Hofstenia Spp., Acoela), with Notes on Color Variation and Genetic Variation Within the Genus

Home , Acoela, Isodiametridae, Meara (genus), Pseudoceros

Hydrobiologia (2007) 592:439–454 DOI 10.1007/s10750-007-0789-0

PRIMARY RESEARCH PAPER

A revision of the systematics of panther worms (Hofstenia spp., Acoela), with notes on color variation and genetic variation within the genus

Matthew Hooge Æ Andreas Wallberg Æ Christiane Todt Æ Aaron Maloy Æ Ulf Jondelius Æ Seth Tyler

Received: 28 July 2006 / Revised: 12 May 2007 / Accepted: 17 May 2007 / Published online: 31 July 2007 Ó Springer Science+Business Media B.V. 2007

Abstract Species of the genus Hofstenia are islands of Bermuda and the Bahamas, and the voracious predators and among the largest and Caribbean and in a variety of habitats includ- most colorful of the Acoela. They are known ing the rocky intertidal, among Thalassia sea from Japan, the Red Sea, the North Atlantic grass, on filamentous algae and decaying mangrove leaves. Certain color morphs associated with each of these habitats seem to have confused the taxonomy of the group. While Handling editor: K. Martens brown-and-white banding and spotting patterns of Hofstenia miamia and Hofstenia giselae are Electronic supplementary material The online version of this article (doi:10.1007/s10750-007-0789-0) contains distinctive for species associated with mangrove supplementary material, which is available to authorized leaves and Thallasia sp. and are likely to be users. cryptic for these specific environments, we find some evidence to suggest that the coloration is M. Hooge (&) S. Tyler Department of BiologicalÁ Sciences, The University of mimicry of a nudibranch with aposematic Maine, Orono, ME 04469-5751, USA coloration. The common plan in these patterns e-mail: [email protected] is one with three variously solid or spotted lighter cross bands on a dark background. Our A. Wallberg Department of Systematic Zoology, Evolutionary examination of museum type material and live Biology Centre, Uppsala University, Norbyva¨gen specimens of Hofstenia collected from Baha- 18D, 752 36 Uppsala, Sweden mas, Belize, Bermuda, and Panama revealed no internal morphological differences between C. Todt Department of Biology, University of Bergen, the Hofstenia species occurring in the Carib- Thormøhlensgate 55, 5007 Bergen, Norway bean. Similarly, our analyses of 18S and 28S molecular sequence data revealed no significant A. Maloy differences among specimens. Accordingly, we Centre of Applied Marine Biotechnology, Letterkenny Institute of Technology, Letterkenny, declare that Hofstenia giselae is a junior syn- County Donegal, Ireland onym of Hofstenia miamia, the three-banded panther worm. U. Jondelius Department of Invertebrate Zoology, Swedish Keywords Platyhelminthes Turbellaria Museum of Natural History, POB 50007, 104 05 Á Á Stockholm, Sweden Mangrove Caribbean Intraspecific variation Á Á

123 440 Hydrobiologia (2007) 592:439–454

Introduction with sclerotized needles. Species of Hofstenia lack a female gonopore, but the sharp needles of the Species of the genus Hofstenia are among the everted penis can penetrate the body wall of the largest and most distinctive of the Acoela. They receiving worm and deposit sperm directly are voracious predators of micrometazoans, suck- beneath its epidermis (Bock, 1923); sperm then ing in prey with a capacious muscular pharynx at apparently migrate through the parenchymal the anterior tip of the body. Mature specimens tissue to the oocytes. are typically 4–9 mm in length and, rather than Such distinctive features made it difficult for being colorless and having the teardrop body early systematists to place Hofstenia among other shape of most acoel species, Hofstenia species are platyhelminths. The first described species, Hof- darkly pigmented, with patterns of white bands stenia atroviridis Bock, 1923, attracted consider- and spots, and nearly cylindrical in shape, often able attention for the primitive features of its with a small pointed tail (Fig. 1). Their carnivory nervous system and gut relative to those of other and color patterns, reminiscent of panthers in platyhelminths and for features of its reproduc- general, inspires their common name. tive system that seemed to be intermediate The large, muscular, anteriorly directed phar- between those of other primitive and the more ynx simplex is one-third to one-half the length of derived flatworms (Bock, 1923; Steinbo¨ ck, 1924; the entire worm (Fig. 2) and is used to suck up Bresslau, 1933; Karling, 1940). At first, similarity copepods, ostracods, and turbellarians. Also dis- of its reproductive organs and pharynx to those of tinctive of Hofstenia is its anteriorly positioned prolecithtophoran and some lecithoepitheliate male copulatory organ (Fig. 2). Located ventral turbellarians was weighed heavily in placing it to the pharynx, the copulatory organ is composed among such so-called ‘‘alloeocoels,’’ even though of a highly muscular, eversible penis equipped its affinity to the Acoela was recognized (Stein-

Fig. 1 Dorsal aspect of eight specimens of Hofstenia miamia from Curac¸ao (from Correˆa, 1963)

