Contributions to Zoology, 70 (3) (2001)Emilia Rota; Patrick Martin; Christer Erséus: Soil-dwelling polychaetes: enigmatic as ever? Some hints on their phylogenetic relationships as suggested by a maximum parsimony analysis of 18S rRNA gene sequences

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Material and methods

Selection of taxa

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The data set analysed in this study consists of: the newly determined 18S rDNA sequences of Hrabeiella periglandulata, Parergodrilus heideri, Stygocapitella subterranea, Aeolosoma hemprichi, Protodrilus purpureus, Ctenodrilidae n. gen. et sp., and Arenicola marina; a compilation of all the sequences of this gene available in EMBL for polychaetes and other taxa assumed to be closely related to, or members of, ‘non-clitellate Annelida’, namely, Sipuncula, Echiura, Pogonophora (including Vestimentifera) and Aphanoneura (Rouse & Fauchald, 1995); and another eleven 18S rDNA sequences, also taken from EMBL, representing the oligochaete, branchiobdellidan, acanthobdellidan and euhirudinean Clitellata (Table I).

 

Table I. List of species used in the 18S rDNA analysis (binomial name, abbreviation of the collecting locality of new material, EMBL accession number).

ARTHROPODA

Chelicerata

Aphonopelma sp. (=“Eurypelma californica”)

X13457

Insecta

Tenebrio molitor Linnaeus, 1758

X07801

MOLLUSCA

Pectinidae

Placopecten magellanicus (Gmelin, 1791)

X53899

Achatinidae

Limicolaria kambeul (Bruguière, 1789)

X66374

Chitonidae

Acanthopleura japonica (Lischke, 1873)

X70210

SIPUNCULA

Phascolosomatidae

Phascolosoma granulatum Leuckart, 1828

X79874

ECHIURA

Echiuridae

Ochetostoma erythrogrammon Rüppell & Leuckart, 1830

X79875

POGONOPHORA

Frenulata

Siboglinum fiordicum Webb, 1963

X79876

Vestimentifera

Ridgeia piscesae Jones, 1985

X79877

POLYCHAETA

?

Hrabeiella periglandulata Pizl & Chalupský, 1984

MO

AJ310501

Aphroditidae

Aphrodita aculeata Linnaeus, 1761

Z83749

Arenicolidae

Arenicola marina (Linnaeus, 1758)

DU

AJ310502

Capitellidae

Capitella capitata (Fabricius, 1780)

U67323

Chaetopteridae

Chaetopterus variopedatus (Renier, 1804)

U67324

Cirratulidae

Dodecaceria concharum Örsted, 1843

U50967

Ctenodrilidae

n. gen. et sp.

EL

AJ310503

Glyceridae

Glycera americana Leidy, 1855

U19519

Magelonidae

Magelona mirabilis (Johnston, 1865)

U50969

Myzostomidae

Myzostoma sp.

AF116916

Nephtyidae

Nephtys hombergii Savigny, 1818

U50970

Nereididae

Neanthes succinea (Frey & Leuckart, 1847) (=”Nereis limbata”)

U36270

Neanthes virens (Sars, 1835) (=”Nereis virens”)

Z83754

Orbiniidae

Scoloplos armiger (Müller, 1776)

U50972

Parergodrilidae

Parergodrilus heideri Reisinger, 1925

CA

AJ310504

?Parergodrilidae

Stygocapitella subterranea Knöllner, 1934

SA

AJ310505

Polynoidae

Harmothoe impar (Johnston, 1839)

U50968

Protodrilidae

Protodrilus purpureus (Schneider, 1868)

EL

AJ310506

Questidae

Questa paucibranchiata Giere & Erséus, 1998

AF209464

Sabellidae

Sabella pavonina Savigny, 1822

U67144

Serpulidae

Protula sp.

U67142

Spionidae

Polydora ciliata (Johnston, 1838)

U50971

Pygospio elegans Claparède, 1863

U67143

Terebellidae

Lanice conchilega (Pallas, 1766)

X79873

APHANONEURA

Aeolosomatidae

Aeolosoma sp.

Z83748

Aeolosoma hemprichi Ehrenberg, 1828

MO

AJ310500

OLIGOCHAETA

Enchytraeidae

Enchytraeus sp. 1

Z83750

Enchytraeus sp. 2

U95948

Lumbricidae

Lumbricus rubellus Hoffmeister, 1843

Z83753

Eisenia fetida (Savigny, 1826)

X79872

Naididae

Stylaria sp.

