copulatory organ musculature in childia (acoela) as revealed by phalloidin fluorescence and confocal...

Y

A

aowdc©

K

1

mcTf2(1s

circst

0d

TICE-444; No. of Pages 14

Tissue and Cell 38 (2006) 219–232

Copulatory organ musculature in Childia (Acoela) as revealed byphalloidin fluorescence and confocal microscopy

O.I. Raikova a,b, Y.I. Tekle a, M. Reuter c, M.K.S. Gustafsson c, U. Jondelius a,∗a Department of Systematic Zoology, Evolutionary Biology Centre, Uppsala University, Norbyvagen 18D, SE-75236 Uppsala, Sweden

b Zoological Institute of the Russian Academy of Sciences, 199034 St-Petersburg, Russiac Department of Biology, Abo Akademi University, Artillerigatan 6, FIN-20520 Abo, Finland

Received 22 November 2005; received in revised form 5 April 2006; accepted 12 April 2006

bstract

Copulatory organs of eight species of the monophyletic taxon Childia were investigated in detail, using phalloidin fluorescence methodnd confocal microscopy. Childia species were shown to have one, two or several tubular stylets, conical to cylindrical in shape, composedf few to numerous needles. The musculature varied greatly, from the absence of seminal vesicle to extensively developed seminal vesiclesith several additional types of specialized muscles. Ten copulatory organ characters were coded and mapped on the total evidence tree. The
ata obtained permitted to follow the evolution of the Childia stylet and to demonstrate that the structure of the stylet apparatus is largelyonsistent with the phylogeny of the group (CI = 0.75). Possible function of different muscle specializations was discussed.
2006 Elsevier Ltd. All rights reserved.

eywords: Muscles; Phalloidin; Copulatory organs; Stylet; Acoela; Childia

aw(JRset

. Introduction

Acoels are small, ciliated, predominantly marine acoelo-ate worms easily recognizable by the absence of a gut

avity and by presence of a statocyst with a single statolith.he Acoela is a relatively large group comprising 20

amilies and about 350 species (Dorjes, 1968; Tyler et al.,005), traditionally classified within the Platyhelminthes
Gegenbaur, 1859; Ehlers, 1985; Rieger et al., 1991; Ax,996). Recently, interest in the Acoela has been revived,ince studies of SSU and LSU rDNA, mitochondrial DNA
Abbreviations: bs, bursa; bw, body-wall muscles; c, sperm canal; cm,ircular muscles surrounding the male opening; fo, female opening; im,nner muscles; m, muscles; mm, mesh of thin muscles; mo, male opening;m, muscles radiating to the body-wall; sa, sperm aggregations; sc, styletap; sn, stylet needles; snm, stylet needles muscles; sr, stylet rings; st, stylet;v, seminal vesicle; svc, seminal vesicle collar; svr, seminal vesicle ring; tm,entacle muscles∗ Corresponding author. Tel.: +46 18 471 6476; fax: +46 18 471 6457.

E-mail address: [email protected] (U. Jondelius).

cA3dtp

nalgt

040-8166/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.tice.2006.04.001

nd nuclear protein coding genes placed them as a basal cladeithin the Bilateria and not members of the Platyhelminthes

Ruiz-Trillo et al., 1999, 2002; Littlewood et al., 1999;ondelius et al., 2002; Telford et al., 2003; Baguna anduitort, 2004). As for the phylogeny within the Acoela, two

tudies based on 18S rDNA appeared recently: by Jondeliust al. (2002) and Hooge et al. (2002). Both studies indicatehat the Acoela is a monophyletic group and are more or lessoncordant regarding the main branches within the taxon.lthough these studies use only few acoel sequences (24 and2, respectively), which is only about 10% of the speciesescribed to-date, they should be considered important stepsowards establishing a well corroborated hypothesis of acoelhylogeny.

Acoel taxonomy still remains largely unresolved and doesot reflect current phylogenetic hypotheses. Acoels have
superficially simple morphology at the light microscope
evel. Westblad (1948) used the positions of male and femaleenital openings and characters of the female genital sys-em (absence/presence of bursa) to subdivide all the Acoela

mailto:[email protected]

dx.doi.org/10.1016/j.tice.2006.04.001

dx.doi.org/10.1016/j.tice.2006.04.001

YTICE-444; No. of Pages 14

2 ue and C

iAggiiTlusftsaTaacipAR2i(lrc

tc2Doh

fihcohtc

2

2

C1(toSRAwaltiwu

tasaw

2

ppfispXla

FSsarmsvstmv

O.I. Raikova et al. / Tiss

nto three tribes: Opistandropora-Abursalia, Proandropora-bursalia and Proandropora-Bursalia. Characters of the maleenital system were then used to further subdivide these threeroups into families and genera. Dorjes (1968) used predom-nately the characters of male copulatory organs, but did notndicate how the 15 families in his classification were related.he emphasis on a single feature (male copulatory organ)

ed to a situation where the bulk of the acoel species werenited into one family, Convolutidae, while almost all thepecies with a copulatory stylet were classified within theamily Childiidae (Dorjes, 1968). Based on observations onhe sperm structure, the muscle patterns and the stylet ultra-tructure, Raikova et al. (2001) and Hooge et al. (2002) havergued that these families are non-monophyletic. Recently,yler and Hyra (1998), Tyler and Rieger (1999), Hoogend Tyler (1999, 2005), Hooge (2001), Hooge et al. (2002)nd Tekle et al. (2005) have shown that certain musculatureharacters are congruent with 18S rDNA trees. Despite thencreasing number of anatomical studies, there are only fourublished formal cladistic analyses of relationships within thecoela using certain morphological characters (Hooge, 2001;aikova et al., 2004; Tekle et al., 2005; Hooge and Tyler,005). Quite recently, Hooge and Tyler (2005) when revis-ng the systematics of the family Convolutidae sensu Dorjes1968), used characters of the male copulatory organ muscu-ature, obtained by the phalloidin fluorescent method. Theyecognized a new family Isodiametridae, for small-bodiedonvolutids.

