).
the subfamily nicomachinae was proposed by Arwidsson (1907) to include three genera: Micromaldane Mesnil, 1897, Nicomache Malmgren, 1865and Petaloproctus Quatrefages, 1865 the genus Petaloproctus Quatrafages, 1865 has nine valid species (De Assis et al., 2007b). the main characters conventionally used to separate species are the form of the anal plate and of the funnel. three of the currently known species have a smooth anal border and the other six have a cirri-rimmed anal plate (De Assis et al., 2007a).
the aim of the present study is to propose the first phylogenetic analysis of Petaloproctus and to describe P. vallejoi n. sp. from shallow shelf waters off the coast of são Paulo, southeastern brazil. We also tested the monophyly of the subfamily nicomachinae, and its genera.
We used illustrations available in the primary literature in order to compare homologous structures throughout the clade.
the analysis was carried out using MacClade 3.05 (Maddison and Maddison, 1992) for editing the data matrix. the trees were constructed with the program Phylogenetic Analysis using Parsimony -PAuP version 3.1.1. (swofford, 1993). heuristic tree searches were executed with PAuP default settings, with tbr, and using the closest stepwise addition sequence with 50 replicates. Zero-length branches were collapsed, MuLPArs was activated, and AC-CtrAn was used for character-state optimisation.
six characters were coded as having two successive states, and eleven were treated as absent/ present. three characters were coded as having more than two states. All characters were treated as unordered (non-additive). Character polarisation was obtained with multiple outgroups (Ax, 1987;nixon and Carpenter, 1993;Christoffersen and Araújode-Almeida, 1994;Von sternberg, 1997;Amorim, 2002). non-applicable states were coded as "-".
specimens to the new species were collected off the coast of são Paulo, in southeastern brazil. the material was anesthetised with menthol, fixed in 10% formalin and preserved in 70% alcohol. the animals were observed with a Zeiss stereoscopic microscope. the setae, rostral hooks and acicular spines were observed with an olympus bX41compound microscope. All illustrations were made with a camera lucida. Measurements are in millimetres.
We propose synapomorphies for the subfamily nicomachinae, and for the genera Micromaldane, Nicomache and Petaloproctus. in the taxonomic remarks we compare the new species to all previously known species. specimens are deposited in the reference collection of invertebrates at the Centro de Estudos do Mar, universidade federal do Paraná, brazil (MCEM-ufPr).
A rounded prostomial anterior is a plesiomorphic character state found in most species of the outgroup and in some species of Petaloproctus (De Assis et al., 2007a). the apomorphic condition of this character is shared by the species P. neoborealis, P. terriculus and P. tenuis.
the prostomium is always very long in the Euclymeninae, all along the cephalic plate. the shorter prostomium is an apomorphic state for the nicomachinae.
the prostomium is keel-shaped and fused to the peristomium in the maldanids (Day, 1967;fauchald, 1977;imajima and shiraki, 1982;De Assis et al., 2007a, b). this keel is straight in the most basal maldanids, and mainly in those with a cephalic plate. the plate is lost and the keel becomes arched. in Petaloproctus the keel is more arched than in Micromaldane and Nicomache. in this paper, this strongly arched keel is interpreted as an apomorphic condition for the genus (Arwidsson, 1907;imajima and shiraki, 1982;De Assis et al., 2007a). 04. two clusters of eyespots: (0) absent; (1) present.
in maldanids the nuchal grooves are usually of the same length as the prostomium. their reduction represents an apomorphic state for the genus Petaloproctus (De Assis et al., 2007a). 07. Cephalic plate: (0) absent; (1) present.
the presence of these spines is a synapomorphy for Euclymeninae, Nicomache and Petaloproctus (De Assis et al., 2007a, b), but it is reverted in the Micromaldane.
in Euclymeninae only Clymenella Verrill, 1873 and Euclymene Verrill, 1900 have acicular spines with a strongly curved apex, looking like a hook. in Nicomache and most species of Petaloproctus the acicular spines are slightly curved. the straight form is found in P. vallejoi n. sp. +P. socialis + P. borealis + P. cirratus + P. ornatus + P. dentatus + P. macrodentatus.