123 Hydrobiologia (2007) 592:439–454 441

Fig. 2 Sagittal histological section of Hofstenia miamia mgp—male gonopore, phm—pharynx musculature, from Belize. cs—Digestive central syncytium, e—egg, st—penis stylets, sv—seminal vesicle gv—granule vesicle, m—mouth, ma—male antrum, bo¨ ck, 1924; Meixner, 1938; Karling, 1940). Not acquired the type material for H. atroviridis, H. until a quarter century after its discovery was its giselae, and H. miamia (Table 2) for comparison. true nature as an acoel established (Papi, 1957). We report here the results of our comparative Currently there are four described species of morphological study of this material. In addition, Hofstenia (see Tyler et al., 2006). The type we document the range in color patterns in species, H. atroviridis, is a dark, blackish green Caribbean specimens, and propose systematic species from the coast of Japan where it is found revision of the genus Hofstenia. Acoels have associated with coralline algae in tide pools and been attributed to possess unusually fast evolving on the holdfasts of Laminaria at subtidal depths nuclear ribosomal genes (Carranza et al., 1997; (Bock, 1923). H. miamia Correâ, 1960, was Ruiz-Trillo et al., 1999), which may make these described from a single specimen found in algae molecular markers useful for studying closely at Miami, Florida (Correâ, 1960); however, addi- related acoel lineages (e.g. Tekle et al., 2005). We tional specimens were subsequently found in therefore used 18S and 28S rDNA genes acquired Antigua as well as Curaçao (Correâ, 1963) and from several specimens of Hofstenia from all four were used to document the variation in the dark- collection sites in an attempt to reconstruct a brown, white-banded color patterns within the molecular phylogeny of this taxon and test the species (Fig. 1). Soon thereafter, Steinbo¨ ck traditional classification. (1966) published a monograph on the Hofstenii- dae in which he established two new species: H. beltagii Steinbo¨ ck, 1966, from specimens collected Materials and methods in the Red Sea and first identified by Beltagi (1958) as H. atroviridis, and H. giselae Steinbo¨ ck, Collection and observation of living specimens 1966, collected from a Thalassia bed in the Bahamas. With his specimens of H. giselae, which Approximately 150 living specimens of Hofstenia were lighter-colored than the reported pigmenta- were collected from sites in Bahamas, Belize, tion of H. miamia, Steinbo¨ ck experimented Bermuda, and Panama (Table 1). The majority of extensively on their regenerative capabilities specimens we collected were taken from sub- (Steinbo¨ ck, 1966, 1967). merged, decaying mangrove leaves found in piles Over a 1-year period from April 2004 to May at the base of living mangroves, and had dark 2005, we collected living specimens of Hofstenia coloration that appeared most similar to Correâ’s from Bahamas, Belize, Bermuda, and Panama (1963) H. miamia specimens (Fig. 1). A single (Table 1). Some of these were light-colored and specimen of H. miamia was also found on associated with Thalassia and filamentous algae, Penicillus attached to a mangrove root at Man- which we identified as H. giselae, and others dark atee Cay, Belize. We collected lightly colored and associated with decaying mangrove leaves, specimens of Hofstenia from a Thalassia sp. bed which we identified as H. miamia. We also at Carrie Bow Cay, Belize, and from filamentous

123 442 123

Table 1 Sampling information for specimens of Hofstenia used in this study Locality Latitude (N) Longitude (W) Date Habitat Depth (m) Specimens

Bahamas Lee Stocking Island 23°46¢26† 76°06¢26† April 2004 ﬁlamentous algae <1 ~10 Belize Manatee Cay 16°39¢ 88°11¢ April 2004 Penicillus on mangrove root 1 1 Twin Cays, Grouper Garden 16°49¢46† 88°06¢10† April 2004 Submerged mangrove leaves 1 ~25 Carrie Bow Cay, North side 16°43¢9† 88°04¢54† April 2004 Sediment underlying Thalassia <1 4 Panama Bocas del Toro, STRI ﬁeld station 9°21¢ 82°15¢ August 2004 Submerged mangrove leaves 1 ~30 Bermuda Walsingham Pond, East side 32°20¢46† 64°42¢32† May 2005 Submerged mangrove leaves 1 ~90

Table 2 Comparison of histological sections of Hofstenia, including those shown in Fig. 4 Specimen Location Habitat Color Body Pharynx Pharynx/ Mouth Stylet Stylet length length body length number (mm) (mm) ratio (lm)

Hofstenia atroviridis Lectotype Japan High-energy intertidal/subtidal Dark green 4.2 2.1 0.5 Subterminal 70 ~25 SMNH 2508a assoc. with Laminaria, Corallina Hofstenia giselae

Lectotype Bahamas Low-energy White/brown 3.7 1.0 0.4 Subterminal 70 ~25 Hydrobiologia NMW 12, III, 5 SMNH Belize Low-energy assoc. with Thalassia White/brown assoc. 3.9 1.7 0.4 Subterminal 65 ~25 89913 with Thalassia Hofstenia miamia Paratype Curaçao Low-energy among Brown/white 2.5 1.0 0.4 Subterminal 40 ~20 (2007) SMNH Thalassia and algae 74878/9 592:439–454 USNM Belize Low-energy submerged Brown/white 4.2 1.3 0.3 Subterminal 75 ~25 1096778 mangrove leaves Hydrobiologia (2007) 592:439–454 443 algae at the surface of a saltwater pond on Lee Gran˜ a’’ (Ferrol, Galiza, Spain) in August 2006 in Stocking Island, Bahamas. These specimens mud sediments obtained by dredging with an matched the coloration described for Hofstenia Ockelmann dredge at 6–10 m depth. giselae by Steinbo¨ ck (1966). Genomic DNA was extracted from most spec- Hofstenia giselae from Thalassia were ex- imens using the Qiagen DNeasyÒ Tissue Kit tracted by placing a large quantity of Thalassia following the manufacturer’s default protocol. sp. (~3 L), including the roots and underlying PCR amplification of the small and large sediment, in a bucket of sea water. Specimens nuclear ribosomal subunit genes (18S and 28S) crawled out of the Thalassia sp. to the water were carried out using Amersham Biosciences surface over a period of 3–8 h. Mangrove-dwell- PuReTaqTM Ready-To-GoTM PCR Beads and an ing specimens of H. miamia were similarly Eppendorf Mastercycler Gradient thermal cycler. extracted by placing submerged mangrove leaves 18S was amplified using a two-step nested PCR and associated detritus in buckets of sea water. approach first using primers TIMA-TIMB, fol- These specimens crawled to the water surface lowed by combining two overlapping fragments over a period of 1–5 h. for a total length of about 1,750 nucleotides Several specimens from each collection site (primers S30-5FK and 4FB-1806R). 28S was were photographed with a digital camera at- amplified using a single step approach combining tached to a dissecting microscope. The specimens three overlapping fragments for a total maximum reacted poorly to isotonic magnesium chloride or length of about 3,000 nucleotides (U178-L1642, magnesium sulfate, often undergoing convulsions 1200F-R2176, U1846-L3449). The typical PCR and thereby distorting their natural habitus. As an regime for each gene and fragment was: 95°C for alternative, drops of ethanol were added to a 5 min, 35· (94°C for 30 s, 50°C for 30 s, 72°C for small dish of seawater until the specimens slowed 30 s), 72°C for 10 min. Samples were sent to enough for photography. Macrogen, Inc. (Seoul, Korea) for sequencing. Total genomic DNA of the Proporus carolin- Histology ensis specimens was extracted using the QIA- ampÒ DNA Micro Kit (Qiagen). Two nearly For histological study, unrelaxed specimens were equal lengths of the 18S rRNA gene were PCR- squirted directly into hot Stefanini’s fixative amplified primers 1F-5R and 5F-9R (Carranza (Stefanini et al., 1967), washed in phosphate et al., 1997). Reaction conditions included 1 ll of buffer (Millonig’s, 0.1 M), fixed in phosphate- template DNA, 2.5 mM MgCl2, 0.75 lM of each buffered 1% (v/v) osmium tetroxide, dehydrated primer, 200 lM of each deoxynucleoside triphos- in an acetone series, and embedded in EMBed/ phate, 1.25 U Taq polymerase (Invitrogen Corp., Araldite epoxy resin. Dehydration was quickened Carlsbad, Calif.), and 2.5 ll 10X PCR buffer. The by microwave radiation according to Giberson & remaining volume was completed with nuclease- Demaree (1995). Serial 2-lm-thick sections were free water. PCR regime was: 95°C for 3 min, 35· made according to Smith & Tyler (1984) using a (95°C for 30 s, 55°C for 30 s, 72°C for 30 s), 72°C Diatome diamond knife mounted in a Butler for 7 min. Amplicons were filter-purified (Mon- trough (Butler, 1979), and stained with toluidine tageTM, Millipore) and directly sequenced at the blue. University of Maine’s DNA sequencing facility using an ABI model 3730 DNA Analyzer DNA analyses (Applied Biosystems, Foster City, Calif.) with the BigDye Version 3.1 Cycle Sequencing Kit per In addition to Hofstenia miamia and H. giselae, manufacturer’s instructions. our molecular sequence analyses used other acoel The sequences were assembled using the GNU/ terminals as well as several non-acoel outgroups Linux-version of the program Staden v1.6.0 (Sta- (Table 3), including two specimens of an uniden- den, 1996); suite enhanced by the Phred/Phrap tified species of Hofsteniidae that were found in base calling/assembly modules (Ewing et al., 1998; the bay near the Marine Biological Station of ‘‘A Ewing & Green, 1998) and manually edited at