As the morphology of the readily visible male copula-ory organ still remains the most important character in theurrent system of the Acoela (Dorjes, 1968; Tyler et al.,005), it has become important to scrutinize its morphology.o closely related species have similar copulatory organs,r do they show random variations? What is the degree ofomoplasy?

In the present study, acoel copulatory stylet and its musclebres are described by modern methods for the first time. Weave carried out a detailed study of the musculature of theopulatory organ in eight species of the genus Childia, six
f them formerly belonging to Paraphanostoma. Our studyas enabled us to discover new stylet characters and to traceheir evolution using an independent data set of molecularharacters.
PTlw

ig. 1. Schematic representation of the organization of the male copulatory organs itylets with proximal sperm aggregations are arranged in a circle around the maletylet needle muscles, incomplete circular muscles surrounding male opening and rasperm aggregation surrounded by weak muscles. (C) C. submaculatum. Long cyl

ing. Note inner muscles, connecting base of stylet with seminal vesicle walls. (D)usculature and wide seminal vesicle ring encircling the opening. Note strong rad

tylet rings, inserted into elongated seminal vesicles with large seminal vesicle collaesicle walls. (F) Viviparous acoel. Conical curved stylet, with stylet rings, insertedtarting from all sides of the seminal vesicle. (G) C. brachyposthium. Long cylindhe main stylet axis. Elongated seminal vesicle with collar of seminal vesicle ringsacroposthium. Large cylindrical stylet composed of numerous parallel thin needesicle ring. Note few stylet rings and numerous tentacle- and radiating muscles. F

Please cite this article as: O.I. Raikova et al., Copulatory organ musculand confocal microscopy, Tissue and Cell (2006), doi:10.1016/j.tice.200

ell xxx (2006) xxx–xxx

. Materials and methods

.1. Animals

Specimens of Childia brachyposthium (Westblad, 1942),. crassum (Westblad, 1942), C. cycloposthium (Westblad,942), C. groenlandica (Levinsen, 1879), C. macroposthiumSteinbock, 1931), C. submaculatum (Westblad, 1942) and C.rianguliferum (Westblad, 1942) were sampled by dredgingn muddy bottoms at 30–60 m depth in the Koster area on thewedish West coast in the vicinity of the Kristineberg Marineesearch Station at the Gullmar Fjord in July 2000 andugust and September 2003. The freshly collected sedimentsere put into large plastic containers, stirred thoroughly by

dding seawater, and then left overnight to settle. The topayer of the sediments was siphoned through a 125 �m sieveo extract the animals. The extracted animals were first stud-ed alive using a Leitz GmbH Wetzlar compound microscopeith Nomarski differential contrast optics. Photos were takensing a Fuji Finepix S1 Pro camera.

A unique viviparous acoel was collected at the same loca-ion as the other species. However, detailed study of styletnd sperm in this species is submitted for publication in aeparate paper along with the species description (Tekle etl., 2006). Here only a brief summary of its stylet structureill be given.

.2. Phalloidin staining of muscles

Worms were fixed in Stefanini’s fixative (2%araformaldehyde and 15% picric acid in 0.1 M Na-hosphate buffer) at pH 7.6. Specimens were storedor several weeks in fixative, then rinsed for 24–48 hn 0.1 M Na-phosphate buffer (pH 7.6) containing 10%ucrose. Prior to staining, the animals were immersed inhosphate-buffered saline (PBS) containing 0.2% Triton-100 (PBS-T). Staining of F-actin fibres with TRITC-

abelled phalloidin (Sigma) (1:200) was performed for 2 ht room temperature (Wahlberg, 1998). After rinsing in
BS, the animals were mounted in 60% glycerol in PBS.he preparations were examined with a confocal scanning
aser microscope (CSLM) LEICA TCS 4D also equippedith Nomarski differential contrast optics. Specimens

n Childia, with special emphasis on muscle patterns. (A) C. cycloposthium.opening. Note a mesh of numerous thin muscles interconnecting stylets,

diating muscles. (B) C. trianguliferum. Single short conical stylet containsindrical stylet inserted into spherical seminal vesicle, with seminal vesicleC. crassum. Conical stylet deeply inserted into seminal vesicle with weak

iating muscles. (E) C. groenlandica. Two thin, curved stylets, encircled byrs. Note stylet cap and inner muscles, connecting base of stylet with seminalinto spherical seminal vesicle. Note strong radiating- and tentacle muscles,rical tapering stylet composed of numerous thin needles, lying at angle tofused with stylet rings. Numerous tentacle- and radiating muscles. (H) C.

les, slightly inserted into compact, spherical seminal vesicle with seminalor abbreviations, see the list of abbreviations.

ature in Childia (Acoela) as revealed by phalloidin fluorescence6.04.001.

dx.doi.org/10.1016/j.tice.2006.04.001

https://www.researchgate.net/publication/11237202_Molecular_systematics_of_the_Acoela_(Acoelomorpha_Platyhelminthes)_and_its_concordance_with_morphology?el=1_x_8&enrichId=rgreq-ce7b2a31-5f92-496e-b1d2-b54a32a37e57&enrichSource=Y292ZXJQYWdlOzY4ODQ4NjM7QVM6OTkwNjc3NzQzNzM4OThAMTQwMDYzMTAwNjUxMA==

https://www.researchgate.net/publication/13754276_The_distribution_of_F-actin_during_the_development_of_Diphyllobothrium_dendriticum_(Cestoda)?el=1_x_8&enrichId=rgreq-ce7b2a31-5f92-496e-b1d2-b54a32a37e57&enrichSource=Y292ZXJQYWdlOzY4ODQ4NjM7QVM6OTkwNjc3NzQzNzM4OThAMTQwMDYzMTAwNjUxMA==

https://www.researchgate.net/publication/35447693_Die_Acoeja_(Turbellaria)_der_Deutschen_Nordseekste_und_ein_neues_System_der_Ordnung?el=1_x_8&enrichId=rgreq-ce7b2a31-5f92-496e-b1d2-b54a32a37e57&enrichSource=Y292ZXJQYWdlOzY4ODQ4NjM7QVM6OTkwNjc3NzQzNzM4OThAMTQwMDYzMTAwNjUxMA==