the acicular spines are modified setae, usually found in the first setigerous segments, in most members of maldanids. this represents an important character for the taxonomy of the family. Acicular spines with obtuse points are an apomorphic character for species P. vallejoi n. sp. + P. socialis + P. borealis + P. cirratus + P. dentatus + P. macrodentatus (Day, 1967;imajima and shiraki, 1982;Lee and Paik, 1986).
spinous capillaries are found in most species of the Euclymeninae and nicomachinae. however, their bases are always smooth. in Petaloproctus vallejoi n. sp. + Petaloproctus socialis, the capillaries basis are ruffled; this is a synapomorphy for both species (De Assis et al., 2007a, b).
the anal cup is an apomorphy for the Euclymeninae and nicomachinae. however, the reduction of the anal cup is the most important apomorphic character for genus Petaloproctus (Arwidsson, 1907;Day, 1967;De Assis et al., 2007a). Petaloclymene Green, 1997 (Euclymeninae) presents a slightly reduced dorsal border of the anal cup, which is laterally flattened (Green, 1997). notwithstanding, the species in this genus have a cephalic plate, an apomorphy to notoproctinae, Euclymeninae and Maldaninae, and herein is interpreted as loss in the nicomachinae. the homology relationship for this character is represented in figure 1.
17. ornamentation of the border of anal cup: (0) absent; (1) present. this character has been often used to separate species of Petaloproctus. this character is interpreted as homoplastic condition arising in P. vallejoi n. sp. + P. socialis + P. borealis and for P. neoborealis (imajima and shiraki, 1982;Lee and Paik, 1986;De Assis et al., 2007a).
this character is an apomorphic condition for Micromaldane, which has no anal cirri (rouse, 1990).
in the genus of analysis, the anal cirri always vary in length. however, in P. dentatus and P. cirratus the anal cirri is of very small. the border is saw-like. imajima and shiraki (1982) compare the two species, agreeing that they are closely related on the basis of the form of the anal cirri, but differ nevertheless because the first has 29 triangular cirri and the second has only 15. 20. Anal pore: (0) opening at apex of anal cone; (1) opening on margin of anal plate.
Most maldanids taxa are diagnosed by characters related to the pygidium. in Euclymeninae and nicomachinae, both having an anal cup, the anus is terminal on the anal cup. however, in Euclymeninae the anal pore is projected beyond the anal plate. in most genera, the anal pore is inserted on a short projection, but in the Maldanella Mcintosh, 1885, Microclymene Arwidsson, 1907 andPraxillella Verrill, 1881, the anal pore is inserted on a large projection and becomes exposed beyond the anal cup (Day, 1967;imajima and shiraki, 1982;Lee and Paik, 1986;fauchald, 1977). in the nicomachinae, the anal pore is placed close to the margin of the anal plate, representing an apomorphic condition of this subfamily. rEsuLts

Phylogenetic analysis
We analysed 20 morphological characters, treated as non-ordered and with equal weighting. Characters resulted in three most parsimonious trees with length = 37, Ci = 0.89, and ri = 0.92. the first tree represents the strict consensus ( fig. 2), in which the ingroup was well-supported in all trees. the second tree represents one of the three trees of same length and same indices, accepted as representative of the phylogeny of genus Petaloproctus ( fig. 3). Character coding is presented in table 1. our proposal for the systematisation of Petaloproctus into subgroups is ((P. tenuis (P. terriculus + P. neoborealis)) (P. ornatus (((P. cirratus + P. dentatus) (P. macrodentatus (P. borealis (P. vallejoi n. sp. + P. socialis)))))).

