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, ): 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- 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. Jean de Luz, France U40049 and U40050 Promonotus ponticus Castiglione della Pescaia, Italy U40199 and U40200 Promonotus spinosissimus Haifa, Israel U40201 and U40202 Duplex-type copulatory bulb, with accessory prostatoid organ Duplominona n. sp. Ia Capo Caccia, Sardinia, Italy U41375 and U41376 Duplominona n. sp. 2a Heron Island, Queensland, Australia U41378 and U41377 Coelgynoporidae Coelogynopora gynocotyla Castiglione della Pescaia, Italy U40207 and U40208 Archimonocelididae Archimonocelis staresoi Lecce, Italy U40209 and U40210

° Complete species descriptions by Curini-Galletti (in prep); proposed species names for Minona n. sp. 1 is Minona ileanae and for Minona n. sp. 2 is Minona concharum. first set of primers used during amplification flanked gel in IX TBE buffer (Sambrook et al, 1989), and the the D3-expansion segment of the 26/28S rRNA gene nucleotide sequence was determined using an auto­ (Litvaitis et ah, 1994). A second set of primers was used mated sequencer (ABI 373A). For each sample, both to amplify an area extending from the D3 expansion strands were sequenced. Sequences have been depos­ segment to the D6 domain. All primers had been de­ ited in GenBank (for accession numbers, see Table 1). signed based on the rDNA of Caenorhabditis elegans Initial alignment of sequences was done by eye and (Ellis et ál., 1986). For primer sequences and corre­ edited using the SeqEd program (vers. 1.0.3; ABI). Fur­ sponding positions in the C. elegans gene, see Table 2. ther alignments were performed using the sequential Five microliters of the double-stranded products were alignment program PILEUP with a gap penalty of 5 run on a 1% SeaKem agarose gel with a DNA molecu- and a gap length penalty of 0.3 (algorithm of Feng and lar-weight-size standard, and the remaining reaction Doolittle, 1987, as implemented by the Genetics Com­ was purified by centrifugation through a Centricon-100 puter Group, Madison, WI). Sequences were analyzed column (Amicon Inc.). Following a onefold dilution, 3 - 5 pi of the amplified DNA’s were used in a cycle se­ quencing reaction using dye-nucleotide terminators TABLE 2 and the original set of primers (protocol according to Applied Biosystems Inc., Foster City, CA). Primers Used in the Amplification and Sequencing of Products were purified by centrifugation through the Proseriate 28/298S rDNA Gene Centri-Sep spin columns (Princeton Separations), Corresponding positions dried, and stored at -20°C. Immediately prior to load­ Primer name Sequence (5' to 3') in the C. elegans gene ing on the gel, the samples were resuspended in 4 pi D3A GACCCGTCTTGAAACACGGA 3304-3323 5:1 formamide/EDTA, heated to 90°C for 2 min, cooled D3B TCGGAAGGAACCAGCTACTA 3628-3647 quickly on ice, and centrifuged. Four microliters of each MKL-D3A.2 CCCGAAAGATGGTGAACTAT 3498-3517 sample were electrophoresed on a 6% polyacrylamide MKL-D6B GGAACCCTTCTCCACTTCAGT 4183-4203 330

NUCLEOTIDE-BASED PHYLOGENY OF THE MONOCELIDIDAE 153 using MEGA vers. 1.0 (1993). This program calculates Pseudomonocelis cetinae * genetic distances according to the Tamura-Nei model 100 i (Tamura and Nei, 1993), which use pairwise distances I— PseudomonocelisI ophiocephala * estimated by assuming a gamma-distributed rate for i ♦ Minona n.sp. 1 * sites (a = 0.75). A distance-based phylogenetic tree was i H simplex type then constructed using the neighbor-joining method -741 » Monocelididae n.g. n.sp. * copulatory bulb (Saitou and Nei, 1987).Coelogynopora gynocotyla and Minona n. sp. 2 * Archimonocelis staresoi were used as the outgroup. In addition, parsimony analyses weighting all bases Monocelis longiceps * equally (Swofford’s PAUP, vers. 3.1, 1993, heuristic Monocelis lineata * search option using the tree bisection-reconnection (TBR) branch-swapping algorithm and with collapsing — Duplominona n.sp. 2 zero-branch lengths option in effect) were performed. 4 —■— Duplominona n.sp. 1 To test for the existence of islands of trees (Maddison, Archilina 1991), sequence addition was set to random (Swofford, ~ deceptoria 1993). Gaps were treated as missing data. Reliability — Promonotus ponticus of internal nodes was ascertained by 2000 bootstrap Archilina israelitica replications. The same outgroups were used for these 100 trees. - Promonotus spinosissimus