O.I. Raikova et al. / Tissue and Cell xxx (2006) xxx–xxx 3

Please cite this article as: O.I. Raikova et al., Copulatory organ musculature in Childia (Acoela) as revealed by phalloidin fluorescenceand confocal microscopy, Tissue and Cell (2006), doi:10.1016/j.tice.2006.04.001.

dx.doi.org/10.1016/j.tice.2006.04.001


4 ue and C

w1CUwmuetow

3

Ti

3

c(ttmsastdfnafited

3

cItsil(m(mdat

3

stsosndiBctcviv

vmn(

3

svTtttd((aaoltp

3

atiaaa


ere viewed either from the dorsal or ventral side. About0–20 specimens for each species were examined, except. cyclopostium, where only 5 specimens were available.sually 10–30 optical sections with a step of 0.5–2 �mere obtained while scanning through the specimen. Twoatching scan series, one using fluorescence light, the other

sing differential contrast option, were usually obtained forvery preparation. The maximum projection option was usedo make reconstructions from all or from several adjacentptical sections in a series. The files obtained were processedith Adobe PhotoShop v. 7.0.

. Results

Eight species of Childia have been investigated (Fig. 1).he stylets are described in the same order, as the correspond-

ng species appear on the phylogenetic tree (Fig. 9).

.1. Childia cycloposthium (Figs. 1A and 2)

This species possesses several (7–10) short (50–55 �m)onical stylets arranged in a circle around the male openingFigs. 1A and 2A and B). Each stylet is composed of 20–30hin needles, lining the central sperm canal. The needles areightly packed together in the distal end of the stylet and lie

ore loosely in its proximal end (Figs. 1A and 2B). Thetylet needles are not stained by phalloidin (Fig. 2A), butre visible in differential contrast (Fig. 2B). The sphericalperm aggregations at the base of each stylet could hardly beermed “seminal vesicles”, as they are enclosed in a commonense mesh of fine muscle fibres (Figs. 1A and 2D). There areew thin muscles running along proximal parts of the styleteedles—stylet needle muscles (Figs. 1A and 2A). The styletsnd the sperm aggregations are immersed in mesh of musclebres. Thin fibres (radiating muscles) extend from the mesh

owards the body wall (Figs. 1A and 2D). The male opening,ncircled by a circular fibre (Figs. 1A and 2C), lies a goodistance away from the posterior end of the animal.

.2. Childia trianguliferum (Figs. 1B and 3)

This species has a single, rather short (about 100 �m),onical stylet with a central sperm canal (Figs. 1B and 3).t is composed of about 50–60 thin needles, closely packedogether in the distal end of the stylet, while lying intrands of 5–6 in the middle of the stylet and in its prox-mal end (Fig. 3C). The stylet needles, unstained by phal-oidin (Fig. 3A and B), are detectable in differential contrastFig. 3C). The aggregation of sperm, lying within the proxi-al part of the stylet, is practically devoid of muscular fibres

Figs. 1B and 3A and B). There are very few thin stylet needle
uscles, running along the proximal parts of the stylet nee-
les, and none are lying in the distal part of the stylet (Fig. 3And B). A few thin radiating muscles extend from the styletowards the body wall and the subterminal male opening.

dbpn


ell xxx (2006) xxx–xxx

.3. Childia submaculatum (Figs. 1C and 4)

This species has a single long (about 240 �m) cylindricaltylet (Figs. 1C and 4). The stylet is composed of numeroushin needles, lining the centrally located sperm canal. Thetylet needles are closely packed together, but not fused, asne can easily detect individual needles detaching from thetylet in a squeezed specimen (Fig. 4C). The stylet needles areot stained by phalloidin (Fig. 4A and B), but are visible inifferential contrast (Fig. 4C). The proximal part of the stylets deeply inserted into a spherical seminal vesicle (Fig. 4A and). The sperm vesicle is well developed and possesses mus-ular outer walls, numerous radiating muscles, connectinghe seminal vesicle with the body wall, strong inner muscles,onnecting the base of the stylet with the walls of the seminalesicle, and two to three strong muscles, encircling the open-ng of the seminal vesicle, that we have termed the seminalesicle muscle ring (Figs. 1C and 4A).

In contrast to the well-developed muscles of the seminalesicle, the stylet itself is practically devoid of associateduscles (Fig. 4A and B). The male opening lies in subtermi-

al position, but quite close to the posterior end of the bodyFig. 4B).

.4. Childia crassum (Figs. 1D and 5)

This species has a single, about 130 �m long, conicaltylet, composed of numerous loosely packed thin needles,ery deeply inserted into the seminal vesicle (Figs. 1D and 5).he stylet needles are closely packed at the distal end of

he stylet, but towards the proximal end they curve outwardso accommodate the aggregation of sperm contained withinhe stylet (Figs. 1D and 5C and D). The stylet needles areevoid of stylet needle muscles and not stained by phalloidinFig. 5A, C and D), but are visible in differential contrastFig. 5B). The seminal vesicle is weakly developed; its wallsre composed of thin, widely spaced muscles (Fig. 5A, Cnd D). A wide seminal vesicle muscle ring encircles thepening (Figs. 1D and 5A, C and D). Numerous thick paral-el muscular fibres radiate from the seminal vesicle towardshe body wall (Fig. 5A). Male opening lies in subterminalosition.