Petaloproctus vallejoi n. sp. (figs. 4 and 5)
Description. holotype complete, with 19 setigerous segments and one asetigerous preanal segment. total body length, 65 mm. Paratype represented by a posterior fragment measuring 3.7 mm in length. head 1.5 mm long ( fig. 4A). first setiger very short, 1.5 mm long and 2 mm wide. fourth setiger 4 mm long and 2 mm wide; setigers 5-16 measure 6 mm long and 2 mm wide. remaining segments decrease gradually in size. Posterior region formed by an preanal asetigerous segment and a funnel with a completely reduced dorsal border. the pygidium is 2 mm long and 3 mm wide.
Prostomium fused to peristomium, forming a well-defined head. Prostomium rounded anteriorly, forming a keel arched, with slightly curved sides ( fig. 4A). nuchal grooves short, deep, and slightly curved outwards. Peristomium with many transverse folds, that become expanded behind the prostomium ( fig. 4b). Mouth located ventrally, with well-developed and rugose lips. setigers 1-3 without a collar. neuropodia of setigers 1-3 with a strong acicular spine, which is obtuse and honey-coloured ( fig. 4C). Posterior to setiger 3, neuropodia have a row of rostrate hooks, which are present up to setiger 19. Each hook has a rostrum and five teeth on the main fringe. A single thick barbule, bent upwards, present below the rostrum ( fig.  4D, E). Each hook is perpendicular to the body wall, with a long and curved posterior shaft, and a prominent manubrium on the posterior half. the tori have Preanal asetigerous segment short. Anal plate with reduced dorsal border and long and smooth ventral border ( fig. 5E). Anus terminal, central and close to anal plate, surrounded by divergent folds (fig. 5f).
tubes. tube fragments composed of fine sand grains and gravel.
Habitat. At 30 m depth, in sand-muddy sediment.
Etymology. the species is named after Dr. sergio i. salazar-Vallejo, who has contributed so much to the knowledge of maldanid polychaetes from the Gulf of Mexico.

DisCussion
De Assis et al. (2007a) reviewed nicomachinae, and recognised seven species for Micromaldane, sixteen valid species for Nicomache, and only nine valid species for Petaloproctus. in this last genus, Petaloproctus crosnieri rullier, 1965 was excluded because of the absence of the posterior part of the body. Petaloproctus crenatus Chamberlin, 1919 was considered incertae sedis, since complete specimens are unknown.
the phylogenetic analysis, based on 20 morphological characters, shows that the subfamily nicomachinae, and the genera Micromaldane, Nicomache and Petaloproctus are monophyletic taxa. nicomachinae was supported by the following synapomorphies: prostomium short and arched (characters 2, 3); nuchal grooves curved and short (characters 5, 6); cephalic plate lost (character 7), anal pore opening on margin of the anal plate (character 20). Euclymeninae is a more basal group and herein considered the sistergroup of nicomachinae (figs. 2-3).
bleidorn et al. (2005) conducted a molecular phylogenetic analysis based on three genes, mitochondrial 16srrnA, nuclear 18srrnA and a small fraction of nuclear 28srrnA, with the aim of testing the monophyly of Maldanidae and Arenicolidae and of the genera of Arenicolidae. both families were considered monophyletic. the genera of Maldaninae appeared as basal and as the sister-group of Euclymeninae. Among the genera of Euclymeninae, Nicomache sp. appeared as the sister-group of Clymenura clypeata (saint-Joseph, 1894). the synapomophies of the genus Micromaldane are two clusters of eyespots on anterior end of the head (character 4), rostrate uncini strongly curved in all setigers (character 11), and the anal cup with crenulated border (character 18) (rouse, 1990;De Assis et al., 2007a).
