Protaeolidiella atra Baba, 1955 versus Pleurolidia juliae Burn, 1966: One or two species?
© Springer-Verlag Berlin Heidelberg and AWI 2015
Received: 23 June 2014
Accepted: 17 December 2014
Published: 10 January 2015
Protaeolidiella atra Baba, 1955 and Pleurolidia juliae Burn, 1966 are two species traditionally regarded as the members of Aeolidiidae but recently attributed to Facelinidae. Because of their apparent similarities, Rudman (J Molluscan Stud 56:505–514, 1990) rendered P. juliae as a junior synonym of P. atra. In this paper, we conducted a review of both species and completed their descriptions with new data regarding the anatomy of the reproductive system. P. atra and P. juliae have differences in their colouration, number of cerata and characteristics of their reproductive system. Based on these differences, we conclude that these species are not conspecific and should be regarded as distinct taxa.
Protaeolidiella atra Baba, 1955 and Pleurolidia juliae Burn, 1966 are two species considered as the only members of these genera within the aeolid families Protaeolidiellidae Odhner, 1968 and Pleurolidiidae Burn 1966, respectively (Baba 1955; Burn 1966; Odhner 1968). Nevertheless, some authors such as Gosliner and Griffiths (1981), Rudman (1990) and Miller (2001) regarded these species as primitive members of Aeolidiidae Gray, 1827. Because of the similar appearance of these two species, differences between P. atra and P. juliae have been controversial and raised doubts about the validity of the two species and genera. According to Burn (1966), the triseriate radula of P. juliae is the main feature that distinguishes this species from P. atra. Almost three decades later, Rudman (1990) did not find any traces of lateral teeth in P. juliae and, therefore, concluded that both were conspecific, rendering P. juliae as junior synonym of P. atra. Baba (1992) rejected this synonymy and maintained both species as valid although he did not examine the radula of P. juliae. Recently, the molecular phylogeny of Aeolidiidae (Carmona et al. 2013) transferred P. juliae from Aeolidiidae to Facelinidae Bergh, 1889. This outcome concurs with the feeding behaviour of P. juliae, since this species, together with P. atra, feeds on hydroids (like most facelinids), whereas species of the Aeolidiidae all prey upon sea anemones and other anthozoans, including zoanthids (Gosliner et al. 2008).
In this contribution, we attempt to clarify the conspecifity of P. atra and P. juliae, using morphology and molecular phylogenetics including specimens from across the geographical range of both species.
Materials and methods
Source of specimens and morphology
Samples were collected by SCUBA diving and obtained from wet collections housed at the California Academy of Sciences. Specimens were dissected by dorsal incision. Their internal features were examined and drawn under a stereoscopic microscope with the aid of a camera lucida. Special attention was given to the morphology of the reproductive system and oral and salivary glands. The buccal mass was removed and dissolved in 10 % sodium hydroxide until the radula was isolated from the surrounding tissue. The radula was then rinsed in water, dried and mounted for examination under a scanning electron microscopy (SEM). The specimens examined are deposited in the California Academy of Sciences, CASIZ (San Francisco, USA) and National Museum of Nature and Science, NSMT-Mo (Ibaraki Prefecture, Japan).
DNA extraction, amplification and sequencing
List of specimens used for phylogenetic analyses
GenBank accession nos.