.5. Childia groenlandica (Figs. 1E and 6)

This species has two conical stylets, about 140 �m longnd slightly curved (Figs. 1E and 6). The stylets are long andhin, progressively tapering to a sharp distal end. Each stylets composed of several very tightly packed thin needles, as nomount of pressure on the squeezed preparation ever detachesneedle from the stylet (Fig. 6D). In the middle of the stylet,sperm canal is localized (Figs. 1E and 6A). The stylet nee-
les are very weekly stained by phalloidin (Fig. 6E and F),ut are clearly visible in differential contrast (Fig. 6D). Theroximal part of the stylet is deeply inserted into a semi-al vesicle. The sperm vesicle is extensively developed; it

dx.doi.org/10.1016/j.tice.2006.04.001



Fig. 2. Male copulatory organ in Childia cycloposthium, CSLM projections of optical sections. (A) General view of copulatory organ, stained by phalloidin.Note stylet needle muscles running along proximal parts of stylet needles. (B) The corresponding scan, using differential contrast. Note circle of several shortconical stylets composed of thin needles. (C) Optical section showing body-wall muscles, male opening, surrounded by irregular circular muscles, and muscularm proximt bbrevia

iwaivcti

oc(n

esh around stylets, not stained by phalloidin. (D) Optical section throughhin muscles in the area, and muscles radiating towards the body wall. For a

s elongate in shape, and possesses strongly muscular outeralls. The walls of the seminal vesicle form a thick collar

round the base of the stylet, which we have termed the sem-nal vesicle collar (Figs. 1E and 6B, C and E). The seminal
esicle ring is absent. There are numerous parallel ring mus-les tightly encircling the proximal part of the stylet, forminghe stylet muscle ring (Figs. 1E and 6C, E and F). A set ofnner muscles, connecting the base of the stylet with the walls
nqs(


al parts of stylets and sperm aggregations at their bases. Note dense net oftions, see the list of abbreviations.

f the seminal vesicle, is extensively developed, forming aap on the proximal end of the stylet, the stylet muscle capFigs. 1E and 6A and C). Only few radiating muscles con-ect the seminal vesicle with the body wall muscles. Stylet
eedle muscles are few and weak (Fig. 6E). Both stylets openuite close to one another and share the single male openingituated in terminal position at the posterior end of the bodyFig. 6B).

dx.doi.org/10.1016/j.tice.2006.04.001


6 O.I. Raikova et al. / Tissue and Cell xxx (2006) xxx–xxx

F s of opt usclest tions, s

3

d(pom

hrra

FNma

ig. 3. Male copulatory organ in Childia trianguliferum, CSLM projectionhin needles, with proximal aggregation of sperm and few weak associated mhe same level as in (B), sperm and stylet needles are revealed. For abbrevia

.6. Viviparous acoel (Fig. 1F)

The description of this new species along with the detailedescription of the copulatory organ is given in Tekle et al.
2006). This acoel has a single conical curved stylet, com-osed of several very tightly packed needles, very similar tone of the paired stylets of C. groenlandica. Stylet needleuscles are weakly stained by phalloidin. Seminal vesicle
ti(t

ig. 4. Male copulatory organ in Childia submaculatum, CSLM projections of optiote seminal vesicle ring and muscles radiating from the seminal vesicle. (B) Phuscles, connecting base of stylet with seminal vesicle walls. Note absence of musc

s in (B), stylet needles are revealed, some are detached from the stylet under press


tical sections. (A and B) Phalloidin staining. Single short conical stylet of, near the subterminal male opening. (C) Scan using differential contrast atee the list of abbreviations.

as thick muscular walls and practically lacks a collar. Styleting muscles are present, while the seminal vesicle muscleing is absent. Numerous strongly stained muscles start fromll sides of the seminal vesicle, some of them radiate towards
he body wall, while many run down along the stylet, towardsts proximal end. These muscles we termed tentacle musclesFig. 1F); the longest tentacle muscles reach down to the sub-erminal male opening.
cal sections. (A) Phalloidin staining of muscular seminal vesicle and bursa.alloidin staining of long cylindrical stylet showing numerous strong innerles lining the stylet needles. (C) Differential contrast scan at the same levelure. For abbreviations, see the list of abbreviations.


dx.doi.org/10.1016/j.tice.2006.04.001



Fig. 5. Male copulatory organ in Childia crassum, CSLM projections of optical sections. (A) Weakly developed seminal vesicle with seminal vesicle ring andstrong radiating muscles, going to the body wall. Phalloidin staining. (B) Scan using differential contrast at the same level as in (A), stylet needles are revealed.(C and D) Two optical sections showing the seminal vesicle opening with seminal vesicle ring. Short conical stylet, with proximal aggregation of sperm andno associated muscles deeply inserted into the seminal vesicle. Phalloidin staining. For abbreviations, see the list of abbreviations.

Fig. 6. Male copulatory organ in Childia groenlandica, CSLM projections of optical sections, phalloidin staining, except (D). (A) View of the posterior endof the animal, showing two seminal vesicles and the proximal ends of the stylets, with stylet caps of inner muscles, perforated by sperm canals. (B) Frontaloptical section of the posterior end of the animal showing two stylets and a common terminal male opening. Note elongated seminal vesicles with collars.(C) Longitudinal section through seminal vesicles and proximal parts of stylets, showing organization of stylet caps and inner seminal vesicle muscles. Noteabsence of seminal vesicle rings and presence of stylet rings. (D) Differential contrast image of stylet needles and sperm in seminal vesicles. (E and F) Twooptical sections of the same preparation as in (D), in fluorescent light. Note stylet needle muscles, stylet rings and seminal vesicle collars. For abbreviations,see the list of abbreviations.