123 444 Hydrobiologia (2007) 592:439–454

Table 3 Taxa used in molecular analyses, GenBank sequence accession numbers, and location of newly collected material Taxon 18S 28S Location

Chordata Mus musculus MMRNA18 – Ctenophora Mnemiopsis leidyi MNESSRRNA – Cnidaria Nematostella vectensis AF254382 – Pennatula sp. AY838561 – Nemertodermatida Meara stichopi AF100191 – Acoela Childiidae Do¨ rjes, 1968 Childia brachyposthium AY297952 AJ849499 Childia crassum AJ845242 AJ849497 Childia cycloposthium AF329178 AJ849494 Childia groenlandica 1 AY078367 – Childia groenlandica 2 CGR012529 AY157603 Childia groenlandica 3 AM701805 AM701806 Galiza, Spain Childia macroposthium AY297953 AJ849500 Childia submaculatum AY297953 AJ849496 Childia trianguliferum AY297950 AJ849495 Childia vivipara AY297954 AJ849498 Hofsteniidae Bock, 1923 Hofstenia giselae 1 AM701807 AM701808 Lee Stocking Island, Bahamas Hofstenia giselae 2 AM701809 AM701810 Lee Stocking Island, Bahamas Hofstenia giselae 3 AM701811 AM701812 Carrie Bow Cay, Belize Hofstenia miamia 1 AM701813 AM701814 Walsingham Pond, Bermuda Hofstenia miamia 2 AM701815 AM701816 Bocas del Toro, Panama Hofstenia miamia 3 AM701817 AM701818 Twin Cays, Belize Hofstenia sp. 1 AM701819 AM701820 Galiza, Spain Hofstenia sp. 2 AM701821 AM701822 Galiza, Spain Paratomellidae Do¨ rjes, 1966 Paratomella rubra AF102892 AY157604 Paratomella unichaeta AY078379 – Proporidae Graff, 1882 Proporus carolinensis 1 AM701825 – Carrie Bow Cay, Belize Proporus carolinensis 2 AM701826 – North Carolina, USA Solenofilomorphidae Do¨ rjes, 1968 Myopea ‘‘callaeum’’ sp. AY078380 – Oligofilomorpha interstitiophilum AM701823 AM701824 Gullmarsfjorden, Sweden Accession numbers in bold denote newly acquired sequences some sites. Small alignments, including only Hof- (Bremer, 1994) assessment as implemented in stenia sequences, were inferred using the default the program TNT v1.0 (Goloboff et al., 2000), settings of the Muscle v3.6 progressive alignment where trees up to 20 steps longer than the most software (Edgar, 2004). These datasets were con- parsimonious tree were included to calculate the catenated. Additional 18S/28S sequences were support. The Hofstenia-only datasets were ana- downloaded from NCBI Genbank and were lyzed using the branch-and-bound algorithm, aligned using the same methods; the alignments while the large datasets were analyzed using 10 were not refined manually. A complete list of random additions, each with TBR branch swap- included sequences is provided in Table 3. ping holding up to 10 trees. Phylogenies were also Phylogenetic trees with branch support were inferred with the Bayesian method as imple- first inferred from each gene separately using the mented in the program MrBayes (Huelsenbeck & parsimony criterion and Bremer support Ronquist, 2001; Ronquist & Huelsenbeck, 2003).

