New tools for resolving phylogenies: a systematic revision of the Convolutidae (Acoelomorpha, Acoela)

Abstract

Molecular sequence data, morphological characters of spermatozoa, and newly obtained morphological characters of penis musculature are used here to revise the systematics of the family Convolutidae (Acoela). Species having isodiametric penes with non-anastomosing longitudinal muscles are transferred to the family Isodiametridae fam. nov. Species with longitudinal penis muscle fibres that anastomose or cross-over each other remain in the Convolutidae. Some species of the genera Convoluta and Conaperta (Convolutidae) are transferred to the genus Isodiametra gen. nov. (Isodiametridae fam. nov.). The genus Stomatricha (Otocelididae) is transferred to the family Convolutidae. Convoluta opisthandropora (Convolutidae) is transferred to the genus Pseudohaplogonaria (Haploposthiidae). Aphanostoma sanguineum (Convolutidae) is transferred to the genus Pseudactinoposthia (Actinoposthiidae). Molekulare Sequenzdaten, Spermien-Morphologie und erstmals ermittelte morphologische Merkmale der Penismuskulatur werden herangezogen, um die Verwandtschaftsverhältnisse innerhalb der Familie Convolutidae (Acoela) neu zu beleuchten. Dabei werden Arten mit isodiametrischen Penes mit nicht anastomosierender Längsmuskulatur in die neue Familie Isodiametridae gestellt, während Arten mit longitudinalen Penis-Muskelfasern, die sich verbunden oder einanderüberkreuzen, innerhalb der Convolutidae belassen. Einige Arten der Gattungen Convoluta und Conaperta (Convolutidae) werden in die neue Gattung Isodiametra gen. nov. (Isodametridae fam. nov.) gestellt. Die Gattung Stomatricha (Otocelididae) wird in die Familie Convolutidae überführt. Convoluta opisthandropora (Convolutidae) wird in die Gattung Pseudohaplogonaria (Haploposthiidae) gestellt und Aphanostoma sanguineum (Convolutidae) in die Gattung Pseudactinoposthia (Actinoposthiidae). The Acoela is an extremely diverse taxon of small, cryptic, almost exclusively marine worms. A monograph of the Acoela by Dörjes (1968) established the use of copulatory-organ morphology as the principle means of dividing the taxon into 15 families. Further refinement of Dörjes's system and the description of additional species has expanded the taxon to 20 families containing over 340 species (see Tyler et al. 2005). The largest of the families, with more than a third of the species of Acoela, is the Convolutidae Graff, 1905. It contains some of the larger and therefore better-known species of acoels, some up to 9 mm in length. Representative of the larger members of the Convolutidae is Convoluta convoluta (Abildgaard, 1806) (Fig. 1a), a well-known European species that was recently recognized as an invasive species in the western North Atlantic Ocean (Rivest et al. 1999). Typically epiphytic, C. convoluta is usually 2–4 mm in length and uses its enrolled lateral sides to create a sort of hood that facilitates the capture of copepods, small molluscs, and diatoms. Due to the presence of endosymbiotic diatoms in its tissue, the body of C. convoluta is very dark, obscuring the pair of red ocelli that flank either side of the statocyst, a simple georeceptor found in nearly all species of acoels. Representatives of the family Convolutidae sensu Dörjes 1968. (a) Ventral view of a typical ‘Large-bodied convolutid,’Convoluta convoluta. From Graff 1882. Scale bar is assumed; average length of C. convoluta is 2–6 mm, but some reach lengths of 9 mm. (b) Ventral view of a typical ‘small-bodied convolutid,’Conaperta earnhardti. (c) Conaperta earnhardti shown at same size scale as A While many species of Convolutidae are similar to C. convoluta with regard to size, shape, and possession of ocelli and photosynthetic endosymbiotic algae, an even greater number are more like Conaperta earnhardti Hooge and Smith, 2004 (Fig. 1b), which lives in the interstitial spaces of marine sediments, is less than 1 mm long, and lacks ocelli or algal symbionts. Despite these morphological differences, all Convolutidae share a feature of the male copulatory organ, namely that it is a muscular, often glandular, tube-shaped penis, usually invaginated into a muscular seminal vesicle. In a phylogenetic investigation of the Acoela using nuclear 18S rDNA gene sequences, Hooge et al. (2002) produced a gene tree that revealed a polyphyletic Convolutidae (Fig. 2) – with small-bodied, meiofaunal convolutids, similar to C. earnhardti, grouping separately from larger, endosymbiont-containing acoels like C. convoluta. Hooge et al. (2002) found support for this apparent polyphyletic grouping of the Convolutidae in ultrastructural characters of spermatozoa, and this was later pursued by Petrov et al. (2004) who examined an