Molecular data for Crenavolva species (Gastropoda, Ovulidae) reveals the synonymy of C. chiapponii

Abstract During fieldwork in Indonesia and Malaysia, eight lots containing 33 specimens belonging to the genus Crenavolva (Ovulidae) were collected. Species were initially identified as Crenavolva aureola, Crenavolva chiapponii, Crenavolva striatula and Crenavolva trailli, respectively. For Crenavolva chiapponii this is the second record. In contrast to the ecological data available from the original description of this species, it was found in shallow water on a gorgonian host coral, i.e. Acanthogorgia sp. A molecular analysis based on COI and 16S mtDNA markers, including sequence data obtained from GenBank, showed that Crenavolva chiapponii should be considered a junior synonym of Crenavolva aureola and that previously identified ovulid specimens are probably misidentified.


Introduction
The nominal taxon Crenavolva was introduced as a subgenus by Cate (1973), together with the subgenera Crenavolva, Cuspivolva and Serratovolva. In the most recent over view regarding Ovulidae these three taxa are considered genera (Lorenz and Fehse 2009). At present 18 nominal species are recognized within Crenavolva (Rosenberg 2014), most of which are considered rare (Lorenz and Fehse 2009). These species are considered rare because few specimens have been collected, probably because they occur at depths greater than standard recreational diving depth of c. 30 m and/or are only known from a limited geographical area, usually just the type locality. This also accounts for C. chiapponii Lorenz & Fehse, 2009, which is only known from Bali casag Isl., Bohol, Philippines, where specimens were trawled from 70-120 m depth and, therefore, were considered rare and confined to deeper water (Lorenz and Fehse 2009). Like almost all other ovulids, species of Crenavolva are associated with octo coral hosts (Schiaparelli et al. 2005;Reijnen 2010) belonging to several families (e.g. Melithaeidae, Ellisellidae, Subergorgiidae and Plexauridae). However, the host species are usually not collected or are disregarded and therefore unknown, which is also the case for C. chiapponii.
Molecular data (16S and COI) obtained from Crenavolva was used by Meyer (2003) to root the phylogeny of the Cypraeidae. Later, the 16S sequence data were used by Schiaparelli et al. (2005) to produce the first molecular phylogenetic reconstruction of the Ovulidae, which included two Crenavolva species: C. cf. rosewateri (Cate, 1973) and C. tokuoi Azuma, 1989. In the present study, material of four additional nominal Crenavolva species, amongst other ovulids, have been used to reconstruct a phylogeny. The newly acquired molecular data are for C. aureola (Fehse, 2002), C. chiapponii Lor enz & Fehse, 2009, C. striatula (Sowerby I, 1828) (type species), and C. trailli (Adams, 1855). In addition to this phylogenetic reconstruction, data on host species and distri butional records are given for this group of rarely recorded ovulid snails.

Collection and identification
During fieldwork in Indonesia (Halmahera, Ternate; Sulawesi, Lembeh Strait) and Malaysia (Borneo, Semporna and Kudat) specimens of Crenavolva species were col lected by SCUBA diving (Table 1). The snails and their octocoral hosts were photo graphed in situ ( Fig. 1) whenever possible and subsequently fixed in 80% ethanol. The holotype of C. chiapponii was studied at the Muséum national d'Histoire na turelle (MNHN) in Paris. For the identification of the other ovulid species, Cate (1973), Fehse (2002b) and Lorenz and Fehse (2009) were used. For the identification of the host species, microscopy slides of their calcareous skeletal parts (sclerites) were made by dissolving the samples in a 4% solution of household bleach. The residual sclerites were rinsed with tap water followed by demineralised water before mounting on a slide or on a stub for Scanning Electron Microscopy (SEM). Stubs with sclerites were coated with Au/Pd before SEM images were made with a JEOL 6480 LV. Iden tification of the octocorals to genus level was based on Stiasny (1947) and Fabricius and Alderslade (2001).

Barcoding Ovulidae
Specimens were barcoded for the COI barcoding region and for additional phylogenet ic research also for the 16S marker. Tissue samples obtained from the foot and/or man tle were extracted with the MacheryNagel DNA extraction kit on a KingFisher Flex. The standard COI barcoding primers by Folmer et al. (1994) and the Palumbi (1996) 16S primers were used. PCR amplification was performed on a C1000 Touch Thermal Cycler (BioRAD). Sequencing of the PCR products was performed at Macrogen Eu rope on an ABI 3730xl Automated Sequencer. Sequences were edited in Sequencher 4.10.1 and aligned with GUIDANCE (Penn et al. 2010) using the MAFFT algorithm (Katoh et al. 2005). Selecting an evolutionary model was done with jModeltest based on the Akaike Information Criterion score. MEGA 6.0.6 (Tamura et al. 2013) was used to perform Maximum Likelihood (ML) and Maximum Parsimony (MP) analyses and to calculate pdistances. Bayesian analyses were performed in MrBayes 3.2.0 (Ron quist and Huelsenbeck 2003). MrBayes was run for 4,000,000 generations with six chains. Data were sampled every 100 generations. Sequence data for Ovula ovum (Lin naeus, 1758) from GenBank was used as an outgroup. GenBank data for Crenavolva cf. rosewateri (Cate, 1973), C. tokuoi Azuma, 1989 and Primovula beckeri (Sowerby III, 1900) was also included in the phylogenetic analyses.