Controversy exists about the validity of including - Archiloa rivularis paired nucleotides in phylogenetic analyses due to as­ sumed selection acting on these nucleotides to main­ ' Coelogynopora gynocotyla tain integrity of secondary structure (Wheeler and —— Archimonocelis staresoi Honeycutt, 1988; Dixon and Hillis, 1993). However, ac­ cording to Noeller and Woese (1981), unpaired loop re­ i------1 gions may actually be more constrained then paired 0 0.05 stem regions. Therefore, there is no a priori reason to FIG. 3. Distance-based phylogeny of the Monocelididae; numbers warrant exclusion of either paired or unpaired nucleo­ at nodes represent bootstrap values (2000 replications), diamonds on tides from a phylogenetic analysis or warrant their dif­ branches represent the character “presence of an accessory prosta­ ferential weighting (Simon, 1991). Thus, we include the toid organ,” and asterisks represent the character “derived karyo­ entire sequences in our analysis, regardless of second­ type.”. ary structure. Furthermore, by using a gamma-distrib­ uted substitution rate in our distance-based phylogeny, we accommodated different substitution rates among latory bulb formed a monophyletic cluster supported in sites. 95% of bootstrap replications (Fig. 3). Even though spe­ cies in this clade all have a simplex-type copulatory RESULTS bulb, only some of them also have an accessory prosta­ toid organ ( Minona n. sp. 1, Monocelidid n. g. n. sp., Amplification using the D3A and D3B primers and Minona n. sp. 2). This clearly supports a division yielded sequence fragments of305-315 base pairs (bp), of the Monocelididae based on the structure of the male and the D3A.2 to D6B primers yielded fragments of copulatory bulb and not based on the presence or ab­ 614-633 bp. The two segments were nonoverlapping. sence of an accessory prostatoid organ. The second No sites exhibited ambiguous sequence information, clade, though containing species with a duplex-type thus, gene copy homogeneity can be inferred. Align­ copulatory bulb, is not monophyletic because Archiloa ments showed considerable similarity between the two rivularis is not included. subfamilies, and even with the sequences of the two Parsimony analysis also resulted in a phylogenetic outgroups which belong to different families. Within tree in which all species with a simplex-type copulatory the amplified product, 255 bp (27%) were phylogeneti- bulb form a monophyletic taxon supported in 94% of cally informative. Base composition was balanced, thus bootstrap replications (Fig. 4). Within this group, the not necessitating tree building algorithms that cor­ two Pseudomonocelis species cluster together, while rected for skewed ratios. Pairwise comparisons re­ the sister group of this clade contains the two Monocelis vealed distances ranging from 0.010 (A. israelitica vs. species and the remaining minonidids. Again, mono- P. spinosissimus) to 0.264 (A. deceptoria vs Duplomi­ phyly of species with duplex-type copulatory bulbs was nona n. sp. 2). The latter value was almost as large as not supported. Specifically, A. rivularis again diverges the distance to the outgroup species. before the other species, and thus has all other mono- The distance-based analysis resulted in a neighbor- celidids as a sister group. Also within the duplex-type joining tree in which species with a simplex-type copu- cluster, the two Duplominona species do not group to- 331

154 LITVAITIS ET AL.

Archiloa deceptoria (1978); presence of an accessory prostatoid organ is evi­ dently a homoplasy. Duplominona n. sp- 1 Monophyly of species with the simplex-type copula­ tory bulb is further supported by karyological data. As Archilina Israelitica mentioned, species with simplex-type copulatory bulbs