additional four species of Convolutidae and confirmed that the Convolutidae fall into two unrelated groups: ‘Large-bodied convolutids’ having 9 + 0 axonemes and axial microtubules, and ‘Small-bodied convolutids’ with 9 + 2 axonemes and cortical microtubules (Table 1). The distribution of spermatozoa ultrastructure characters superimposed on the 18S rDNA tree of Hooge et al. (2002). Species names in bold denote those taxa for which sperm morphology is known Clearly, 18S rDNA molecular sequences and features of spermatozoa ultrastructure are phylogenetically useful characters for dividing the Convolutidae into natural groupings that better reflect their evolution; however, from the standpoint of a taxonomist these characters are overly expensive and time-consuming to collect. In the present investigation, we remedy the polyphyly of the Convolutidae by revising the systematics of the family with characters that are more easily obtainable – specifically, newly identified morphological characters of male copulatory organs. Most examined specimens (Table 2) were extracted from sediment using magnesium-chloride anesthetization (Sterrer 1971); however, Aphanostoma sanguineum was removed directly from the gut of its holothuroidean host, C. convoluta was collected from filamentous algae growing in the rock-covered shallow subtidal, Convoluta henseni was collected from the plankton, and Convoluta marginalis, of which histological sections were borrowed from the Russian Academy of Sciences, St Petersburg, Russia. For histological study, specimens were relaxed in isotonic magnesium chloride, fixed in phosphate-buffered 2.5% (v/v) glutaraldehyde, washed in phosphate buffer (Millonig's, 0.1 M), fixed in phosphate-buffered 1% (v/v) osmium tetroxide, dehydrated in acetone, and embedded in EMBed/Araldite epoxy resin. Dehydration was quickened by microwave radiation (Samsung oven, two 7-s irradiations at 650 W separated by a 20-s interim, with specimen-vial on ice and with water ballast of two filled 300-ml beakers; Giberson and Demaree 1995). Serial thick sections of 1.5 μm were made according to Smith and Tyler (1984) and stained in toluidine blue. Copulatory musculature of worms was revealed through F-actin staining of whole mounts with fluorescently labeled phalloidin (Alexa 488; Molecular probes, Eugene, OR, USA) according to Hooge (2001). The specimens were first relaxed in magnesium chloride or magnesium sulphate solution isotonic to sea water, fixed for 1 h in 4% formaldehyde, rinsed in phosphate-buffered saline (PBS), attached to a coverslip with poly-l-lysine, permeabilized for 1 h with 0.2% Triton X-100 in PBS, stained for 1 h with phalloidin-Alexa, mounted under a second coverslip with Fluoromount-G, and viewed with a Leica TCS SP2 confocal microscope, or with an epifluorescence Leitz Ortholux microscope. The male copulatory organ of C. convoluta is located mid-ventrally in the posterior half of the body, immediately behind the female gonopore (1, 3). Several longitudinal fibres of the body wall extend into the male gonopore, and are continuous with the longitudinal musculature of the penis (Fig. 3b). The muscular penis of C. convoluta is much wider at its base and tapers towards the narrow tip; it is clearly not isodiametric over its length. It is invaginated into a seminal vesicle composed of a network of widely spaced muscle fibres (Fig. 3c). The penis musculature is made up of thin rings of inner circular fibres surrounded by an outer layer of longitudinal fibres that anastomose or cross over each other (3, 9). Confocal microscopy also reveals the actin-containing zonula-adherentes of the cilia-bearing epithelium that extends the entire length of the penis lumen (Fig. 3d). Convoluta convoluta. Confocal microscopy images of Alexa-488-labelled phalloidin-stained muscles associated with the male copulatory organ. (a) View of ventral body wall. (b) Ventral body-wall musculature surrounding male gonopore. (c) Sagittal view of seminal vesicle and penis. (d) Sagittal view of everted penis. Inset: Magnified view of longitudinal penis musculature. Arrowhead marks cell web. cop, male copulatory organ; fgp, female gonopore; m, mouth; mgp, male gonopore; p, penis; po, prostatoid organ; sv, seminal vesicle Photomicrographs of sagittal histological sections showing penis musculature of male copulatory organs. (a) Convoluta convoluta. (b) Convoluta marginalis. p, penis; sv, seminal vesicle The penis of C. henseni is surrounded by a muscular seminal vesicle (Fig. 4a,b), and comprises many thin circular fibres that surround approximately 25 thick longitudinal fibres (Fig. 4b). As in C. convoluta, individual longitudinal fibres connect with other longitudinal fibres to form an anastomosing pattern (Fig. 