Aphanostoma, ORSTED 1845

Atherton, Sarah & Jondelius, Ulf, 2022, Phylogenetic assessment and systematic revision of the acoel family Isodiametridae, Zoological Journal of the Linnean Society 194, pp. 736-760 : 750-753

APHANOSTOMA ØRSTED 1845 , ISODIAMETRA HOOGE & TYLER, 2005 AND PRAECONVOLUTA DÖRJES, 1968

Species of Aphanostoma , Isodiametra and Praeconvoluta are united by the possession of a single bursa with some type of bursal appendage, the morphology of which, following the classification systems of Dörjes (1968), Faubel (1974) and Hooge & Tyler (2005), in large part distinguishes each genus (for a list of bursa terminology, see also: Petrov et al., 2006). Isodiametra encompassed those species of Isodiametridae with a single sclerotized bursal nozzle, and Aphanostoma comprises species with a cellular bursal cap, which may also include small actin-sclerotized bodies ( Petrov et al., 2006; Hooge & Tyler, 2008). For Praeconvoluta, Dörjes (1968) and later Faubel (1974) specified a ‘simple bursa’ in the genus diagnosis. However, all species, except P. tigrina Hooge & Tyler, 2003 , were described with a bursal cap, and one species, P. bocasensis Hooge & Tyler, 2008 , further possesses a bursal cap with several spots of concentrated actin, and Hooge & Tyler (2005) listed Praeconvoluta and Aphanostoma as morphologically indistinguishable in their identification table. There was some discussion of combining Aphanostoma and Praeconvoluta ( Nilsson et al., 2011; Zauchner et al., 2015), since the general morphologies of the species of the two genera are the same, although no formal synonymization ever actually occurred.

All species of Praeconvoluta in our analyses form a clade with Aphanostoma bruscai Dörjes, 1968 and A. collinae Hooge & Tyler, 2008 , a result that is not surprising, given that the genera are morphologically indistinguishable from each other ( Hooge & Tyler, 2005; Zauchner et al., 2015). This clade also included Isodiametra norvegica ( Westblad, 1946) , the type species of Isodiametra , and I. hortulous ( Hooge & Tyler, 2003) ( Fig. 1 ). Thus, this clade includes species with all forms of bursa morphology ( Fig. 5 ): simple without adornments ( P. tigrina ), with a cellular cap reported with ( A. bruscai , A. collinae and P. bocasensis ) or without ( P. castinea Hooge & Tyler, 2003 and P. tornuva Hooge & Tyler, 1999 ) spots of actin and with a fully sclerotized nozzle ( I. norvegica and I. hortulous ). The type species of Aphanostoma , A. virescens , was not included in this group, but formed a clade with Otocelis erinae , Raphidophallus actuosus and seven other species of Isodiametra ( Fig. 1 ). The bursal appendages in the species of this clade are more similar to each other, since all species, except for A. virescens , posses a bursa with a sclerotized nozzle ( Fig. 5 ), and the morphology of the bursal cap of the latter is somewhat unique. Aphanostoma virescens has a large and highly muscular bursal cap with numerous (six to eight according to Steinböck, 1931, although Graff, 1905, recorded as many as 14) sclerotic spines. Both clades were maximally supported.

Our results indicate that, while the presence or absence of a walled/true bursa may be useful to distinguish genera of Isodiametridae , the details and composition of the bursal appendage is not ( Fig. 5 ). This is perhaps not so surprising given the similarities in the composition of the different bursal appendages. Each sclerotized bursal nozzle in Acoela is composed of a sperm duct with actin-reinforced inner edges, while the bursal cap of at least some species includes disjunct spots of intracellular actin localized along a sperm duct ( Petrov et al., 2006). Further, because intracellular actin is not always visible in squeeze preparations and living specimens, it can easily be missed if the bursal cap of a species has not been properly examined with phalloidin-labelled probes and fluorescence microscopy ( Hooge & Tyler, 2008). Petrov et al. (2006) warned that actin-sclerotized sections of bursal caps could be confused with muscle fibres following conventional staining by iron haematoxylin and, in fact, they were highly skeptical that muscles were a component of any bursal appendage in Acoela at all. Thus, the analysis of bursal morphology may be incomplete or incorrect in some species considered to have only muscular or cellular caps, especially for species with older descriptions.

Below, we attempt to update the current classification system to reflect as best as possible their phylogenetic relationships as represented by the results of our molecular analyses ( Fig. 1 ), and we reassign species to, and emend, the genera accordingly. Information gathered from live observations and original descriptions of each species included in the analyses form the basis for the morphological summary of each genus, while those species for which sequence data are currently unavailable, were classified based on morphology. Specifically, our results supported the existence of two clades representing two genera with walled bursas ( Figs 1 , 5 ), which appeared to be best distinguished morphologically based on the presence or absence of one or more distinct muscular vaginal sphincters ( Fig. 9 ). This result notably concurs with the findings of Jondelius et al. (2011) that details of the musculature have higher potential to be taxonomically informative at the genus level for Isodiametridae . Nevertheless, we must underscore the need for further detailed morphological study using modern methodology to clarify any potential discrepancies that may exist in the reproductive anatomy and musculature, as well as the importance of obtaining DNA sequences of the remaining species of the genera.