Promonotua spinosissimus are also characterized by a derived karyotype in which all chromosomes are of equal length (Martens and Cur­ — Promonotus ponticus ini-Galletti, 1987). Moreover, there is at least one other morphological feature, namely the structure of so- Duplominona n. sp. 2 called “eye spots,” which also supports the observed monophyly. Among the species with simplex-type copu­ Pseudomonocelis cetinae latory bulbs, there are numerous species belonging to P.ophiocephala different genera ( Monocelis, Minona, Pseudomonocelis, Preminona) that exhibit characteristic pigment spots Minona n. sp. 2 in the cephalic area. Pigment spots of this type are not present in other monocelidids. These pigment spots Monocelis longiceps simplex type copuiatory bulb have been studied in detail in Monocelis fusca and Pseudomonocelis agi¿is(Sopott-Ehlers, 1984,1993), and Monocelis lineata in both species they form a multicellular pigment shield, located close to the photoreceptors. Differences between these two species in the ultrastructural fea­ -e- Monocelididae n.g. n.sp.4 tures of the eyespots are slight. According to Sopott- Ehlers (1993), these pigment shields are not known in *—— Archiloa rivularis other proseriates and have evolved within the Monocel- lididae. Thus these pigment shields are considered ho­ Coelogynopora gynocotyla mologous structures, and since they are only known in

Archimonocelis staresoi species with simplex-type copulatory organs, they rep­ resent another synapomorphy uniting these species. FIG. 4. Single most parsimonious tree (length 960; consistency Relationships among monocelidids having the du­ index = 0.668; homoplasy index = 0.332) based on two nonoverlap­ plex-type copulatory organs are not as clear. Among the ping segments of the 26/28S rDNA gene. Branch lengths are propor­ duplex-type species, Archiloa rivularis occupies the po­ tional to the number of steps on each branch. Numbers at nodes rep­ resent bootstrap values (2000 replications), diamonds on branches sition of a sister group to all other members of the Mo­ represent the character “presence of an accessory prostatoid organ,” nocelididae, and thus the duplex group appears pa- and asterisks represent the character “derived karyotype.”. raphyletic. The most parsimonious explanation of the relationship between species with a simplex- and du­ plex-type copulatory bulb postulates the presence of a gether. Instead, Duplominona n. sp. 1 from Capo Cac­ duplex-type copulatory bulb in the stem species of the cia, Sardinia, is much closer to a species of another ge­ Monocelididae. A simplex-type copulatory bulb derived nus, namelyArchilina deceptoria from Capraia Island, from the duplex-type then constitutes a synapomorphy Italy. Similarly, the twoPromonotus spp. do not cluster for the taxon Monocelidinae (sensu Midelburg, 1908). monophyletically with respect to Archilina israelitica The accessory prostatoid organ is a complex musculo- in the parsimony tree. glandular structure, armed with a stylet. It is highly unlikely that such a complex structure would have DISCUSSION evolved several times independently within the Mono­ celididae. A more parsimonious assumption is its pres­ The topologies of both the distance- and the parsi­ ence in the stem species of the Monocelididae with sub­ mony-based tree are in agreement, providing strong sequent multiple events of secondary loss. Whether support for the monophyly of species possessing the this character is an apomorphy of the Monocelididae is simplex-type copulatory bulb (Figs. 3 and 4). Species still unresolved though, given the presence of similar with a duplex-type copulatory bulb, to the contrary, do structures in related families (e.g., the prostatoid or­ not form a monophyletic clade. Archiloa rivularis re­ gans of Nematoplanidae, the accessory glandular organ mains outside the cluster of other species characterized of Archimonocelididae). Circumstantial supporting evi­ by a duplex-type copulatory bulb. The character “pres­ dence comes from developmental observations that ence of an accessory prostatoid organ” is widespread show that the accessory prostatoid organ is the last fea­ in the cladogram and not limited to any one particular ture of the copulatory structures to develop, often when clade. Consequently, our results do not support the di­ sperm is already present in the seminal vesicle (Mar­ vision of the Monocelididae as proposed by Karling cus, 1952; Curini-Galletti, 1991). Therefore, since the 332