4b). Confocal microscopy images of Alexa-488-labeled phalloidin-stained muscles associated with the male copulatory organs of acoels with a 9 + 0 pattern of spermatozoa axonemes. Projections of musculature of Convoluta henseni (a, b) and Wulguru cuspidata (c–f). (a) Dorsal view of copulatory organs. (b) Dorsal view of male copulatory apparatus. (c) Ventral body-wall musculature surrounding gonopores. (d) Ventral view of penis musculature. (e) Projection of copulatory organ showing seminal vesicle and penis. Inset: Magnified view of longitudinal penis musculature. (f) Optical section of male copulatory organ showing longitudinal and circular muscles of penis. bn, bursal nozzle; cm, circular muscles; fgp, female gonopore; ffgp, false female gonopore; lm, longitudinal muscles; mgp, male gonopore; p, penis; pl, penis lumen; sv, seminal vesicle Wulguru cuspidata has a male copulatory organ similar to that of C. convoluta (Fig. 4c–f). The male gonopore opens directly to a rostrally directed penis that is not isodiametric, and is invaginated into a muscular seminal vesicle. Only the distal half of the penis lumen is ciliated. The penis musculature is composed of outer longitudinal fibres that anastomose or cross-over each other (Fig. 4e,f) and closely spaced inner anastomosing circular fibres (Fig. 4f). The same general pattern of copulatory musculature found in C. convoluta and W. cuspidata was also found in Amphiscolops sp. (Fig. 5a,b), Stomatricha hochbergi (Fig. 5c,d), and Polychoerus caudatus (data not shown). The seminal vesicle musculature of Amphiscolops sp. is diffuse and less organized than that of C. convoluta and W. cuspidata (Fig. 5a), while in S. hochbergi the musculature is well developed and tightly spaced (Fig. 5c). In both Amphiscolops sp. and S. hochbergi the penis musculature is composed of inner circular muscle fibres and outer longitudinal fibres that anastomose or cross over each other (Fig. 5b,d). Confocal microscopy images of Alexa-488-labelled phalloidin-stained muscles associated with the male copulatory organs of acoels with a 9 + 0 pattern of spermatozoa axonemes. (a) Projection of musculature associated with the copulatory organs of Amphiscolops sp. (b) Projection of penis musculature of Amphiscolops sp. Inset: Magnified view of longitudinal penis musculature. (c) Projection of ventral body-wall musculature and seminal vesicle of Stomatricha hochbergi. (d) Optical section of seminal vesicle and penis of Stomatricha hochbergi. (e) Optical section of adenodactyls and seminal vesicle of Anaperus gardineri. (f) Optical section of seminal vesicle of Anaperus gardineri. ad, adenodactyls, bn, bursal nozzle; fgp, female gonopore; lm, longitudinal muscles; p, penis; sv, seminal vesicle For comparison with an outgroup of the Convolutidae we examined Anaperus gardineri (Graff, 1911), a large interstitial acoel that has spermatozoa with 9 + 0 axonemes and axial microtubules. The male copulatory organ of A. gardineri is composed of actin-sclerotized needles surrounding a muscular seminal vesicle that is similar to that of the other ‘large-bodied convolutids’ we examined (Fig. 5e,f); however, the lack of an invaginated penis in A. gardineri precludes a comparison with the penis musculature of the other examined taxa. Convoluta karpredi has a thick muscular seminal vesicle that surrounds a curved, tubular, isodiametric penis (Fig. 6) with a lumen containing glandular secretions. The penis musculature is composed of c. 40 thick outer longitudinal fibres that do not anastomose or cross over each other (Fig. 6d) and that anchor to the distal side of the seminal vesicle (Fig. 6b) and extend the entire length of the penis, surrounding numerous inner circular muscle fibres (Fig. 6e). Convoluta karpredi. Through-series of selected optical sections of male copulatory organ in whole-mount stained with Alexa-488-labelled phalloidin and viewed with confocal microscopy. mgp, male gonopore; p, penis, sv, seminal vesicle In addition to C. karpredi, we examined the male copulatory organ musculature in 17 species of ‘small-bodied convolutids’ (Table 2): C. pulchra (Fig. 7a), Praeconvoluta castinea (Fig. 7b), Praeaphanostoma wadsworthi (Fig. 7c), cf. Pseudaphanostoma divae (Fig. 7d), Conaperta westbladi (Fig. 7e), Conaperta vexillaria (Fig. 7f), Conaperta divae (Fig. 8a), Conaperta norvegica (Fig. 8b), Aphanostoma bruscai (Fig. 8c), Diatomovora amoena (Fig. 8d), Praeconvoluta tornuva (Fig. 8e), C. marginalis (Fig. 9b), as well as Avagina marci, C. earnhardti, Conaperta hortulus, Praeconvoluta tigrina, and Raphidophallus actuosus (data not shown). The copulatory organs of these species differed with regard to spacing and thickness of muscle fibres, as well as the size and shape of seminal vesicles