Platyhelminthes, Proseriata): Inferences from Rdna Sequences

327 MOLECULAR PHYLOGENETICS AND EVOLUTION Vol. 6, No. 1, August, pp. 150-156, 1996 ARTICLE NO. 0067 A Reappraisal of the Systematics of the Monocelididae (Platyhelminthes, Proseriata): Inferences from rDNA Sequences M . K . L it v a it is , * M . C . C u r in i -G a l l e t t i ,+ P . M . M a r t e n s , * a n d T . D . K o c h e r * * Department o f Zoology, University o f New Hampshire, Durham, New Hampshire; t Istituto d i Zoología, Université degii Studi di Sassari, Sassari, Italy; and * Department SBM, Limburgs Universitair Centrum, Diepenbeek, Belgium Received August 11, 1995 on the presence (Minoninae) or the absence (Monoceli The current classification system for the Monocelidi dinae) of the accessory prostatoid organ, a musculo- dae which is based on the character “presence or ab glandular structure armed with a stylet (Fig. 1), and sence of an accessory prostatoid organ” divides the he attributed less phylogenetic weight to the structure family into two subfamilies, namely the Minoninae and of the male copulatory bulb. Within the Monocelididae, the Monocelidinae. However, other characters re the copulatory bulb is of the conjuncta type (Karling, lating to the structure of the male copulatory bulb and 1956), and two subtypes can be recognized. The sim to karyotypes do not support this division. Monoceli- plex-type consists of a single muscular wall, which de d id m ale c o p u la to ry b u lb s c an b e e ith e r o f th e sim plex rives from the last portion of the ductus ejaculatorius or the duplex-type, and if this character is mapped (Fig. 2A). The duplex-type is surrounded by two muscu onto the current classification, then both subfamilies lar walls, an outer and an inner one, termed “septum” contain species w ith either type of copulatory bulb. We therefore decided to construct an independent phylog- and “axial canal,” respectively (Karling, 1956). The eny for the Monocelididae using nucleotide-sequence axial canal corresponds to the last portion of the ductus d a ta o f th e g en e co d in g fo r th e 26/28S rDNA. D istance- ejaculatorius (Fig. 2B). In both types, many differentia and parsimony-based analyses resulted in phyloge tions are present. A conflict arises when the character netic trees that strongly supported a division of the “simplex- or duplex-type copulatory bulb” is added to Monocelididae based on characters of the male copula Karlingi (1978) phylogeny. Both subfamilies then con tory bulb and not on the accessory prostatoid organ. tain species with copulatory bulbs of simplex and du Thus, all species possessing a simplex-type copulatory plex-type. Thus, multiple origins need to be evoked to bulb cluster together into one monophyletic group, the explain this distribution, as one of the types needs to Monocelidinae (sensu Midelburg), whereas species have evolved at least twice. characterized by a duplex-type copulatory bulb consti Furthermore, our karyological observations (Curini- tute a paraphyletic assemblage. © 1996 Academic Press, Inc. Galletti et al., 1989; Martens, and Curini-Galletti, 1987; Martenset al., 1989) also are in conflict with the present subdivision of the family Monocelididae. In a study based on more than 150 proseriate species, we INTRODUCTION reconstructed the basic karyotype of the Monocelididae as n = 3, with chromosomes of distinctly differing sizes. Currently, the systematics of the family Monocelidi The largest and middle-sized chromosomes are meta- dae (Platyhelminthes, Proseriata) is based mostly on centric, the smallest one is markedly heterobrachial. the presence and position of structures associated with This set of different-sized chromosomes is found exclu the reproductive system. Features used for classifica sively in species with the duplex-type copulatory bulb. tion include the bursa, the vagina, the accessory prosta Species containing a simplex-type copulatory organ are toid organ, the ovaries, and the male copulatory bulb. characterized by chromosome sets comprising chromo In this extremely species-rich group, virtually any pos somes of nearly equal length (n = 3). This set can be sible combination of character states can be found. At derived from the basic set of the Monocelididae through present, each of the observed patterns is considered a a translocation involving chromosomes I and III (Mar distinct genus. Therefore, any reconstruction of phylo tens and Curini-Galletti, 1987). It is found in species genetic relationships among these genera necessarily with the simplex-type copulatory organ, regardless of needs to take multiple events of parallelism and/or re their placement in the two subfamilies. duction into consideration (Martens, 1983). Karling Therefore, on the basis of the characters “presence of (1978) based his widely accepted division of the familya simplex-type copulatory organ” and a “derived karyo- 150 1055-7903/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in ahy form reserved. 