NUCLEOTIDE-BASED PHYLOGENY OF THE MONOCELIDIDAE 155

reduction affects one of the last features formed during secondary structure format. Nucleic Acids Res. (suppl.) 18: r2319- ontogenesis, absence of the prostatoid organ may be a r2330. result of a progenetic mechanism (Gould, 1977). Pro­ Hassouna, N., Michot, B., and Bachellerie, J.-P. (1984). The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the genesis may have happened many times independently process of size increase of the large subunit RNA in higher eukary­ within the Monocelididae, and thus, assemblages of otes. Nucleic Acids Res. 12: 3563-3583. species without an accessory prostatoid organ are prob­ Hillis, D M., and Dixon, M. T. (1991). Ribosomal DNA: Molooular ably paraphyletic taxa. evolution and phylogenetic inference. Quart. Rev. Biol. 66: 411- The current molecular phylogeny based on sequences 453. of the 26/28S rDNA gene indicates that the classifica­ Karling, T. G. (1956). Morphologisch-histologische Untersuchungen tion of the Monocelididae has no phylogenetic basis and an den männlichen Atrialorganen der Kalyptorhynchia (Turbella­ that all species with a simplex-type copulatory bulb ria). Ark. Zool. 9:187-279. form a monophyletic taxon. For this monophyletic Karling, T. G. (1978). Anatomy and systematics of marine Turbella­ ria from Bermuda. Zool Ser. 7: 225-248. group, the subfamily-rank taxon Monocelidinae (Midel- Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Pääbo, S., burg, 1908), whose type genus isMonocelis, may apply. Villablanca, F. X., and Wilson, A. C. (1989). Dynamics of mitochon­ It is here redefined as including all monocelidid genera drial DNA evolution in animals: Amplification and sequencing with a simple-type copulatory bulb: Monocelis, Minona, with conserved primers. Proc. Natl. Acad. Sei. USA 86:6196-6200. Pseudomonocelis, Preminona, Peraclistus, Necia, Kumar, Sa- S., Tamura, K , and Nei, M. (1993). MEGA: Molecular Evolu­ batius, and Ectocotyla. We place the remaining genera' tionary Genetics Analysis, vers. 1.0. The Pennsylvania State Uni­ of Monocelididae, namely Achilina, Archiloa, Inaloa, versity, University Park, Pennsylvania. Mesoda, Archilopsis, Tajikina, Monocelopsis, Litvaitis, Boreo­ M. K., Nunn, G., Thomas, W. K., and Kocher, T. D. (1994). A molecular approach for the identification of meiofaunal turbel- celis, Paramonotus, Promonotus, Duploperaclistus, larians (Platyhelminthes, Turbellaria). Mar. Bio. 120: 437-442. Pseudominona, and Duplominona in the Duplomono­ Maddison, D. R. (1991). The discovery and importance of multiple celidinae subfam. n., which is probably a paraphyletic islands of most-parsimonious trees. Syst. Zool. 40: 315-328. group. Marcus, E. (1952). Turbellaria Brasileiros (10). Boletins da Facul- dade de Filosofía, Ciencias e Letras, Universidad'e de S. Paulo, Zoo­ logía 17: 5-188. ACKNOWLEDGMENTS Martens, P. M. (1983). Three new species of Minoninae (Turbellaria, Proseriata, Monocelididae) from the North Sea, and remarks on We thank Dr. W.-J. Lee for his help with the software program the of the subfamily. Zool. Ser. 12:153-160. MEGA. The present study was supported in part by NSF Grant BSR 9108757 to M.K.L. Martens, P. M. (1984). Comparison of three diffrent extraction meth­ ods for Turbellaria. Mar. Ecol. Prog. Ser. 14: 229-234. Martens, P. M., and Curini-Galletti, M. C. (1987). Karyological study REFERENCES of three MonoceZis-species, and the description of a new species from the Mediterranean, Monocelis longistyla sp. n. (Monocelidi­ dae, Platyhelminthes). Microfauna Marina 3: 297-308. Cedergren, R., Gary, M. W., Abei, Y., and Sankoff, D. (1988). The evolutionary relationships among known life forms. J. Mol. Evol. Martens, P. M., Curini-Galletti, M. C., and van Oostveldt, P. (1989). 28: 98-112. Polyploidy in Proseriata (Platyhelminthes) and its phylogenetical implications. Evolution 43: 900-907. Curini-Galletti, M. C. (1991). Monocelididae (Platyhelminthes: Pro­ Midelburg, A. (1908). Zur Kenntnis der Monocelididae.Z. wiss. Zool. seriata) from Puerto Rico. I. Genera Minona and Monocelis. Proc. 89: 81-108. Biol. Soc. Washington 104: 229-240. Noeller, H. F., and Woese, C. R. (1981). Secondary structure of 16S Curini-Galletti, M. C., Puccinelli, I., and Martens, P. M. (1989). Kary- ribosomal RNA. Science 212: 403-411. ometrical analysis of ten species of the subfamily Monocelidinae (Proseriata, Platyhelminthes) with remarks on the karylogical evo­ Nunn, G. B. (1992). Nematode molecular evolution. An investigation lution of the Monocelididae. Genetica 78: 169-178. of evolutionary patterns among nematodes based upon DNA se­ quences. Ph.D. dissertation, University of Nottingham, UK. Dixon, M. T., and Hillis, D. M. (1993). Ribosomal RNA secondary structure: Compensatory mutations and implications for phyloge­ Nunn, G. B., Theisen, B. F., Christensen, T. B., and Arctander, P. netic analysis.Mol. Bio. Evol. 10: 256-267. (1996). Simplicity correlated with size growth of the D3 ribosomal RNA expansion segment in the order Isopoda. J. Mol. Ellis, R. E., Sulston, J. E., and Coulson, A. R. (1986). The rDNA of Evol. 42: 211-223. C. elegans: Sequence and structure. Nucleic Acids Res. 14: 2345- 2364. Saitou, N., and Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees, Mol. Biol. Evol. 4: Feng, D.-F., and Doolittle, R. F. (1987). Progressive sequence align­ 406-425. ment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25: 351-360. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning. A Laboratory Manual,” 2nd ed, Cold Spring Harbor Labo­ Field, K. G., Olsen, G. J., Lane, D. J., Giovannonoi, S. J., Ghiselin, ratory Press, Cold Spring Harbor, New York. M. T., Raff, E. C., Pace, N. R., and Raff, R. A. (1988). Molecular phylogeny of the kingdom. Science 239: 748-753. Simon, C. (1991). Molecular systematics at the species boundary: Ex­ ploiting conserved and variable regions of the mitochondrial ge­ Gould, S. J. (1977). “Ontogeny and Phylogeny,” Belknap Press of Har­ nome of animals via direct sequencing from enzymatically ampli­ vard University Press, Cambridge, Massachusetts. fied DNA. In “Molecular Techniques of Taxonomy” (G. M. Hewitt, Gutell, R. R., Schnare, M. N., and Gray, M. W. (1990). A compilation A. W. B. Johnston, and J. P. W. Young, Eds.), pp. 33-71, Springer of large subunit (23S-like) ribosomal RNA sequences presented in a Verlag, New York. 333