dx.doi.org/10.1016/j.tice.2006.04.001




dx.doi.org/10.1016/j.tice.2006.04.001



Fig. 7. Male copulatory organ in Childia brachyposthium. (A) Differential contrast image of stylet in a live specimen, showing seminal vesicle and conicalstylet, with needles arranged at an angle to stylet axis. (B) CSLM optical section of a phalloidin stained specimen, showing stylet needle muscles in distalpart of stylet. (C) Scan using differential contrast at same level as in (B), stylet needles are revealed. (D–F) Optical sections of copulatory organ stained byphalloidin. Cylindrical stylet with conical distal end deeply inserted into elongated seminal vesicle. Note numerous tentacle muscles, stylet- and stylet needlemuscles present all along stylet. For abbreviations, see the list of abbreviations.


dx.doi.org/10.1016/j.tice.2006.04.001



Fig. 8. Male copulatory organ in Childia macroposthium. Phalloidin staining, except (D). (A) Large cylindrical stylet with prominent sperm canal and numerousstylet needle muscles present all along the stylet. Note seminal vesicle ring and stylet ring. (B) Optical section showing strongly stained compact seminal vesicleand tentacle muscles running down to male opening. (C) Stylet slightly inserted into the compact seminal vesicle with tentacle muscles and radiating muscles.( es and sp l semins fine fibrt

3

s(icntTsI(stenTs

iwlTe(itocs

3

D) Scan using differential contrast at the same level as in (C), stylet needlroximal part of stylet. (E) Projection of the whole series, showing sphericatylet ring. (G) Section showing seminal vesicle ring. (H) Section showinghe list of abbreviations.

.7. C. brachyposthium (Figs. 1G and 7)

The relatively long (140 �m) single stylet of thispecies is cylindrical in shape, tapering at the distal endFigs. 1G and 7D–F). Sometimes the stylet appears to be con-cal (Fig. 7A–C). The stylet is composed of numerous thinlosely packed needles. In the proximal part of the stylet, theeedles lie at an angle of about 40◦ to the main stylet axis; inhe distal parts the needles run practically parallel to the axis.herefore, the needles in the proximal part of the stylet arehorter than those in the distal part (Figs. 1G and 7B, C and F).n the middle of the stylet, the sperm canal is localizedFigs. 1G and 7F). The stylet needles are clearly visible inqueezed live specimens (Fig. 7A) or with differential con-rast (Fig. 7C). The needles are stained by phalloidin, to an
xtent, likely due to the extensive development of the styleteedle muscles, incorporated in the stylet (Fig. 7B and F).he proximal part of the stylet is deeply inserted into theeminal vesicle. The sperm vesicle is extensively developed;
crit


perms revealed. (E–H). CSLM optical sections of seminal vesicle and theal vesicle with tentacle muscles and base of the stylet. (F) Section showinges interconnecting stylet- and seminal vesicle rings. For abbreviations, see

t is elongate in shape, and possesses strongly muscular outeralls and a long muscular collar. The distal part of the col-

ar is composed of many ring muscles, seminal vesicle rings.he latter seem to be fused with stylet muscle rings, tightlyncircling the proximal and the middle region of the styletFigs. 1G and 7D–F). Well-developed tentacle muscles orig-nate from the seminal vesicle and run down along the stylet,owards its proximal end (Figs. 1G and 7D and F). Numer-us radiating muscles, starting at the seminal vesicle walls,onnect it with the body wall muscles. The male opening isituated in subterminal position.

.8. Childia macroposthium (Figs. 1H and 8)

This species has a single, big (250 �m long), cylindri-
al stylet (Figs. 1H and 8). The stylet is very large, withespect to the body size, about 30% of the body length. Its composed of very numerous, thin parallel needles, lininghe wide central sperm canal (Figs. 1H and 8A and C). The

dx.doi.org/10.1016/j.tice.2006.04.001


ue and Cell xxx (2006) xxx–xxx 11

ninbdssiiTvess(cNcRwa

3a

t(oitcmtsc

as

Table 2Data matrix of the copulatory organ characters in Childia

Taxa Characters

1 2 3 4 5 6 7 8 9 10

Childia cycloposthium 1 0 0 0 0 0 0 0 0 0C. trianguliferum 0 0 0 0 0 0 0 0 0 0C. submaculatum 0 2 1 0 1 0 0 1 0 1C. crassum 0 0 0 0 1 0 0 1 0 1C. groenlandica 2 1 0 0 2 1 1 0 0 0Viviparous acoel 0 1 0 0 2 0 1 0 1 1CC

aosTptmcsswm(Cmtoplcpc

TC

N

1


eedles are stained by phalloidin, though most of the stainings probably due to numerous stylet needle muscles, lining theeedles all along the stylet (Fig. 8A). The needles also coulde detected by using differential contrast (Fig. 8D). The nee-les are closely packed, but by squeezing a live specimen,ome could be detached. The proximal part of the stylet islightly inserted into the seminal vesicle. The spherical sem-nal vesicle is compact and strongly muscular; it seems smalln comparison with the length of the stylet (Figs. 1H and 8C).he vesicle lacks a seminal vesicle collar, but the seminalesicle muscle ring, composed of numerous ring fibres, isxtensively developed (Figs. 1H and 8C and E–H). A fewtylet muscle rings tightly encircle the proximal part of thetylet, at the level of the opening of the seminal vesicleFigs. 1H and 8A and D–F). Stylet rings and the seminal vesi-le ring are interconnected by thin muscle fibres (Fig. 8H).umerous tentacle muscles originate from the seminal vesi-

le and run down along the stylet (Figs. 1H and 8A, B and E).adiating muscles, starting at the seminal vesicle walls, areell developed (Figs. 1H and 8C). The male opening is situ-

ted in subterminal position (Fig. 8B).