123 Hydrobiologia (2007) 592:439–454 445

MrAIC v1.4.2 (Nylander, 2004) and Phyml v2.4.4 SMNH 74869-74880, which bear markings from (Guindon & Gascuel, 2003) were used to select the University of Saõ Paulo, Brazil, and are likely one of the evolutionary models available for the Correâ’s (1963) specimens from Curaçao. For H. Bayesian analysis. The method consistently sug- giselae, we examined Steinbo¨ ck’s (1966) material, gested the two models GTR + G and GTR + including NMW 12-III-4, 5, 6, 7. There is appar- I + G with very similar likelihoods (see Lanave ently no deposited type material of H. beltagii et al., 1984; Tavare´, 1986 for formal description of (see Steinbo¨ ck, 1966). the GTR model). We used GTR+G for the small datasets and GTR + I + G for the large datasets. All analyses used four independent runs of four Results MCMC chains of several millions of generations. Average standard deviation of split frequencies of Morphological analysis 0.005 or lower were used to deduce when param- eters had been adequately sampled from the Mature specimens of Hofstenia collected from stationary distribution. The burn-in fraction was Bahamas, Bermuda, Belize, and Panama were set to 50%. Trees were saved every 500th gener- approximately 4 mm long when contracted, but ation. The two small Hofstenia-only datasets were could elongate up to 6–7 mm. The coloration of also concatenated and analyzed using the same H. miamia specimens collected from mangroves methods. The majority rule consensus tree result- varied considerably, and no two specimens had ing from this analysis was rooted using the two exactly the same pattern of coloration (Fig. 3). Hofstenia specimens from Galiza as outgroups. All specimens of H. miamia were predominately The patristic distances between terminals brown to blackish brown, but they varied with inferred by the bayesian method were calculated regard to the occurrence of additional white by summing the branch lengths separating termi- coloration. Most common was the pattern of nals and inspected to try addressing within/ three white cross bands that were either solid or between species divergence. somewhat discontinuous. Other white dapples or flecks of white often occurred, particularly at the Type material anterior tip of the animal and also along the dorsal midline. While the coloration of speci- We examined all the type material available for mens collected from Panama were predomi- the genus Hofstenia from the Swedish Museum of nately spotted (Fig. 3: 1–8) and those from Natural History (SMNH) and the Naturhistoris- Belize were mostly striped (Fig. 3: 25–28), we ches Museum Wien (NMW). For H. atroviridis observed a whole gamut of coloration in Ber- we examined Bock’s (1923) original material muda, from specimens that nearly lacked any including the lectotype (SMNH 2508, 2508a), as white coloration, to those with solid white well as several paratypes (SMNH 74901-74915). stripes and additional white blotches (Fig. 3: 9– For H. miamia we examined SMNH 2744, 2744a. 24). These slides were designated ‘‘Holotype’’ by Specimens of Hofstenia giselae collected from Do¨ rjes & Karling (1975). However, the slides Thalassia at Carrie Bow Cay, Belize (Fig. 3: 30– are marked ‘Antigua’, M56 and M57 whereas the 31), and from filamentous algae from a saltwater type specimen collected in Virginia Key, Miami pond on Lee Stocking Island, Bahamas (Fig. 3: Florida was originally marked M2 (Correâ, 1960). 32–33) had a distinctly different coloration than We therefore regard SMNH 2744 and 2744a as specimens of H. miamia. These were predomi- the neotype of Hofstenia miamia designated by nately covered with overlapping white flecks, so Do¨ rjes & Karling (1975). The specimen is prob- that they appeared mostly white. In the posterior ably one of those collected by PW Hummelinck half of the body was a wide brown band that as mentioned by Correâ (1963). We could not covered the lateral regions of the dorsal side but locate the original holotype specimen from did not cross over the dorsal midline, so as to Miami, Florida. Also for H. miamia, we examined leave a white stripe in that region.

123 446 Hydrobiologia (2007) 592:439–454

Fig. 3 Color variation in Hofstenia miamia and Hofstenia sediment with Thalassia (30–31) and on ﬁlamentous algae giselae from four geographic locations. All specimens of (32–33). (1–8) Bocas del Toro, Panama. (9–24) Walsingham H. miamia (1–29) were collected from submerged man- Pond, Bermuda. (25–29) Twin Cays, Belize. (30–31) Carrie grove leaves. Specimens of H. giselae were found in Bow Cay, Belize. (32–33) Lee Stocking Island, Bahamas

Despite differing external body coloration, revealed only minor differences (Fig. 4, Table 2), specimens of Hofstenia have matching internal none of which appear to be useful diagnostically. morphology. Our comparison of the internal The mouth in all specimens examined was posi- morphology in serial histological sections of tioned subterminally. Nearly all of the sectioned Hofstenia atroviridis, H. giselae, and H. miamia specimens we examined were approximately

123 Hydrobiologia (2007) 592:439–454 447

Fig. 4 Photomicrographs of histological sections of Hof- Bahamas. (d) H. miamia, sagittal section, Carrie Bow Cay, stenia, showing morphology of male copulatory organ (left Belize from Thalassia. (e) H. miamia, sagittal section, side), and close-up of penis stylets (right side). (a) H. Twin Cays, Belize from mangrove leaves. Scale bars at atroviridis, sagittal section, Lectotype SMNH 2508a, bottom of ﬁgure apply to all images on right and left sides Misaki, Japan. (b) H. miamia, frontal section, Paratype respectively. gv—granule vesicle, ma—male antrum, SMNH 74878, Piscadera Baai, Curac¸ao. (c) H. giselae, pb—penis bulb, phe—pharynx epithelium, sp—penis sagittal section, Lectotype NMW 12, III, 5, North Bimini, sperm, st—penis stylets, sv—seminal vesicle

4 mm long and had a pharynx that was one-third Behavior to one-half the body length. There was also similarity with regard to the penis stylets, with Living specimens of H. miamia collected from most specimens having approximately 25 stylets mangroves in Bermuda were successfully kept in that were 65–75 lm in length. The exception to the laboratory in Petri dishes at room tempera- these measurements was H. miamia from Cura- ture for as long as four weeks. During this time c¸ao, which had a shorter body length, 2.5 mm, the specimens tended to lie unmoving at the was proportionally smaller with regard to pharynx bottom of the dish unless disturbed by vibrations and stylet length, and had fewer stylet needles or light. No changes in the coloration of the (~20) (Fig. 4b, Table 2). specimens could be discerned during this period.

123 448 Hydrobiologia (2007) 592:439–454

Squeeze preparations and histological sections separated from the unidentified Hofstenia revealed that the worms fed upon copepods and sequences; but neither H. miamia or H. giselae ostracods. Additionally, we observed the animals are recovered as distinctly separate species when feeding upon other prey items we provided them, the two genes are analyzed separately (Figs. 5, 6) including small acoels, rhabdocoel turbellarians, or concatenated (Fig. 7). Instead, the H. miamia and polychaetes. Using the dissecting microscope and H. giselae sequences are mixed with respect we also observed some copulation events. The to the traditional classification. The 18S alignment worms occasionally everted their penes in the limited to only Hofstenia sequences recovers a presence of another specimen, and there ap- slightly different topology compared to the com- peared to be a short period of penis ‘probing’ plete 18S alignment (Fig 5), where the two before the penis stylets were finally stabbed into Hofstenia specimens from Belize make up the the epidermis at the posterior end of the receiving sister group to the other H. miamia and H. giselae worm. (not shown). The corresponding 28S alignment recovers the same phylogeny (not shown) Molecular analysis as the complete and concatenated alignments (Figs. 6, 7). Analysis of both 18S and 28S sequences support a Analyses of the concatenated genes recover monophyletic Hofstenia clade as the sister taxon two sister groups: an ‘‘Atlantic’’ clade uniting to members of the Solenofilomorphidae (Figs. 5, specimens from Bahamas and Bermuda, and a 6). Sequences of H. miamia and H. giselae form a ‘‘Caribbean’’ clade of specimens from Panama well-supported group within that clade, clearly and Belize (Fig. 7). There are very few variable or