156 LITVAITIS ET AL.

Smith, A. B. (1989). RNA sequence data in phylogenetic reconstruc­ Turbeville, J. M., Field, K. G., and Raff, R. A. (1992). Phylogenetic tion: Testing the limits of its resolution. Cladistics 5: 321-344. position of phylum Nemertini, inferred from 18S rRNA sequences: Sopott-Ehlers, B. (1984). Feinstruktur pigmentierter und unpig- Molecular data as a test of morphological character homology. Mol. mentierter Photoreceptoren bei Proseriata (Plathelminthes). Zool. Biol. Evol. 9: 235-249. Ser. 13*. 9-17. Wheeler, W. C., and Honeycutt, R. L. (1988). Paired sequence differ­ Sopott-Ehlers, B. (1993). Ultrastructural features of the pigmented ence in ribosomal RNAs: Evolutionary and phylogenetic implica­ eye spul in Pseudomonocelis agilis (Platyhelminthes, Proseriata). tions. Mul. Biol. Evol. 5: 90-96. Mikrofauna Marina 8s 77-88. Xiong, B., and Kocher, T. D. (1993). Phylogeny of sibling species of Swofford, D. L. (1993). Phylogenetic analysis using parsimony Simulium venustrum and S. verecundum (Diptera: Simuliidae) (PAUP), vers. 3.1. University of Illinois, Champaign. based on sequences of the mitochondrial 16S rRNA gene. Mol. Phy- Tamura, K., and Nei, M. (1993). Estimation of the number of nucleo­ log. Evol. 2:293-303. tide substitutions in the control region of the mitochondrial DNA in human and chimpanzees. Mol. Biol. Evol. 10: 512-526. 334