Tritoniidae Lamarck, 1809
Tritonia antarctica Pfeffer, 1886
Bouvetoya (EA, GB)
30 Jun 04
Dendronotidae Allman, 1845
Dendronotus venustus MacFarland, 1966
Santa Monica (California, GB)
Proctonotidae Gray, 1853
Janolus mirabilis Baba and Abe, 1970
19 May 09
Aeolidiidae Gray, 1827
Aeolidia papillosa (Linnaeus, 1761)
14 Sep 11
Berghia coerulescens (Laurillard, 1830)
03 Dec 04
ZSM Mol 20041584
Spurilla neapolitana (delle Chiaje, 1841)
17 Mar 09
Babakinidae Roller, 1973
Babakina anadoni (Ortea, 1979)
Babakina indopacifica Gosliner, González-Duarte and Cervera, 2007
20 Mar 08
Facelinidae Bergh, 1889
Cratena peregrina Gmelin, 1791
30 May 05
Facelina annulicornis (Chamisso and Eysenhardt, 1821)
Azores Is. (Portugal)
11 Jun 02
Favorinus branchialis (Rathke, 1806)
26 Jun 07
Favorinus elenalexiarum García and Troncoso, 2001
Costa Rica (EP, GB)
17 Apr 07
Godiva quadricolor (Barnard, 1927)
South Africa (EA, GB)
09 Jan 08
Hermosita hakunamatata (Ortea, Caballer and Espinosa, 2003)
17 Feb 06
Moridilla brockii Bergh, 1888
29 Apr 11
Noumeaella isa Marcus and Marcus, 1970
01 May 11
Phidiana lynceus Bergh, 1867
21 Jul 08
Phyllodesmium horridum (Macnae, 1954)
South Africa (EA, GB)
03 Jan 08
Pleurolidia juliae Burn, 1966
05 May 05
Protaeolidiella atra Baba, 1955
17 Feb 04
18 Feb 04
Pruvotfolia longicirrha (Eliot, 1906)
Pruvotfolia pselliotes (Labbé, 1923)
05 Sep 04
Sakuraeolis enosimensis (Baba, 1930)
13 Dec 07
Fionidae Alder and Hancock, 1855
Fiona pinnata (Eschscholtz, 1831)
22 Dec 10
Flabellinidae Bergh, 1881
Flabellina affinis (Gmelin, 1791)
Balearic Is. (Spain, MED)
14 Jul 07
Flabellina ischitana Hirano and Thompson, 1990
07 Mar 08
Piseinotecidae Edmunds, 1970
Piseinotecus gabinieri (Vicente, 1975)
13 Oct 07
Sequence alignment and phylogenetic analyses
Sequences were assembled and edited with Geneious Pro v. 4.7.6 (Drummond et al. 2009), aligned in MAFFT (Katoh et al. 2009) and further checked using MacClade v. 4.06 (Maddison and Maddison 2005). Uncorrected pairwise p-distance values between each taxon were calculated for the COI gene. The most variable regions from the 16S rRNA alignment were removed using the default settings in Gblocks (Talavera and Castresana 2007). Excluding “indel-rich” regions, the tree was in general poorly resolved with lower node support. Therefore, final analyses were performed including all bases. Sequences of COI, 16S and H3 were trimmed to 658, 442 and 327 base pairs, respectively.
The best-fit evolutionary model (GTR + I+G for the three genes) was determined in MrModeltest v. 2.3 (Nylander 2004), using the Akaike information criterion (Akaike 1974). MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003) was used for Bayesian inference analysis and to estimate posterior probabilities (PP) for node support with two runs of 5,000,000 generations each. Convergence was checked in TRACER v. 1.5 (Drummond and Rambaut 2007) with a burn-in of 25 %. Maximum likelihood (ML) analyses were performed using the software RAxML v7.0.4 (Stamatakis 2006), and node support was assessed with nonparametric bootstrapping (BS) with 5,000 replicates, random starting trees, and parameters estimated from each dataset under the model selected for the original dataset. Only nodes supported by BS ≥70 (Hillis and Bull 1993) and PP ≥0.95 (Alfaro et al. 2003) are discussed.
Diagnosis of Protaeolidiella following Baba (1955)
Kasajima, Sagami Bay, Japan (Fig. 1).
To our knowledge, Baba (1955) designates two syntype lots (NSMT-OpR 0872, NSMT-OpR 0884) at the National Museum of Nature and Science.