1.00/13 Childia macroposthium 1.00/3 Childia brachyposthium 0.98/3 Childia vivipara 0.99/4 Childia crassum Childia groenlandica1 USA 1.00/20+ 1.00/3 Childia groenlandica 3 Spain Childia groenlandica 2 Europe 1.00/20+0.68/0 Childia submaculatum Childia trianguliferum 1.00/5 Childia cycloposthium 1.00/20+ Proporus carolinensis 1 Belize Proporus carolinensis 2 USA 0.68/0 Hofstenia giselae 1 Bahamas 1.00/3 Hofstenia giselae 2 Bahamas 1.00/4 1.00/1 Hofstenia miamia 1 Bermuda 1.00/4 Hofstenia miamia 2 Panama 1.00/17 Hofstenia miamia 3 Belize 1.00/20+ Hofstenia giselae 3 Belize 1.00/20+ 1.00/20+ Hofstenia sp 1 Spain 1.00/4 Hofstenia sp 2 Spain 1.00 /20+ Myopea sp “callaeum” 1.00/20+ Oligofilomorpha interstitiophilum 1.00/20+ Paratomella unichaeta Paratomella rubra 1.00/5 Mus musculus Meara stichopi 1.00/9 Pennatulasp Nematostella vectensis Mnemiopsis leidyi

0.1

Fig. 5 Phylogenetic analyses of nuclear ribosomal small lected from four runs of 6 million MCMC generations subunit 18S rDNA sequences indicate that Hofstenia is using the GTR+I+G model in MrBayes. Bremer support closely related to Solenoﬁlomorphidae. Topology, branch- (italic values above branches) for the corresponding most lengths and bayesian posterior probabilities (bold values parsimonious tree(s) (2 trees, 2,961 steps, CI = 0.55, above branches) were estimated from 24,000 trees col- RI = 0.78) were calculated using TNT

123 Hydrobiologia (2007) 592:439–454 449

1.00/2 Hofstenia giselae 1 Bahamas 0.54/0 Hofstenia giselae 2 Bahamas Hofstenia miamia 1 Bermuda 1.00/20+ 0.97/1 Hofstenia giselae 3 Belize 0.99/1 Hofstenia miamia 3 Belize 1.00/20+ Hofstenia miamia 2 Panama 1.00/20+ 1.00/20+ Hofstenia sp 1 Spain Hofstenia sp 2 Spain Oligofilomorpha interstitiophilum 1.00/10 Childia macroposthium 1.00/6 Childia brachyposthium Childia vivipara 0.92/-- 0.83/1 Childia trianguliferum 0.99/--1.00/-- Childia cycloposthium Childia submaculatum 1.00/5 Childia crassum 1.00/20 Childia groenlandica Childia groenlandica Spain Paratomella rubra 0.1

Fig. 6 Phylogenetic analyses of nuclear ribosomal large lected from four runs of 8 million MCMC generations subunit 28S rDNA sequences indicate that Hofstenia is using the GTR + I + G model in MrBayes. Bremer closely related to Solenoﬁlomorphidae. Topology, branch- support (italic values above branches) for the correspond- lengths and bayesian posterior probabilities (bold values ing most parsimonious tree(s) (1 tree, 1,498 steps, CI above branches) were estimated from 32,000 trees col- = 0.79, RI = 0.90) were calculated using TNT

1.00/2 Hofstenia giselae 1 Bahamas once inside the clade (CI = 1.0). In the longer 0.96/2 Hofstenia giselae 2 Bahamas 28S sequence, only 14 sites are parsimony infor- Hofstenia miamia 1 Bermuda mative out of 45 variable sites. 1.00 /20+ 1.00/3 Hofstenia giselae 3 Belize The average patristic distance or sequence 1.00/3 Hofstenia miamia 3 Belize divergence between a member of one geographic Hofstenia miamia 2 Panama clade and a member of the other is low; only Hofstenia sp 1 Spain 0.0089 for 18S. Since analysis of that gene did not Hofstenia sp 2 Spain recover the monophyletic ‘‘Caribbean’’ clade 0.01 when analyzing all data at once, its monophyly Fig. 7 Phylogenetic analyses of concatenated ribosomal was enforced as a topological constraint. The data (nuclear small and large subunit 18S/28S rDNA) fail corresponding average distance for 28S is 0.0098. to recover separate H. miamia and H. giselae clades. These distances translate into an expected Topology, branch-lengths and bayesian posterior proba- number of substitutions between the members bilities (bold values above branches) were estimated from 120,000 trees collected from four runs of 20 million of different clades of approximately 15 in a MCMC generations using the GTR + G model in 1,750-nucleotide long stretch of 18S and 27 in a MrBayes. Bremer support (italic values above branches) 3,000-nucleotide long stretch of 28S. The molec- for the corresponding most parsimonious tree(s) (1 tree, ular divergence within the H. miamia/H. giselae 229 steps, CI = 0.98, RI = 0.97) were calculated using TNT. The majority rule consensus tree resulting from this clade is thus lower than or about equal to that in analysis was rooted using the two Hofstenia specimens the Hofstenia sp. clade (18S: 0.0144; 28S: 0.0077), from Galicia as outgroups higher than or about equal to that within the Childia groenlandica clade (18S: 0.0030; 28S: parsimony informative sites in the 18S and 28 S 0.0099), lower than that within the Proporus sequences within the H. miamia / H. giselae clade carolinensis clade (18S: 0.026212) and lower than (Table 4). For instance, only 11 sites in the 18S that between the two most closely related but sequence are actually variable among the samples distinctly classiﬁed Childia species, Childia mac- and only 7 of those are parsimony informative; roposthium and Childia brachyposthium (18S: they seem to have changed character states only 0.0117; 28S: 0.0139).

123 450 Hydrobiologia (2007) 592:439–454

Discussion SD (from avg <0.005 <0.005 <0.003 <0.001 <0.002 <0.005 Distinguishing Hofstenia species freq.) In his monograph of the Hofsteniidae, Steinbo¨ ck split million; million; million; million; million; million; (1966) detailed the morphological differences 4 5 5 5 3 3 Stationarity generation; of among species of all 6 species of the Hofsteniidae he recognized, namely four Hofstenia species and

inference two species in monotypic genera, Hofsteniola pardii Papi, 1957, and Marcusiola tinga (Marcus, million million million million million million 1957). In his key to Hofstenia species, Steinbo¨ ck 8 6 Bayesian 10 20 6 20 MCMC (generations) (1966: 137) distinguishes H. miamia and H. beltagi from H. atroviridis and H. giselae by a proportionally shorter antrum and smaller body size, and H. beltagi speciﬁcally by its antrum having a wider support