and penes; however, in all the species examined, a muscular seminal vesicle surrounded an isodiametric tubular penis, with the penis musculature composed of inner circular fibres surrounded by longitudinal fibres that did not anastomose or cross over each other. In some cases, the longitudinal muscle fibres of the penis could be seen to branch (Fig. 7e,f), but this occurred only at the base of the penis, where the penis muscles connected to muscles of the seminal vesicle and is clearly different than the anastomosing muscles found in the ‘large-bodied convolutids’. Confocal microscopy images of Alexa-488-labeled phalloidin-stained muscles associated with the male copulatory organs of ‘Small-bodied convolutids’. (a) Convoluta pulchra. (b) Praeconvoluta castinea. (c) Praeaphanostoma wadsworthi. (d) Pseudaphanostoma divae. (e) Conaperta westbladi. (f) Conaperta vexillaria. Arrowheads mark branching longitudinal penis muscles at connection between penis and seminal vesicle. p, penis; sv, seminal vesicle Confocal and epifluorescence microscopy images of Alexa-488-labelled phalloidin-stained muscles associated with the male copulatory organs of ‘Small-bodied convolutids’. (a) Conaperta divae. (b) Conaperta norvegica. (c) Aphanostoma bruscai (d) Diatomovora amoena. (e) Praeconvoluta tornuva Along with 18S rDNA sequence data and ultrastructural characters of spermatozoa, the morphology of longitudinal penis musculature provides further evidence that the family Convolutidae is a non-natural group in need of revision. Confocal microscopy is a valuable tool for providing clear images of the penis muscles of acoels, but these same features of penis musculature – anastomosing longitudinal fibres and non-anastomosing longitudinal muscle fibres – can also be discerned in easily prepared conventional epifluorescence images (Fig. 8e) as well as standard histological sections (Fig. 9). As such, we now have a relatively simple means of distinguishing ‘large-bodied convolutids’ from ‘small-bodied convolutids’ without acquiring sequence data or ultrastructural images of sperms. Based upon the evidence listed above, we propose systematic revisions to the family Convolutidae as detailed in Appendix 1. This revision includes emended diagnoses for two genera within the Convolutidae, Convoluta and Conaperta, and the erection of a new family and genus, Isodiametridae fam. nov. and Isodiametra gen. nov., respectively. Morphological characters important for the classification of genera within Convolutidae and Isodiametridae fam. nov. are tabulated in Tables 3 and 4. Acoels with a ventral mouth opening. Body-wall musculature with circular, longitudinal, and longitudinal cross-over muscle fibres in both the dorsal and ventral body wall, and U-shaped fibres in the ventral body wall. Male copulatory organ with muscular, often glandular, tubular penis. Penis musculature with inner circular fibres and outer longitudinal fibres that anastomose or cross over each other. When present, a muscular seminal vesicle surrounds all of, or a portion of, the penis. Male gonopore ventral, subterminal, or terminal at posterior end; usually behind to female seminal with 9 + 0 axonemes and axial microtubules. often present, in the cell and lack or as with bursal male and female male gonopore posterior to Male copulatory organ of a tubular, muscular penis that is often glandular and have inner Penis invaginated into a muscular seminal vesicle. and mouth with or without prostatoid organs. organ usually usually with algal symbionts. with with a bursal male and female gonopore, to male copulatory organ to to seminal Male copulatory organ of a muscular, often glandular, tubular penis, surrounded by a muscular seminal vesicle. organs often with algal symbionts. with Acoels with a ventral mouth opening. Body-wall musculature with circular, longitudinal, and longitudinal cross-over muscle fibres in both the dorsal and ventral body wall and U-shaped fibres in the ventral body wall. Male copulatory organ with muscular, isodiametric, tubular penis, often very Penis musculature with inner circular and outer non-anastomosing longitudinal Penis is invaginated into a muscular seminal Male gonopore ventral, subterminal, or terminal at posterior with 9 + 2 axonemes and cortical microtubules. with present, do not in the The from the isodiametric of the penis – that having approximately the same its entire length. Isodiametra gen. nov. with a bursal Male and female or Male copulatory organ with muscular, glandular, isodiametric, tubular penis, without inner surrounded by a muscular seminal vesicle. without enrolled lateral penis algal or to the family The is Isodiametra norvegica gen. nov., nov. The diagnoses the of morphological in sperm ultrastructure