328 NUCLEOTIDE-BASED PHYLOGENY OF THE MONOCELIDIDAE 151 nonmorphological data to test our hypothesis. The use of rDNA’s in the assessment of phylogentic relation ships has been applied successfully in a wide variety of -aog studies (Smith, 1989; Cedergrenet al., 1988; Fieldet al., 1988). Many such studies were intended to resolve conflicts in morphology-based trees (Smith, 1989; Tur- beville et al., 1992; Xiong and Kocher, 1993). The 26/ 28S rDNA is ideally suited for this purpose since differ ent regions of the gene evolve at different rates (Hillis and Dixon, 1991) and thus it can be used to investigate ms relationships at almost any level of taxonomic hierar chy (Litvaitis et al., 1994). In eukaryotes, the 26/28S rDNA gene contains 12 intermittent regions variously called the expansion segments (D1-D12) or variable re gions (Hassouna et al., 1984). These expansion seg aop ments vary greatly in size and exhibit high variability FIG. 1. Schematic representation of an accessory prostatoid or as compared to the core areas (Gutell et al., 1990). In gan present in the subfamily Minoninae (sensu, Karling, 1978); b, fact, the D3 expansion segment has been used to iden bulb; aog, accessory organ glands; ms, muscle sheath; s, stylet; aop, tify different meiofaunal turbellarians (Litvaitis et al, accessory organ pore. 1994). It also has been used for identifications and phy logeny reconstructions of nematodes (Nunn, 1992) and type” (i.e., chromosomes of equal lengths), we hypothe isopods (Nunn et al, 1996). Our objectives were to size that all species with a simplex-type copulatory apply sequence data of the 26/28S rDNA gene to the bulb have a common ancestor and hence should be phylogentic analysis of the Monocelididae, specifically placed into a monophyletic taxon. Consequently, the (1) to test the hypothesis of a proposed monophyly of current systematic division of the Monocelididae into monocelidids with a simplex-type copulatory bulb, and two subfamilies would not reflect the phylogeny of the (2) to compare the resulting molecular phylogeny with group. Second, the character “presence/absence of an the existing classification scheme of Karling (1978). accessory prostatoid organ” upon which the current classification scheme is based, can only be included if MATERIALS AND METHODS its origin is evoked at least twice or its loss is implied multiple times. Proseriates used in this study were collected from Because of the structural uniqueness of the monoceli- various intertidal and sublittoral marine and brackish did copulatory bulb and the subsequent difficulty of as habitats of the Mediterranean, Red Sea, and Australia signing polarity to character states, we decided to use (Table 1). One species (Archiloa rivularis) was collected from a freshwater stream on the Atlantic side of the Pyrenees. To avoid phylogenetic bias, we included ei _vd ther noncongeneric species or congeners from widely separated geographic areas, representing all four possi ble morphological states of male copulatory bulbs and accessory prostatoid organs (i.e., simplex-type copu latory bulb/accessory organ absent; simplex-type co -pg- pulatory bulb/accessory organ present; duplex-type copulatory bulb/accessory organ absent; duplex-type copulatory bulb/accessory organ present). cis- Animals were extracted from the sediment using the -pp MgCl2-decantation technique (Martens, 1984), and live animals, gently squeezed under coverslips, were used for species identifications. All unnamed species are new mp mp and will be described in later publications. Animals were stored in 95% ethanol at room temperature. DNA FIG. 2. Schematic representation of (A) simplex vs (B) duplex extractions were performed according to Litvaitis et al, type male copulatory bulbs; vd, vas deferens; vs, vesicula seminalis; (1994). Total genomic DNA was resuspended in 100- ms, muscle sheath; pg, prostate glands; pp, penis papilla; ma, male antrum; mp, male pore; vg, vesicula granulorum (prostatic vesicle); 200 pi TE buffer (pH 7.6) (Sambrook et al, 1989). ims, inner muscle sheath; oms, outer muscle sheath; ci, eversible cir Double-stranded amplifications from genomic DNA rus; cis, cirrus spine. were performed according to Kocher et al. (1989). The 329 152 LITVAITIS ET AL. TABLE 1 Specimens, Collection Localities, and GenBank Accession Numbers Taxon Locality Accession Numbers Monocelididae Simplex-type copulatory bulb, no accessory prostatoid organ Monocelis lineata Leece, Italy U40203 and U40204 Monocelis longiceps Akziv, Israel U40205 and U40206 Pseudomonocelis ophiocephala Kanoni, Corfu, Greece U40197 and U40198 Pseudomonocelis cetinae Giglio Island, Italy U40051 and U40052 Simplex-type copulatory bulb, with accessory prostatoid organ Minona, n. sp. Ia Eilat (Red Sea), Israel U42000 and U42001 Minona n. sp. 2a Caloundra, Queensland, Australia U42002 and U42003 Monocelididae n.g. n. sp.0 Eaglehawk Neck, Tasmania U42004 and U42005 Duplex-type copulatory bulb, no accessory prostatoid organ Archilina deceptoria Capraia Island, Italy U40047 and U40048 Archilina israelitica Atlit, Israel U39927 and U39928 Archiloa rivularis St.

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