MPT+20 distal part opening in a section perpendicular to the ventral body surface. He distinguished 7156 26406 561 – 112 80 Bremer (trees steps) H. atroviridis from H. giselae by shape of the penis bulb and pouch. b b

b b Contrary to the ﬁndings of Steinbo¨ ck (1966), 0.91)

0.91) our investigation of the histological sections of 1.0) 1.0) Hofstenia species revealed the morphology of the (1.0; (1.0; (0.96; copulatory organs to be quite similar (Fig. 4, Table 2). We suggest that minor differences in the 0.90 0.78 0.98 0.98 0.97 0.97(0.97; RI) inference length or shape of muscular structures such as the 0.79; 0.55; 0.97; 0.97; 0.97; 0.98; Consistency (CI; penis pouch and male antrum are more likely due to differences in contraction during ﬁxation,

parsimony rather than true diagnostic differences. Most a steps) specimens we examined had similar body length, 76 77

152 229 pharynx length, stylet length, and stylet number 1489 2961 1; 1; 2; 1; 1; 2; (trees; Maximum MPT(s) (Fig. 4, Table 2). Although the H. miamia spec- topology sites performed imen from Curac¸ao is proportionally smaller with 28S b b b b

+ regard to these features (Fig 4B), the holotype of 7) 7) 813 871 14) 21) H. miamia was reported by Correâ (1960) to be analysis 18S of (11; (11; sites (45; (56; more similar at 4 mm long and with penis stylets informative 1473; 1080; 73 73 the pars. 70 lm in length. We are, however, perplexed that 144 217 to 89; 89; the H. miamia holotype was also reported to have 262; 351; Variable (variable; which informative) clade 50 penis stylets (Correâ, 1960), far more than any phylogenetic parsimony of the specimens we examined (Table 2), even according each giselae those prepared by Correâ (1963). H. for The lack of any available type material for including 3445 2994 4767 1988 1773 1773 +

enforce H. beltagii precludes us from verifying the validity 8; 8; 8; 8; 19; 29; Dataset Dimensions (terminals; sites)

only of this species. The diagnostic characters Stein- details

miamia bo¨ ck (1966) provides – namely, in the shape of the c steps H.

constraint male antrum and canal – seem rather weak, since of

the agonistic contraction upon ﬁxation could easily Technical

4 cause such shape differences. He, himself, cau- constrained

Inside tions that the material is poorly preserved and Number Topology 18S 28S 28S 18S+28S 18S 18S Table Gene a b c comprises just two sets of sections. In all other

123 Hydrobiologia (2007) 592:439–454 451 characters that Steinbo¨ ck (1966) mentions (struc- solely from the descriptions of Correâ (1960, ture of the nervous system and pharynx, arrange- 1963) and not from his own personal examination ment of ovaries and testes) H. beltagii is like of the type material. Our investigation of the H. miamia and H. giselae, and its color is morphology of H. giselae and H. miamia revealed described as mottled brown, also perhaps like virtually no difference between these two species that of H. miamia. Lacking type specimens and with regard to body size, shape, the position of adequately documented distinguishing features, the mouth, or the morphology of the copulatory we declare H. beltagi to be a species inquirenda. organs (Fig. 4, Table 2). As for H. atroviridis, although its internal Our molecular analysis also does not support morphology seems to match closely that of other H. miamia and H. giselae being classified as Hofstenia species, there are other characteristics separate species. Specifically, the molecular phy- that seem to secure its position as a distinct logeny reconstructed here has a topology that is species. H. atroviridis can be superficially distin- incongruent with the traditional classification and guished from other species by its dark blackish- instead suggests a geographic partitioning of green coloration and by its lack of a tail (although lineages. Although such patterns should perhaps perhaps this feature was simply overlooked by be expected in benthic fauna with little known Bock, 1923). More important is the habitat capability of long distance pelagic dispersal, it has preference of H. atroviridis, which is distinct from not been demonstrated in acoels before. that of the Caribbean species. Bock (1923) A comparison of patristic distances in non- collected H. atroviridis from wave-washed inter- Hofstenia acoel clades do not yield consistent tidal and subtidal sites where the oxygen content results regarding within-species or between-spe- of the water is frequently replenished. Bock cies variation. However, the molecular sequence (1923) found that specimens kept in dishes in data available for such studies in acoels are too the laboratory were often attached to the water limited to bring any clear insight into these surface and only lived a short time; he hypothe- matters at this point. Molecular divergence in sized that their deaths were due to a lack of an the H. miamia + H. giselae clade is very low, adequate supply of oxygen. By contrast, H. mi- about 1% in the two ribosomal genes, and a large amia and H. giselae are found in quiet subtidal proportion of the variation is not parsimony water in rather stagnant conditions among dead informative. We question whether this low vari- and decaying vegetable material. We were able to ation and degree of molecular synapomorphies keep specimens of H. miamia alive in Petri dishes approach that which might be randomly gener- for as long as a month, doing little to maintain ated when base calling and assembling chroma- them other than occasionally changing their water tograms with some low level of ambiguity. Not all and feeding them specimens of the acoel Isodi- chromatograms were perfect in our case, but it is ametra pulchra (Smith & Bush, 1991). unlikely that a minor number of errors would create such a phylogenetic bias that the two genes Synonomization of Hofstenia giselae under independently recover very similar phylogenies, Hofstenia miamia assuming that chromatogram errors are randomly distributed to begin with. Such errors would most In his description of H. giselae, Steinbo¨ ck (1966) likely simply inflate the proportion of variable but reported many differences with H. miamia, stat- uninformative sites and we might therefore actu- ing that H. giselae was longer, wider, taller, had a ally overestimate the molecular divergence in this distinct tail that was lacking in H. miamia and was group, which in a way further supports the finding without the distinct ‘‘head’’ of H. miamia. In of H. miamia and H. giselae not being separate addition, Steinbo¨ ck also mentioned differences in species. If an unknown biological mechanism is mouth position, pharynx length, and characters of indeed responsible for generating elevated evo- the male copulatory organ. The findings of lutionary rates in acoel sequences, its effect seems Steinbo¨ ck (1966) may have been askew in part to go unnoticed in this sample of geographically because his information on H. miamia came separated Hofstenia specimens.