EMENDED DIAGNOSIS OF APHANOSTOMA ØRSTED, 1845 AND REASSIGNMENT OF SPECIES ; SYNONYMIZATION OF RAPHIDIOPHALLUS KOZLOFF, 1965 AND APHANOSTOMA ØRSTED, 1845

Aphanostoma is hereby emended to include the species that form a clade with its type species, A. virescens . Isodiametra bajaensis Hooge & Eppinger, 2005 , I. cuernos Hooge & Tyler, 2008 , I. divae ( Marcus, 1950) , I. finkei Kånneby & Jondelius, 2013 , I. nicki Hooge & Tyler, 2008 , I. pulchra ( Smith & Bush, 1991) , Otocelis erinae and Raphidophallus actuosus are all transferred to Aphanostoma .

Raphidophallus was distinguished from other genera solely based on the delicate cuticularized rods that occurred in the lumen of the penis. Otherwise, Kozloff (1965) noted that its morphology was highly similar to other species of Isodiametra , particularly with reference to the bursa with nozzle, which the majority of the species in the clade do also possess (although see notes above). Raphidophallus actuosus is the type and only species of its genus and, therefore, Raphidophallus is considered a junior synonym of Aphanostoma .

All species in this clade are united by the possession of a muscular bursa with evident actin in the form of a nozzle or spines, a distinct male or common atrium and one or more well-developed vaginal sphincters. Of the species for which DNA is currently unavailable, Isodiametra earnhardti ( Hooge & Smith, 2004) , I. helgolandica ( Dörjes, 1968) , I. karpredi ( Hooge & Tyler, 2003), I. vexillaria ( Marcus, 1948) and I. westbladi ( Marcus, 1949) all also possess these characters and are, therefore, transferred to Aphanostoma . In addition, Aphanostoma album and A. rhomboides Jensen, 1878 both possess strong vaginal sphincters that are characteristic of the genus, although neither have the evident spots of actin in their bursal caps. Neither species has been examined with phalloidin-labelled probes.

SYNONYMIZATION OF PRAECONVOLUTA DÖRJES, 1968 AND ISODIAMETRA HOOGE & TYLER, 2005 ; EMENDED DIAGNOSIS OF PRAECONVOLUTA DÖRJES, 1968 AND REASSIGNMENT OF SPECIES

All four species of Praeconvoluta group with Aphanostoma bruscai Hooge & Tyler, 2003 , A. collinae Hooge & Tyler, 2008 , Isodiametra norvegica and I. hortulous in our analyses to form a second clade of species of Isodiametridae with true/walled bursas ( Fig. 5 ). The species of this clade are united together and distinguished from Aphanostoma through a lack of well-defined vaginal sphincter muscles ( Fig. 9 ). Nine of the 16 species with true bursas without DNA sequences currently available all lack well-defined vaginal sphincters, including all remaining four species of Praeconvoluta , Aphanostoma piscae Zauchner et al., 2015 , Isodiametra colorata ( Ehlers & Dörjes, 1979) , I. marginalis ( Ivanov, 1952) , I. urua ( Marcus, 1954) and I. variomorpha ( Dörjes, 1968) .

Isodiametra norvegcia is the type species of Isodiametra , which was proposed by Hooge & Tyler (2005) for species with a single bursa nozzle. According to our phylogenetic hypothesis, this genus is polyphyletic with the majority of the species grouping separately from the type species. Isodiametra norvegica groups with the four species of Praeconvoluta included in the analyses. Although DNA sequences are not available for the type species Praeconvoluta karinae , it and other species of the genus lack well-defined vaginal sphincters. Based on our phylogenetic hypothesis, Praeconvoluta and Isodiametra are synonymized such that Isodiametra is a junior synonym of Praecovoluta.

Isodiametra was the type genus of Isodiametridae . Nevertheless, no name replacement for the family is necessary in this instance since replacing Isodiametridae with another name would be exclusively due to the type genus being considered a junior synonym (see ICZN section 40.1). Hence Isodiametridae is retained and Praeconvoluta becomes the type genus.

Figures

Figure 1. Concatenated 28S, 18S and COI gene tree summary. Percent bootstrap values are given at each node. Sequences from six species of Isodiametridae and Actinoposthiidae represented only by 18S sequences downloaded from GenBank (Table S1) were excluded.

Figure 5. Bursal morphology of Isodiametridae mapped onto the concatenated tree. The bursal morphology of each species was coded following original descriptions and illustrations, as well as photos and live observation of species where possible.

Figure 9. Vaginal sphincter presence or absence in species of Isodiametridae mapped on to the concatenated tree. The morphology of each species was coded following original descriptions and illustrations, as well as photos and live observation of species where possible.? indicates that the presence or absence ofa vaginal sphincter was not examined/could not bedetermined.