(NSMT-Mo 78850), one specimen, adult, mature, dissected, 40 mm in length alive, Place Issai, Ohtsuki town Hata-gun, Kochi prefature, Japan, collected by Rie Nakano, 17.iii.12; (NSMT-Mo 78851), one specimen, adult, mature, dissected, 20 mm in length alive, Place Issai, Ohtsuki town Hata-gun, Kochi prefature, Japan, collected by Rie Nakano, 17.iii.12; (NSMT-Mo 78852), one specimen, adult, mature, dissected, 40 mm in length alive, Zushi, Japan, collected by Michiaki Homma, 18.ii.12;
External morphology (Fig. 1a, b):
The cerata densely packed along the edge of the mantle, forming a transverse row. They extend from behind the rhinophores to the posterior end of the body. The ceratal length is variable, but all are slender, cylindrical, with a round apex and uniform diameter throughout most of length. There are around 50–60 cerata per side. They have the same background colour as the body with hyaline white apices. On the right side of the body, the gonopore is situated anteriorly below the beginning of the ceratal row. The anus is on the right side of the body, in a pleuroproctic position.
Anatomy (Fig. 1c–e):
The radula is uniseriate (26 × 0.1.0, NSMT-Mo 78850). The radular teeth are pectinate with 24–60 acutely pointed denticles (Fig. 1c). The latter are flattened laterally, with a similar length. The masticatory border of the jaws is smooth (Fig. 1d). Salivary and oral glands were not observed.
The reproductive system is diaulic (Fig. 1e). The hermaphroditic duct widens into an elongate and wide ampulla, which has moderately thick walls. The ampulla narrows again before dividing into the oviduct and vas deferens. The short vas deferens, which lacks a morphologically well-differentiated prostate, enters the wider proximal portion of the penial sac, which contains the unarmed penial papilla. The oviduct is moderately elongated and connects to a well-developed receptaculum seminis. The remaining portion of the oviduct separates from the base of the receptaculum and enters the female gland. The vagina opens ventrally relative to the penis.
Diagnosis of Pleurolidia following Burn (1966)
“The Brook”, Lord Howe Island, Australia (Fig. 2).
According to Burn (1966), the material was deposited in the Australian Museum, Sidney. The registration number of the holotype of P. juliae is C. 65661.
CASIZ 167988, one specimen, adult, mature, dissected, 7 mm in length preserved, Maui, Hawaii, collected by Pauline Fiene-Severns, 12.ix.03; CASIZ 065416, one specimen, adult, mature, dissected, 11 mm in length preserved, Papua New Guinea, collected by Terrence M. Gosliner, 21.i.88.
This species was originally described from Lord Howe Island, Australia (Burn 1966), but it can also be found in Madagascar (Gosliner et al. 2008), Papua New Guinea (this study), Indonesia (Gosliner et al. 2008), the Philippines (Gosliner et al. 2008), Palau (Gosliner et al. 2008), and Japan (Baba 1992; Ono 1999, 2004 under the name of P. atra; Gosliner et al. 2008).
External morphology (Fig. 2a):
The cerata are arranged along the edge of the mantle, forming small groups that constitute a transversal row. Sometimes these groups are not symmetrical, having a zigzag arrangement. They extend from behind the rhinophores to the posterior end of the body. The length of the cerata may vary, but all are slender, cylindrical, with round apex and uniform diameter throughout most of their length. There are around 10–20 cerata per side. They have the same background colour of the body with hyaline white speckles over their surface. The apices are translucent white. On the right side of the body, the gonopore is placed anteriorly below the beginning of the ceratal row. The anus is on the right side of the body, in a pleuroproctic position.
Anatomy (Fig. 2b–d):
The radula is uniseriate (14 × 0.1.0, CASIZ 167988). The radular teeth are pectinate with 10–12 quite long and acutely pointed denticles (Fig. 2b). The denticles are flattened laterally, with a similar length. The masticatory border of the jaws is finely denticulate (Fig. 2c). The oral glands are relatively large, consisting of clusters of small and rounded acini. Salivary glands were not found.