123 452 Hydrobiologia (2007) 592:439–454

The phylogeny recovered within the H. miamia of body color is uncommon in the Acoela. + H. giselae clade displays a curious geographic Intraspecific color variation comparable to that pattern, but is based on very little informative of Hofstenia occurs in the Polycladida and also in data and may be subject to change if more data is the Nudibranchia [e.g., Roboastra gracilis (Bergh, added. We suggest a posteriori that further studies 1877)]. The polyclad genera Pseudoceros and of very closely related lineages of acoels focus on Pseudobiceros can be identified by their color more quickly evolving markers such as mitochon- patterns, but in several species the details of the drial genes or the ITS-region, as done in many coloration are inconstant among individuals (e.g., other taxa. Pseudoceros dimidiatus von Graff, 1893; P. gratus Given the failure of our morphological and Kato, 1937; P. scintillates Newman & Cannon, molecular analyses to support H. miamia and 1994; P. zebra Leuckart, 1828). In Pseudoceros H. giselae being classified as separate species, we dimidiatus, the body always has a black back- herein designate H. giselae as a junior synonym of ground with an orange margin and two yellow H. miamia. longitudinal stripes; however, the thickness and spacing of these longitudinal stripes are not fixed, Systematic position of the Hofsteniidae and some specimens may have additional trans- verse yellow stripes that can be quite variable Although not widespread, species possessing (Newman & Cannon, 1994, 2003). pharynges simplices do occur in several acoel In the case of Pseudoceros dimidiatus, the families, including Convolutidae (Oligochoerus), bright yellow and black coloration is thought to Diopisthoporidae, Hallangiidae, Hofsteniidae, be a classic example of aposematic coloration that Isodiametridae (Isodiametra helgolandica), Nadi- warns potential predators of the worms’ toxicity. nidae, Proporidae, and Solenofilomorphidae. Polyclads also use mimicry to deter predators. Morphological studies suggest that pharynges Some palatable species use Batesian mimicry to were derived independently in multiple lineages match the coloration of noxious nudibranchs, within the Acoela (Riedl, 1954; Doe, 1981), and while some unpalatable species have coloration molecular sequence data supports this conclusion closely matching other unpalatable organisms, so (Hooge et al., 2002). Our finding that 18S and 28S called Mu¨ llerian mimicry (Newman & Cannon sequence data supports a close relationship 2003). The contrasting white and brown banding between Hofsteniidae and Solenofilomorphidae pattern in H. miamia may be a case of aposematic may indicate that the pharynges in these groups coloration or a case of mimicry of the similarly have a common origin. The male copulatory colored nudibranch Navanx aenigmaticus (Bergh, organ of hofsteniids is distinctly different from 1893), an unpalatable species that is sometimes that of other acoels, including solenofilomorphids, found in Thallasia sp. beds in the Caribbean and does not appear to provide characters useful (Gundersen, 2003). Not all individuals are dis- for elucidating the phylogenetic position of the tinctly banded, however, and we have no exper- Hofsteniidae within the Acoela. However, we imental data to show that H. miamia is expect that on-going studies on body-wall mus- unpalatable to fish or other potential predators. culature, as well as sperm and pharynx ultrastruc- More than likely, the coloration of H. miamia ture may be helpful in this regard. is cryptic for its particular habitat. From our A full account of synonyms for the family mangrove samples, which consisted of brown Hofsteniidae is provided in Electronic supple- leaves spotted with white patches of sulfur bac- mentary material Appendix 1. teria and the sessile ciliate Zoothamnium niveum Ehrenberg, 1838, we found that several of the Patterns of body coloration associated small invertebrates had similar brown and white banding, including a small amphipod The color patterns of H. miamia are unusual in that appeared superficially quite similar to H. part because most acoels do not have opaque miamia, particularly if viewed from the rostral coloration, but also because intraspecific variation apex. If Hofstenia coloration is adapted to their

123 Hydrobiologia (2007) 592:439–454 453 particular habitat, this could explain the distinct National Science Foundation under grant no. 0118804 color differences we found between specimens (MH, ST) and the Swedish Research Council (UJ). Wallberg was kindly funded by Inez Johanssons stiftelse. collected from mangrove leaves and Thalassia beds. True to its name, H. atroviridis is dark black- References green in color and relatively conspicuous among the coralline algae in the tidepools from which it Beltagi, S. M., 1958. Hofsteniidae und neue Acoela aus was collected (Bock, 1923: 3, 4). Hofsteniola dem Roten Meer. Dissertation, Institut fu¨ r Zoologie pardii is reddish to pink in color, the most heavily Wien, Nr. 333. Bock, S., 1923. Eine neue marine Turbellariengattung aus pigmented around the pharynx and paler caudally Japan. Uppsala Universitets A˚ rsskrift, Mathematik (Papi, 1957: 133). The last hofsteniid, Marcusiola och Naturvetenskap 1: 1–52. tinga, is colorless (Marcus, 1957: 170). Bremer, K., 1994. Branch support and tree stability. Cla- distics 10: 295–304. Bresslau, E., 1933. Erster Klasse des Cladus Plathelmin- Future studies thes: Turbellaria. In Kukenthal, W. & Th. Krumbach (eds), Handbuch der Zoologie, Vol. 2, Part 1, 54–320. Our investigation shows Hofstenia miamia to be a Butler, J. K., 1979. Methods for improved light microscope widespread and abundant species in the Carib- microtomy. Stain Technology 54: 53–69. Carranza, S., J. Bagun˜ a & M. Riutort, 1997. Are the bean, and we foresee its use in future ecological Platyhelminthes a monophyletic primitive group? An and experimental investigations. That it has assessment using 18S rDNA sequences. Molecular apparently escaped any mention in studies of Biology and Evolution 14: 678–679. Caribbean ecology, particularly the diversity of Correâ, D. D., 1960. Two new marine Turbellaria from Florida. Bulletin of Marine Science of the Gulf and mangrove assemblages, is surprising, especially Caribbean 10: 208–216. given its voracious appetite for other small Correâ, D. D., 1963. The turbellarian Hofstenia miamia in invertebrates. Its relatively large size compared the Caribbean Sea. Studies Fauna Curaçao 17: 38–40. to that of other acoel species, along with its Do¨ rjes, J. & T. G. Karling, 1975. Species of Turbellaria Acoela in the Swedish Museum of Natural History, apparent basal position in the Acoela, its ability with remarks on their anatomy, taxonomy and distri- to regenerate (Steinbo¨ ck, 1966, 1967), and its bution. Zoologica Scripta 4: 175–189. potential for being easily cultured in the labora- Doe, D. A., 1981. Comparative ultrastructure of the tory, make H. miamia a suitable candidate for use pharynx simplex in Turbellaria. Zoomorphology 97: 133–193. in experimental biology, or perhaps even as a Edgar, R. C., 2004. MUSCLE: Multiple sequence align- model organism of basal bilaterians. ment with high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797. Acknowledgements We are grateful to Klaus Ru¨ tzler for Ewing, B., L. Hillier, M. C. Wendl & P. Green, 1998. Base- the opportunity to work at Carrie Bow Cay and we thank calling of automated sequencer traces using phred. I. Wolfgang Sterrer and Jo¨ rg Ott for their help in collecting Accuracy assessment. Genome Research 8: 175–185. specimens in Belize. We thank Rachel Collin of the Ewing, B. & P. Green, 1998. Base-calling of automated Smithsonian Tropical Research Institute (STRI) for sequencer traces using phred. II. Error probabilities. inviting us to be a part of the taxonomic survey at Bocas Genome Research 8: 186–194. del Toro, Panama and for providing financial assistance Giberson, R. T. & R. S. Demaree Jr., 1995. Microwave and logistical support. We also extend our gratitude to fixation: Understanding the variables to achieve rapid Wolfgang Sterrer of the Bermuda Natural History reproducible results. Microscopy Research and Museum for his invaluable help in the collection of Techniques 32: 246–254. specimens in Bermuda and to the highly altruistic staff of Goloboff, P., S. Farris & K. Nixon, 2000. TNT (Tree the Perry Institute for Marine Science in Lee Stocking analysis using New Technology) (BETA) ver. 1.0 Island, Bahamas. Type material was generously made Published by the authors, Tucumań, Argentina. available to us by the Swedish Museum of Natural History Guindon, S. & O. Gascuel, 2003. A simple, fast, and the Naturhistorisches Museum Wien. This is and accurate algorithm to estimate large phylogenies contribution no. 797 of the Caribbean Coral Reef by maximum likelihood. Systematic Biology 52: Ecosystems Program, National Museum of Natural 696–704. History, Smithsonian Institution, and contribution no. Gundersen, R. W., 2003. Navanax aenigmaticus from 125 of the Bermuda Biodiversity Project (BBP), Jamaica. [Message in] Sea Slug Forum. Australian Bermuda Aquarium, Natural History Museum and Zoo. Museum, Sydney. Available from http://www. This material is based upon work supported by the seaslugforum.net/find.cfm?id=11126.