.9. Coding of the male copulatory organ charactersnd mapping them on the tree

The variable characters of the male copulatory organ andheir states are given in Table 1. Out of 10 coded charactersTable 2), only character No. 5 was treated as ordered; thether nine were treated as unordered. Characters of the styletn the viviparous acoel (Tekle et al., 2006) were also addedo the matrix. Transformations in the male copulatory organharacters are shown below nodes in Fig. 9. The selectedorphological characters were mapped on a total evidence

ree (Tekle et al., 2005). Five of the 10 characters have con-istency index CI = 1 (1, 4, 5, 7, 9, see Table 1). The ensemble
onsistency index was 0.75.
The monophyly of Childia is supported by the appear-nce of the unique copulatory organ with a stylet. The ple-iomorphic state of Childia’s copulatory organ (marked by

si(d

able 1haracters of the copulatory organ and their states

o. Character

1 Number of stylets2 Shape of stylet3 Number of needles forming the stylet4 Stylet needle muscles

5 Seminal vesicle6 Seminal vesicle collar

7 Stylet rings, tightly encircling the proximal part of the stylet8 Seminal vesicle ring, encircling the opening of the seminal vesicle9 Tentacle muscles, running from the seminal vesicle down along the

stylet needles0 Muscles radiating from the seminal vesicle towards the body-wall


. brachyposthium 0 2 1 1 2 1 1 1 1 1

. macroposthium 0 2 1 1 2 0 1 1 1 1

n asterisk in Fig. 9) seems to be a single short conical styletf a few needles, without developed stylet needle muscles,eminal vesicle, stylet rings, tentacle- and radiating muscles.his state is found in C. trianguliferum, while C. cyclo-osthium acquired several stylets (1:1), each of them sharinghe above mentioned plesiomorphic features. There are noale copulatory organ synapomorphies for the clade of C.

ycloposthium + C. trianguliferum. The rest of the Childiapecies form a monophylum, characterised by presence of theeminal vesicle with numerous muscles radiating to the body-all (5:1, 10:1). C. submaculatum has a number of autapo-orphic features: cylindrical stylet (2:2) of numerous needles

3:1) and the presence of the strong seminal vesicle ring (8:1).. groenlandica + viviparous acoel + C. brachyposthium + C.acroposthium form a monophyletic clade, characterised by

he strongly muscular seminal vesicle (5:2) and presencef stylet rings (7:1). C. groenlandica has three autapomor-hies: two stylets (1:2), a prominent seminal vesicle col-ar (6:1) and the absence of radiating muscles (10:0). Thelade of viviparous acoel + C. brachyposthium + C. macro-osthium is characterised by the presence of tentacle mus-les (9:1). C. brachyposthium and C. macroposthium are
trongly supported (100%) sister-taxa sharing the follow-ng morphological synapomorphies: large cylindrical stylets2:2) of numerous needles (3:1), with prominent stylet nee-le muscles (4:1) and the presence of the strong seminal
Coding of character states

0: one; 1: several; 2: two0: short conical; 1: long, thin conical, slightly curved; 2: cylindrical0: few; 1: numerous0: absent, weak or confined to the proximal part of the stylet; 1:well developed, running along the stylet needles to the distal end0: absent; 1: with weak musculature; 2: strongly muscular0: seminal vesicle spherical, without muscular collar; 1: seminalvesicle elongated; the walls form a collar around the base of thestylet0: absent; 1: present0: absent; 1: present0: absent; 1: present

0: few; 1: numerous


dx.doi.org/10.1016/j.tice.2006.04.001



F n optimt ekle et ac e pointa

vvl

cCa

ov(

ig. 9. Distribution of 10 morphological characters of male copulatory orgaaxa). The tree is based on 18S, 28S, H3 and 50 morphological characters (Thanges are denoted by numbers below the tree branches. Asterisk marks thre at 0.

esicle ring (8:1). C. brachyposthium acquired a seminalesicle collar (6:1), seemingly independently from C. groen-andica.

Four of the clades are supported by unambiguous maleopulatory organ apomorphies (Fig. 9): the clade including. submaculatum, C. crassum, C. groenlandica, viviparouscoel, C. brachyposthium and C. macroposthium (presence

cmCn


ized on a total evidence tree of the Childiidae (mecynostomids as outgroupl., 2005). Bootstrap frequencies are shown above branches. Character statewhere the Childia stylet appeared. At this point all the 10 character states

f seminal vesicle); the clade including C. groenlandica,iviparous acoel, C. brachyposthium and C. macroposthiumstrongly muscular seminal vesicle and stylet rings); the
lade including viviparous acoel, C. brachyposthium and C.acroposthium (tentacle muscles) and the clade including. brachyposthium and C. macroposthium (prominent styleteedle muscles).

dx.doi.org/10.1016/j.tice.2006.04.001


ue and C

dstpspsp

4

slistiouduiDwwndo(

suceCsNbb

iidCttwcttdtC

sob

sitohc

u(t2pfslAl

taveagsfgcsnmtcorrTsa

t1tcvfPt


Several cases of parallel evolution are revealed. Cylin-rical stylet (2:2) of numerous needles (3:1) and the strongeminal vesicle ring (8:1) were acquired in C. submacula-um independently from the similar features of C. brachy-osthium + C. macroposthium clade. Therefore, apparentlyimilar stylets in C. submaculatum (Fig. 1C) and in C. macro-osthium (Fig. 1H) proved to be homoplasious, as well as longeminal vesicle collars in C. groenlandica and C. brachy-osthium (Fig. 1E and G).

. Discussion

Detailed studies of acoel copulatory organs are few,trange as it may seem, considering the fact that the copu-atory organs presently constitute one of the main charactersn acoel classification (Dorjes, 1968; Tyler et al., 2005). Thetylets of the species described here were previously inves-igated by Westblad (1942, 1948), using traditional histolog-cal methods. His studies gave fairly accurate descriptionsf the copulatory organs, though several small details werendetected, due to the method limitations. Westblad (1948)escribed a hypothetical transformation series of acoel cop-latory organ starting with the so-called basal type, commonn Proporus, Paraproporus, Haploposthia, Paranaperus andiopisthoporus. This is a long terminal ciliated male antrum,hich can be everted inside out during the copulation. Thealls of the antrum often bear simple stimulatory organs, likeeedles. From this basal type of copulatory organs, Westbladeduced many other types, common in Abursalia, the groupf acoels without a bursa, and several species of BursaliaWestblad, 1948).