The reproductive system is diaulic (Fig. 2d). The preampullary duct widens into a large and wide ampulla, which has moderately thick walls. The ampulla narrows again before dividing into the vas deferens and the oviduct. The short and wide vas deferens, which lacks a morphologically well-differentiated prostate, enters the wider proximal portion of the penial sac, which contains the unarmed penial papilla. The female gland mass exits at the female genital aperture, adjacent to the bursa copulatrix, which is large and exits via a relatively long, wide duct.
Protaeolidiella atra and P. juliae were nested among different facelinid species although this relationship was not supported (PP = 0.78, BS = 26). P. atra and P. juliae formed a single clade (PP = 1, BS = 100) with Hermosita hakunamatata as its sister species (PP = 0.94, BS = 59). The minimum uncorrected p-distance for COI between P. atra and P. juliae was 17.93 %.
Despite differences in radular morphology, ceratal arrangement and specialised diet, Protaeolidiella and Pleurolidia have been considered as primitive aeolidiids (Tardy 1965; Burn 1966; Gosliner 1985). In addition, Rudman (1990) regarded P. juliae as a junior synonym of P. atra. Nevertheless, after reassessing Rudman’s paper, we are strongly inclined to consider that he only studied specimens of P. juliae because none of these specimens were from Japan, the type locality of P. atra. More recently, the molecular study conducted by Carmona et al. (2013) placed P. juliae within the non-monophyletic Facelinidae, rejecting its traditional placement within the Aeolidiidae. Our anatomical and molecular results agree with those of Carmona et al. (2013) and demonstrated that the presence of morphological and genetic differences between P. atra and P. juliae. The two species can be distinguished by differences in colouration, since P. atra lacks the transverse and dorsal line found in P. juliae, as well as by the number of cerata. From an internal anatomical point of view, these species are quite similar but there is an important difference: P. atra has a receptaculum seminis, which opens into the proximal region of the gonoduct (Ghiselin 1966; Schmekel 1985; Gosliner 1994), while P. juliae posses a bursa copulatrix that opens into the distal region of the vaginal duct (Ghiselin 1966; Schmekel 1985; Gosliner 1994). Since Baba (1955, 1992) never depicted the reproductive system of P. atra and Rudman (1990), likely studied only P. juliae (see above), this difference in the reproductive anatomy has not been reported previously. In addition, we only found oral glands in P. juliae. Nevertheless, according to previous studies, oral glands can show some intraspecific variation in other aeolids (Carmona et al. 2014c, d), and therefore, the systematic relevance of this character should be regarded with caution. Considering the anatomical, morphological and genetic differences, we conclude that both genera are valid and monotypic. On the other hand, in order to decide about the validity of the families Protaeolidiellidae and Pleurolidiidae further information about the phylogeny of the non-monophyletic Facelinidae is needed.
The close relationship among P. atra, P. juliae and H. hakunamatata represents an intriguing case that warrants further study, given that these species are the only aeolids that feed on the hydroid Solanderia fusca (Gosliner personal observation). Furthermore, according to Ghiselin (1966), Gosliner (1981), Schmekel (1985) and Mikkelsen (1996), the presence of both, receptaculum semins and bursa copulatrix, represents the plesiomorphic state in the traditional “Opisthobranchia” and it is interesting to notice that H. hakumanatata, the only species with both anatomical structures (Millen and Hermosillo 2012), has a position as sister to the other two species within this clade. The only other species of aeolid nudibranch that has been observed feeding on species of Solanderia is Hermosita sangria Gosliner and Behrens, 1986. Future studies should examine the phylogenetic relationships of this species to the species discussed above.
We are deeply grateful to R. Nakano who helped to collect and provide specimens and images for this study. Advisements of two anonymous referees contributed to improve the final version of the manuscript. This work was supported by the research grants (CGL2010-17187), Spanish Ministry of Economy and Competitiveness (includes the early Ministry of Sciences and Innovation) to J. L. Cervera. This is CEI MAR journal publication 80.
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