123 454 Hydrobiologia (2007) 592:439–454

Hooge, M. D., P. Haye, S. Tyler, M. K. Litvaitis & I. Ronquist, F. & J. P. Huelsenbeck, 2003. MRBAYES 3: Kornfield, 2002. Molecular systematics of the Acoela Bayesian phylogenetic inference under mixed models. (Platyhelminthes) and its concordance with mor- Bioinformatics 19: 1572–1574. phology. Molecular Phylogenetics and Evolution 24: Ruiz-Trillo, I., M. Riutort, D. T. J. Littlewood, E. A. 333–342. Herniou & J. Bagun˜ a, 1999. Acoel flatworms: Earliest Huelsenbeck, J. P. & F. Ronquist, 2001. MRBAYES: extant bilaterian metazoans, not members of the Bayesian inference of phylogeny. Bioinformatics 17: Platyhelminthes. Science 283: 1919–1923. 754–755. Smith, J. P. S. & S. Tyler, 1984. Serial sectioning and Karling, T. G., 1940. Zur Morphologie und Systematik der staining of resin-embedded material for light micros- Alloeocoela Cumulata und Rhabdocoela Lecitho- copy: Recommended procedures for micrometazoans. phora (Turbellaria). Acta Zoologica Fennica 26: 1– Mikroskopie 41: 259–270. 260. Staden, R., 1996. The Staden sequence analysis package. Lanave, C., G. Preparata, C. Saccone & G. Serio, 1984. A Molecular Biotechnology 5: 233–241. new method for calculating evolutionary substitution Stefanini, M., C. De Martino & L. Zamboni, 1967. Fixation rates. Journal of Molecular Evolution 20: 86–93. of ejaculated spermatozoa for electron microscopy. Marcus, E. D. B., 1957. On Turbellaria. Anais da Acade- Nature 216: 173–174. mia Brasileira de Cieˆncias 29: 153–191. Steinbo¨ ck, O., 1924. Die Bedeutung der Hofstenia atro- Meixner, J., 1938. Turbellaria (Strudelwu¨ rmer). I. All- viridis Bock fu¨ r die Stellung der Alloeocoela im Sys- gemeiner Teil. In Grimpe, G., E. Wa¨gler & A. Re- tem der Turbellarien. Zoologischer Anzeiger (Jena) mane (eds), Die Tierwelt der Nord- und Ostee 59: 156–166. 33(IVb), 1–146. Steinbo¨ ck, O., 1966. Die Hofsteniiden (Turbellaria Aco- Newman, L. J. & L. R. G. Cannon, 1994. Pseudoceros and ela). Grundsa¨tzliches zur Evolution der Turbellarien. Pseudobiceros (Platyhelminthes, Polycladida, Pseu- Zeitschrift fu¨ r Zoologische Systematik und Evolu- docerotidae) from Eastern Australia and Papua New tionsforschung 4: 58–195. Guinea. Memoirs of the Queensland Museum 37: Steinbo¨ ck, O., 1967. Regenerationsversuche mit Hofstenia 205–266. giselae Steinb. (Turbellaria Acoela). Roux’ Archiv fu¨ r Newman, L. J. & L. R. G. Cannon, 2003. Marine Flat- Entwicklungsmechanik der Organismen 158: 394–458. worms: The World of Polyclads. CSIRO Publishing, Tavare´, S., 1986. Some probabilistic and statistical prob- Collingwood, Victoria, 1–112. lems in the analysis of DNA sequences. Lectures on Nylander, J. A. A., 2004. MrAIC.pl. Program distributed Mathematics in the Life Sciences 17: 57–86. by the author. Evolutionary Biology Centre, Uppsala Tekle, Y. I., O. I. Raikova, A. Ahmadzadeh & U. Jonde- University. lius, 2005. Revision of the Childiidae (Acoela), a total Papi, F., 1957. Sopra un nuovo Turbellario arcooforo di evidence approach in reconstructing the phylogeny of particulare significato filetico e sulla posizione della acoels with reversed muscle layers. Journal of fam. Hofsteniidae nel sistema dei Turbellari. Pubb- Zoological Systematics and Evolutionary Research licazioni Della Stazione Zoologica di Napoli 30: 132– 43: 72–90. 148. Tyler, S., S. Schilling, M. Hooge & L. F. Bush, 2006. Riedl, R., 1954. Neue Turbellarien aus dem mediterranen Turbellarian taxonomic database. Version 1.5. Avail- Felslitoral. Zoologische Jahrbucher Abteilung fu¨ r able via http://turbellaria.umaine.edu. Systematik, O¨ kologie und Geographie der Tiere 82: 157–244.

123