C. groenlandica has no bursa and in Westblad’s (1948)ystem belonged to Abursalia. However, Westblad wasnable to deduce its tubular stylet from the basal type ofopulatory organ, as the latter never comprised any tubularlements. Westblad never suggested a close relationship of. groenlandica with Paraphanostoma (all the other Childia

pecies in the present classification, see Tekle et al., 2005).evertheless, he commented on the numerous similaritiesetween the stylets in C. groenlandica, Paraphanostomarachyposthium and P. macroposthium (Westblad, 1948).

The use of the features of the female copulatory organn Westblad’s classification of the Acoela proved mislead-ng (Dorjes, 1968; Tekle et al., 2005). Recent molecularata permitted to re-assign all Paraphanostoma species tohildia (Tekle et al., 2005). Westblad (1948) believed that

he copulatory organs of C. cycloposthium, C. crassum, C.rianguliferum and, to a lesser degree, C. submaculatum,ere similar to the stimulatory organs of the basal type of

opulatory apparatus. However, our studies show that all ofhese stylets contain a sperm canal; they are tubular struc-
ures, much like the stylet of C. groenlandica, and cannot beeduced from scattered needles. Westblad (1948) restrictedhe use of the term “stylet”, reserving it for C. groenlandica,. brachyposthium and C. macroposthium only. We have no
ptst


ell xxx (2006) xxx–xxx 13

uch reservations, being sure that all the Childia copulatoryrgans are homologous. The homology is further supportedy our new ultrastructural data.

Westblad (1948) believed that the evolution of the acoeltylet is distinct from that of the stylets of “other turbellaria”,.e. the Platyhelminthes Rhabditophora in modern defini-ion. Considering the modern views on the basal positionf the Acoela within the Bilateria, separate from the Platy-elminthes (Ruiz-Trillo et al., 1999), he was undoubtedlyorrect.

There are some acoels with stylets, or sclerotized cop-latory organs, outside of Childia, e.g. Paratomella rubraParatomellidae), which is one of the most basal clades ofhe Acoela, according to recent molecular data (Hooge et al.,002; Jondelius et al., 2002). Several species of the Actino-osthiidae and the Otocelididae have conical stylets with aew needles (Tyler et al., 2005). It is unlikely, that any of theirtylets are homologous with Childia stylets. It is more thanikely that the evolution of the copulatory organ within thecoela took place parallelly, within different monophyletic

ines.Tekle et al. (2005) published a total-evidence phylogenetic

ree of Childia, based on 18S rRNA, 28S rRNA, Histone H3nd 50 morphological characters. We were able to find 10ariable morphological characters and map them on the totalvidence tree (Fig. 9). The copulatory organ with a styletppeared within Childia, while mecynostomids, the sister-roup of Childia, lack stylets (Fig. 9). The plesiomorphictate of the stylet apparatus, weak stylet(s) composed of aew needles without seminal vesicle, is visualised in C. trian-uliferum and C. cycloposthium. The evolution of the maleopulatory organ within Childia led to the formation of aingle big stylet composed of numerous needles, with styleteedle muscles running all along the stylet needles, and styletuscle rings at the base of the stylet. It was possible to follow

he formation of a strongly muscular, compact seminal vesi-le with a muscle ring at the opening. The male copulatoryrgan became firmly attached to the body wall by muscles,adiating from the seminal vesicle, and by tentacle muscles,unning from the seminal vesicle down to the male opening.he new characters of the copulatory stylet are largely con-istent with the previous phylogenetic hypothesis (Tekle etl., 2005) with a CI of 0.75.

We can make some suggestions about the functioning ofhe muscles of the male copulatory organ (see also Hyman,951). Contraction of the seminal vesicle muscles pusheshe sperm, contained in the seminal vesicle, into the spermanal. The presence of strong inner muscles of the seminalesicle, in the case of C. submaculatum and C. groenlandica,acilitates the contraction of the seminal vesicle even more.erhaps, the presence of a sphincter around the opening of

he seminal vesicle, as well as stylet muscle rings, could
revent sperm from leaking through the sperm canal beforehe moment of impregnation. The muscle fibres, linking theeminal vesicle ring with stylet rings, could assure the simul-aneous relaxation of both sets of sphincters, opening the

dx.doi.org/10.1016/j.tice.2006.04.001


1 ue and

slwsosittams

A

ihbA

R

A

B

D

E

G

H

H

H

H

H

J

L

L

R

R

R

R

R

S

T

T

T

T

T

T

W

561–570.

4 O.I. Raikova et al. / Tiss

perm canal for the passage of the sperm. C. groenlandicaacks the seminal vesicle muscle ring, but has a sphincterithin the seminal vesicle – stylet cap – with an opening for

perm. Contraction of the stylet cap and the inner musclesf the seminal vesicle during the copulation likely causes thetylet needles to protrude out of the body, while the openingn the contracted stylet cap widens, letting sperm pass intohe sperm canal. The radiating muscles anchor the big styleto the body wall and, probably, also enable to change thengle of the stylet during the copulation. As for the tentacleuscles, their contraction likely assures the protrusion of the

tylet during the copulation.

cknowledgements

The authors are grateful to the staff of the marine biolog-cal stations at Klubban and Kristineberg (Sweden) for theirelp with material collection. Financial support was providedy the Swedish Research Council (UJ), the Royal Swedishcademy (UJ/OR) and RFBR grant 06-04-48053-a (OR).

eferences

x, P., 1996. Multicellular Animals. A New Approach to the PhylogeneticOrder in Nature, vol. I. Springer, Berlin, 225 pp.

aguna, J., Ruitort, M., 2004. Molecular phylogeny of the Platy-helminthes. Can. J. Zool. 82, 168–193.