and penis morphology for distinguishing but this revision also to the distribution of two other ocelli and algal symbionts. are found in numerous species in the Convolutidae (Table and but than or as found in et al. and the cell is in containing as well as et al. and only two members of the Isodiametridae fam. nov. are known to have and Aphanostoma but these are diffuse of like that for – large of that are composed of a of containing and The ocelli of the Convolutidae and are therefore almost not to the of the Isodiametridae fam. nov., providing useful for distinguishing members of those families. The presence of algal in the of acoels to be useful in members of the Convolutidae (Table but are not known to in members of the Isodiametridae fam. nov. have also found in the and Pseudohaplogonaria and It has recognized that the genus Convoluta was in need of revision Smith and In the of the genus Convoluta could be into groups: containing species in which the penis is an a containing taxa in which the penis is from musculature and that has a glandular and third group with Smith and that revision was a first a more recognized that the could not be a as it is only the longitudinal muscles of the body wall that could to the penis and not the circular muscles (see for In the of the Convoluta species by of their or remain in the only the species of Convoluta that have non-anastomosing longitudinal penis muscles were to the Isodiametridae fam. nov. useful for distinguishing genera within the Convolutidae, and Isodiametridae fam. nov. are in Tables 3 and respectively. The number of is used as the distinguishing for the genera Convoluta and Conaperta Some species of these genera have transferred into the new genus Isodiametra gen. nov., without regard for gonopore it is the presence of a bursal that is the of the species in Isodiametra gen. nov., karpredi nov., and pulchra nov., have that are closely that are easily for a gonopore in histological In the species description of S. Hooge that its enrolled sides and possession of photosynthetic algal were characters not found in the and were more of species in the Convolutidae and the species was in the of the of the female gonopore behind the male copulatory organ, a feature of that investigation of acoel spermatozoa ultrastructure et al. revealed that the sperm of S. hochbergi has a 9 + 0 pattern of a pattern not found in the but for the families Convolutidae, and In the sperm of S. hochbergi is nearly to that of two members of the Convolutidae, C. and C. (see Petrov et al. Stomatricha possession of a copulatory organ with anastomosing longitudinal penis muscles (Fig. is also evidence that it be transferred to the family Convolutidae. the phylogenetic of S. hochbergi within the Convolutidae is that the of its copulatory organs from that of other members of the The of S. hochbergi to the Convolutidae the morphological posterior to the male copulatory organ either through a or through a female a in the Acoela – a for the – and as such, the is now in need of revision. In with systematic classification of the Acoela, systematic revision greater on the seminal than the male copulatory organ. As such, the Acoela into two and upon the presence or of a seminal to this C. which has a seminal as well as a bursal with other recognized the of C. copulatory organ to those of The of C. opisthandropora are but at to form an that extends to the of a tubular male that to a terminal gonopore. The sperm in this are in and in a that a The sperm as well as the of the male is surrounded by a of that extend into the lumen of the to this are additional glandular that also the half of the In the classification system used greater systematic is to the male copulatory organ. As such, C. tubular male and lack of an invaginated penis is with members of the The morphology of C. opisthandropora is with the genus which contains species having a seminal and bursal but an or developed seminal vesicle. now C. opisthandropora to the genus A of the holothuroidean A. sanguineum has a copulatory organ that has to Aphanostoma sanguineum has male and female in some specimens the are closely that be as a as was the in of this 18S rDNA sequence data a between A. sanguineum and members of the et al. and this is further by A. possession of a penis needles (Fig. than a muscular A. sanguineum to the genus a group that is in having a seminal that is not with a bursal Pseudactinoposthia sanguineum nov. of histological showing needles of male copulatory organ. penis needles are to for specimens of C. convoluta, and to Smith, and who in the of other to for on and into the to Petrov for Russian specimens were collected with support from the of within the – The This is of This is upon by the under and