U95946

Tubificidae

Tubifex sp.

U67145

BRANCHIOBDELLIDA

Branchiobdellidae

Xironogiton victoriensis Gelder & Hall, 1990

AF115977

Cambarincola holti Hoffman, 1963

AF115975

ACANTHOBDELLIDA

Acanthobdellidae

Acanthobdella peledina Grube, 1851

AF099948

EUHIRUDINEA

Glossiphoniidae

Glossiphonia complanata (Linnaeus, 1758)

AF099943

Hirudinidae

Hirudo medicinalis

Linnaeus, 1758

The choice of an outgroup to Polychaeta is problematic because recent molecular studies failed to recover the monophyly of this taxon (McHugh, 1997; Kojima, 1998; Westheide et al., 1999) and because putatively valid outgroups, such as Mollusca, proved to be scattered among annelids or even appeared as an ingroup within polychaetes (Winnepenninckx et al., 1995, 1998; Siddall et al., 1998). For this reason, not only three Mollusca but also two Arthropoda were included in the data set. Arthropods were designated as an unambiguous outgroup (Table I), in accordance with their molecular placement among the Ecdysozoa, the sister group of the Lophotrochozoa to which all others taxa herewith considered belong (Aguinaldo et al., 1997; Adoutte et al., 2000).

Collection of new specimens

The newly sequenced material was collected at the following localities (abbreviations used in Table 1): (MO) Montalbuccio, 43°20‘N 11°15‘E, Siena, Italy (in 1998; coll. E. Rota); (CA) Camugnano, 44°10‘N 11°10‘E, near Bologna, Italy (in 1999; coll. C. Jacomini and E. Rota); (SA) Costa Paradiso, 41°03‘N 8°55‘E, Sardinia (in 1999; coll. E. Rota); (DU) Monkstown, 53°18‘N 6°10‘E, Dublin, Ireland (in 1999; coll. O. Schmidt); (EL) Capo S. Andrea, Elba Island, 42°48‘N 10°09‘E, Tyrrhenian Sea (in 2000; coll. E. Rota and C. Erséus). All the material was fixed and preserved in 95-99 % alcohol.

DNA extraction, amplification and sequencing

DNA of A. hemprichi, H. periglandulata and S. subterranea was extracted according to a standard Chelex™ procedure (Singer-Sam et al., 1989; Hillis et al., 1996). DNA extraction of P. heideri and A. marina was made using “High Pure PCR Template Preparation” from Boehringer-Mannheim Biochemicals, and that of Ctenodrilidae n. gen et sp. and P. purpureus using “QIAamp DNA Mini Kit” from Quiagen, following the instructions of the manufacturers. 18S rRNA gene fragments of A. hemprichi and H. periglandulata were amplified and sequenced according to Martin (2001), using the 16 primers of Winnepenninckx et al. (1994), kindly granted us by the authors. Specimens of the other taxa were analysed following the protocol described in Erséus et al. (2000).

Alignment

Since it has repeatedly been shown that the sequence alignment may influence the phylogenetic relationships inferred from ribosomal genes (Wägele & Stanjek, 1995; Winnepenninckx & Backeljau, 1996; Erséus et al., 2000; Martin et al., 2000), we studied 18S rDNA sequence data aligned in two different ways: (1) using DCSE (De Rijk & De Wachter, 1993; De Rijk, 1995), which considers a secondary structure model; (2) using Clustal W (Thompson et al., 1994), default settings, without manual corrections. Whatever the alignment method used, some hyper-variable regions of the gene (domain 23 in particular; Van de Peer et al., 1996) were virtually impossible to align and were discarded from the final alignment.

Phylogenetic analyses

Maximum parsimony analyses of the resulting alignments (EMBL accession numbers ALIGN-000074 for ClustalW and ALIGN-000096 for DCSE) were performed using PAUP*, version 4.0b4a (Swofford, 1998) with the following settings: unweighted characters, heuristic search, random addition of sequences with 100replicates, tree-bisection-reconnection (TBR) branch-swapping algorithm, ‘Multrees’ option in effect, gaps treated as missing. Bootstrap analyses were performed using a heuristic search and TBR branch-swapping on 100 replicates. The Bremer support index was calculated with PAUP* in connection with AutoDecay 4.0 (Eriksson, 1998).