orjes, J., 1968. Die Acoela (Turbellaria) der deutschen Nordseekusteund ein neues System der Ordnung. Z. Zool. Syst. Evolutionsforsch.6, 56–452.

hlers, U., 1985. Das phylogenetische System des Platyhelminthes. Gus-tav Fischer, Stuttgart, 317 pp.

egenbaur, C., 1859. Grundzuge der Vergleichenden Anatomie. Engel-mann, Leipzig.

ooge, M.D., 2001. Evolution of body-wall musculature in the Platy-helminthes (Acoelomorpha, Catenulida, Rhabditophora). J. Morphol.249, 171–194.

ooge, M.D., Haye, P.A., Tyler, S., Litvaitis, M.K., Kornfield, I.,2002. Molecular systematics of the Acoela (Acoelomorpha, Platy-helminthes) and its concordance with morphology. Mol. Phylog. Evol.24, 333–342.

ooge, M.D., Tyler, S., 1999. Body-wall musculature in Praeconvolutatornuva, n. sp., and the use of muscle patterns in taxonomy of acoelturbellarians. Invertebr. Biol. 18, 8–17.

ooge, M.D., Tyler, S., 2005. New tools for resolving acoel phylogenies:
a systematic revision of the Convolutidae (Acoela, Acoelomorpha). J.Zool. Syst. Evol. Res. 43 (2), 100–113.
yman, L.H., 1951. The Invertebrates. II. Platyhelminthes and Rhyn-chocoela. The Acoelomate Bilateria. McGraw-Hill, New York, 550pp.

W

W


Cell xxx (2006) xxx–xxx

ondelius, U., Ruiz-Trillo, I., Baguna, J., Ruitort, M., 2002. The Nemer-todermatida are basal bilaterians and not members of the Platy-helminthes. Zool. Scr. 31, 201–215.

evinsen, G.M.R., 1879. Bidrag til Kundskab om Gronlands Turbellar-iefauna. Vidensk Meddr Dansk Naturhist Foren 31, 165–204.

ittlewood, D.T.J., Rohde, K., Clough, K.A., 1999. The interrelationshipsof all major groups of Platyhelminthes: phylogenetic evidence frommorphology and molecules. Biol. J. Linn. Soc. 66, 75–114.

aikova, O.I., Reuter, M., Gustafsson, M.K.S., Maule, A.G., Halton,D.W., Jondelius, U., 2004. Evolution of the nervous system in Para-phanostoma (Acoela). Zool. Scr. 33, 71–88.

aikova, O.I., Reuter, M., Justine, J.-L., 2001. Contributions to the phy-logeny and systematics of the Acoelomorpha. In: Littlewood, T.J.,Bray, R.A. (Eds.), Interrelationships of Platyhelminthes. Taylor andFrancis, London, pp. 13–23.

ieger, R.M., Tyler, S., Smith III, J.P.S., Rieger, G.E., 1991. Platy-helminthes: Turbellaria. In: Harrison, F.W., Bogitsh, B.J. (Eds.),Microscopic Anatomy of Invertebrates, vol. 3: Platyhelminthes andNemertinea. Wiley/Liss, New York, pp. 7–140.

uiz-Trillo, I., Paps, J., Loukota, M., Ribera, C., Jondelius, U., Baguna,J., Riutort, M., 2002. A phylogenetic analysis of myosin heavy chaintype II sequences corroborates that Acoela and Nemertodermatida arebasal bilaterians. Proc. Natl. Acad. Sci. 99, 11246–11251.

uiz-Trillo, I., Riutort, M., Littlewood, T.J., Herniou, E.A., Baguna, J.,1999. Acoel flatworms: earliest extant Bilaterian Metazoans, not mem-bers of Platyhelminthes. Science 283, 1919–1923.

teinbock, O., 1931. Marine Turbellaria. In: Jensen S., Lundbeck W.,Mortensen Th. (Eds.), Zoology of the Faroes, vol. 8, Copenhagen, 26pp.

ekle, Y.I., Raikova, O.I., Ahmadzadeh, A., Jondelius, U., 2005. Revisionof the Childiidae (Acoela), a total evidence approach in reconstructingthe phylogeny of acoels with reversed muscle layers. J. Zool. Syst.Evol. Res. 43 (1), 72–90.

ekle, Y.I., Raikova, O.I., Jondelius, U., 2006. A new viviparous acoelChildia vivipara sp. n. with observations on the developing embryos,body wall, stylet musculature and sperm ultrastructure. Acta Zool. 87,121–130.

elford, M.J., Lockyer, A.E., Cartwright-Finch, C., Littlewood, D.T.J.,2003. Combined large and small subunit ribosomal RNA phylogeniessupport a basal position of the acoelomorph flatworms. Proc. R. Soc.London B 270, 1077–1083.

yler, S., Hyra, G.S., 1998. Patterns of musculature as taxonomic char-acters for the Turbellaria Acoela. Hydrobiologia 383, 51–59.

yler, S., Rieger, R.M., 1999. Functional morphology of musculature inthe acoelomate worm Convoluta pulchra (Platyhelminthes). Zoomor-phology 119, 127–141.

yler, S., Schilling, S., Hooge, M.D., Bush, L.F. (comp.), 2005. Turbellar-ian taxonomic database. Version 1.4. http://devbio.umesci.maine.edu/styler/turbellaria/.

ahlberg, M.H., 1998. The distribution of F-actin during the develop-ment of Diphyllobothrium dendriticum (Cestoda). Cell Tiss. Res. 291,

estblad, E., 1942. Studien uber skandinavische Turbellaria Acoela II.Ark. Zool. 33A, 1–48.

estblad, E., 1948. Studien uber skandinavische Turbellaria Acoela V.Ark. Zool. 41A, 1–82.


http://devbio.umesci.maine.edu/styler/turbellaria/

dx.doi.org/10.1016/j.tice.2006.04.001

copulatory organ musculature in childia (acoela) as revealed by phalloidin fluorescence and confocal...

Documents