Nicomache has also been supported as a monophyletic taxon, with the following synapomorphy: helicoidal capillaries with bifurcated tip in all setigers (character 12). this kind of seta has not been found in other maldanid polychaetes (Day, 1967;imajima and shiraki, 1982;Lee and Paik, 1986;De Assis et al., 2007a, b). Although not resolved in the strict consensus, Nicomache is considered as a sister-group of the Petaloproctus because of the presence of acicular spines on the first three setigers (character 8) (De Assis et al., 2007a). finally, the genus Petaloproctus is herein treated as a valid group in the analysis, having been well supported by three apomorphies: prostomium strongly arched (character 3(2)), capillaries long and pinnate (character 13), and an anal cup with dorsal border reduced (character 16) (Day, 1967;imajima and shiraki, 1982;De Assis et al., 2007a) (figs. 2-3).
the most basal clade of Petaloproctus is formed by the species P. terriculus, P. tenuis, and P. neoborealis, which are grouped by the anteriorly pointed prostomium (character 1). the plesiomorphic condition of this character is a rounded prostomium, found both in the outgroup and in the majority of maldanids. the synapomophy of the clade formed by P. terriculus and P. neoborealis is the loss of the pre-anal asetigers (character 15). the next more derived clade was supported by the presence of straight acicular spines (character 9). some genera of Euclymeninae have strongly curved acicular spines, hook-like. in Nicomache and in the most basal species of Petaloproctus, these spines become slightly curved, and in the clade formed by species P. ornatus, P. dentatus, P. cirratus, P. macrodentatus, P. borealis, P. socialis, and P. vallejoi n. sp. the acicular spines become straight, a derived condition not observed in other species of maldanids (Day, 1967;imajima and shiraki, 1982;Lee and Paik, 1986;De Assis et al., 2007a).
A more nested clade is formed by the species P. dentatus, P. cirratus, P. macrodentatus, P. borealis, P. socialis and P. vallejoi n. sp., having as an apomorphy acicular spines with obtuse points (character 9(2)). both in the outgroups, with the exception of Micromaldane, that does not have acicular spines, and the most basal species of Petaloproctus, these spines have fine tips (imajima and shiraki, 1982; Lee and Paik, 1986).
A distal end of the acicular spines with obtuse points (character 10) is only present in P. vallejoi n. sp., P. socialis, P. borealis, P. cirratus, P. dentatus and P. macrodentatus, representing a synapomorphy uniting these species (Day, 1967;imajima and shiraki, 1982;Lee and Paik, 1986). the acicular spines are modified setae with a single tooth, usually found in the first setigerous segments in most members of maldanids. this is an important character for the taxonomy of the family. the ornamentation of the border of the anal plate (character 17) is a character that has been used to differentiate many species. in our analysis, this character is interpreted as homoplastic for P. vallejoi n. sp. + P. socialis + P. borealis and for P. neoborealis (imajima and shiraki, 1982;Lee and Paik, 1986;De Assis et al., 2007a). however, many species of Euclymeninae and nicomachinae have cirri of different sizes and forms. We hypothesise that crenulations represent reduced cirri, which retain the same sensory function. these cirri are further reduced to a crenulated border in Micromaldane (rouse, 1990). the clade formed by species P. cirratus and P. dentatus was supported by the diminute, symmetrical, saw-like, anal cirri (character 19 (1)).
the clade containing species P. macrodentatus, P. borealis, P. socialis, and P. vallejoi n. sp. was sustained by the presence of strongly curved nuchal grooves (character 5(2)) (hartman, 1969;imajima and shiraki, 1982;Lee and Paik, 1986;De Assis et al., 2007a), herein considered to represent an apomorphic character. in species of Micromaldane and Nicomache, and the most basal species of Petaloproctus, the nuchal organs form slightly curved sinuous lines, sometimes with hooks on anterior and posterior ends.
the derived clade containing the species P. borealis, P. socialis, and P. vallejoi n. sp. is supported by the absence of ornamentation on the ventral border of the anal cup. this character is considered to be homoplastic for P. neoborealis. P. socialis and P. vallejoi n. sp. are unique within Maldanidae for having the base of the spinulous setae ruffled (character 14).
We chose the most resolved topology among our three phylogenetic trees of equal length, Ci, and ri, as representative of the phylogeny of the nicomachinae ( fig. 3). the relationships among the species of Petaloproctus are identical to those appearing in the strict consensus phylogenetic tree ( fig. 2).