Journal o f Fish Biology (1996) 49, 573-583

Reprinted with kind permission from Academic Press

Dietary ascorbic acid requirements during the hatchery production of turbot larvae

G. Merchie*, P. LAVENS*t, Ph. Dhert*, M. García U lloa Gómez*J, H. N e lis § , A. De Leenheer^ and P. Sorgeloos* *Laboratory o f Aquaculture & Artemia Reference Center, University o f Gent, Rozier 44, 9000 Gent, Belgium; §Laboratory of Pharmaceutical Microbiology, University of Gent, Harelbekestraat 72, 9000 Gent, Belgium and ^Laboratory of Medical Biochemistry and Clinical Analysis, University o f Gent, Harelbekestraat 72, 9000 Gent, Belgium

(Received 23 October 1995, Accepted 25 January 1996)

The effect of high ascorbic acid (AA) levels transferred through enriched live food was evaluated for tu rb o t Scophthalmus m aximus larvae in two consecutive feeding experiments. The same feeding strategy was applied to all treatments, except for the AA content of the live food which was manipulated through bioencapsulation with ascorbyl palmitate. This resulted finally in a low, medium and high-AA treatment. The AA incorporation levels in the turbot larvae (up to 1400 gg AA g DW “ 1) were correlated with the AA content of the live food administered. However, feeding the high AA concentration resulted in the same values as for the medium treatment, indicating a saturation of the body AA reserves. Under standard culture conditions, no differences in growth nor overall survival could be detected among the different groups, illustrating that the dietary AA requirements of larval turbot are met by non-enriched live food containing already 500 gg AA g DW ~ '. The larvae of the high-AA treatment, however, showed a better pigmentation rate (47 and 32% for experiments 1 and 2, respectively) compared to the other groups (35 and 25%, respectively). Evaluation of the physiological condition applying a salinity stress test revealed an improvement by feeding extra AA, significantly in the medium-AA treatment. Though not significantly different, cumulative mortalities after chal­ lenge with Vibrio anguillarum amounted to 50% for the control v. 40% for the fish fed medium and high-AA diets, respectively. Moreover, the onset of mortalities in this study was slower (not significantly) for the fish fed the extra AA. © 1996 The Fisheries Society of the British Isles

Key words: ascorbic acid; enrichment; larviculture; Scophthalmus maximus; turbot; vitamin C.

INTRODUCTION

It is estimated that in 1993 over 2-5 million turbotScophthalmus maximus (L.) fry were produced by intensive hatcheries, mainly in Galicia (Spain). Average survival rates in commercial hatcheries reach 10% (varying from 0 to 50%) over the first month of development. This largely unpredictable output might be related to the nutritional value of the live prey organisms. No particular dietary needs for turbot larvae are reported apart from a specific requirement for the fatty acid 22 ; 6n-3 (docosahexaenoic acid, DHA) and more specifically the ratio 22 : 6n-3/20 : 5n-3 (eicosapentaenoic acid, EPA). The current opinion favours a DHA/EPA ratio of 2/1 (Minkoff & Broadhurst, 1994). However, larval require­ ments with respect to DHA/EPA appear to change as functions of the egg tAuthor to whom correspondence should be addressed. Tel: +32-9-2643754; fax: +32-9-2644193; email: [email protected] . JPresent address: Laboratorio de Ciencias Marinas, Universidad Autonoma de Guadalajara, M. Lopez de Legazpi #235, Ap. Postal 3, C.P. 48987, Barra de Navidad, Jalisco, Mexico. 573 0022-1112/96/100573 + 11 $25.00/0 © 1996 The Fisheries Society of the British Isles