Collecting and morphology
Eight lots, containing 33 specimens representing four nominal Crenavolva species (C. aureola, C. chiapponii, C. striatula and C. trailli) were collected in Indonesia and Ma laysia (Table 1; Fig. 2). For C. chiapponii this is the first record from shallow water. The specimens were assigned to these nominal species based on shell shape (rhomboid, in flated or slender) and the colour bands on the dorsum, which in case of C. striatula were also present on the labrum. For C. aureola and C. chiapponii the absence or presence of a white dorsal band on the shell is allegedly the most obvious character to distinguish the species. After examination of the illustrations presented by Lorenz and Fehse (2009) and the newly collected material, minor morphological differences (strongly or weakly pronounced dentation, keeling angle, strongly or weakly produced funiculum, position of the widest part of the shell) do not clearly separate between C. aureola and C. chiapponii and can be considered morphological variation in a single species. The soft tissue colouration of both C. aureola and C. chiapponii is very similar (e.g. Fig 1; Lorenz and Fehse 2009: A106, A107 p. 527). Both have a semitransparent mantle which is entirely covered with small, irregularly placed, white dots, and both have a completely black or white foot, black tentacles with white tips, and a black siphon.

Molecular data
Nine specimens representing five species were sequenced for COI and 16S. For one sample of C. chiapponii (RMNH.MOL.164211) the 16S marker could not be am plified. Sequences were concatenated and aligned (GUIDANCE alignment score: 0.965034) which resulted in an alignment length of 1080 base pairs per specimen including indels. Sequences obtained from GenBank are slightly shorter (~40 base pairs), these missing base pairs were coded as 'missing data'. The program jModeltest yielded in HKY+G as most optimal evolutionary model. This evolutionary model was implemented in the Bayesian and ML analysis. The results from the different phylo genetic reconstructions were congruent, therefore only the ML tree is shown (Fig. 3).
In the phylogenetic reconstructions, specimens of Crenavolva striatula and C. tokuoi form an unresolved trichotomy with the other Crenavolva specimens. The two Primovula species cluster together and are wellsupported sister species to all the Crenavolva species (with C. striatula as type species for the genus). This implies that the Crenavolva species used herein form a monophyletic group. The clustering of two C. trailli specimens is highly supported. Another wellsupported clade holds three nominal spe cies: Crenavolva aureola, C. chiapponii and C. cf. rosewateri. The pairwise pdistances between these three species are very low (16S: 0.2%; COI: 0.7%; concatenated: 0.9%).  Table 1. In contrast, the sequence divergence between C. trailli and the C. chiapponii / C. aureola clade is almost ten times larger (16S: 5.2%; COI: 8.7%; concatenated: 8.2%). The sequence divergence between the two C. trailli specimens (16S: 0.6%; COI: 0.8%; concatenated: 0.8%) is almost equal to that between C. aureola and C. chiapponii. With the help of the Automatic Barcode Gap Discovery tool (ABGD) (Puillandre et al. 2011), the data were analysed to identify the MOTU's within the dataset. The results of this analysis showed that the barcode gap to identify the different species is 5-6% sequence divergence. This resulted in five groups containing the following species: 1, C. aureola, C. chiapponii, C. cf. rosewateri; 2, C. trailli; 3, C. tokuoi; 4, C. striatula; 5, P. rosewateri. One of the samples obtained from GenBank, viz. Crenavolva cf. rosewateri (= Primovula cf. rosewateri), clusters surprisingly within the clade containing C. aureola and C. chiapponii and not with the other Primovula rosewateri specimen. Instead, Primovula beckeri proves to be identical to the newly sequenced specimen of Primovula rosewateri from Malaysia.

Octocoral hosts
Almost all Ovulidae species are associated with Octocorallia hosts. By examining the sclerites and the habitus of the host corals, several new host species for ovulids of the genus Crenavolva could be identified. An overview of previously identified host species and new records is provided in Table 2. Some of the former host identifications were published with obsolete generic names, and therefore their names in the current lit erature are also provided. Before C. chiapponii was synonymised, Acanthogorgia would have been a new host record. Yet, Reijnen (2010) already recorded Acanthogorgia sp. as a host for C. aureola and therefore it is not a new host record. Morphologically at least two different species of Acanthogorgia could be distinguished but these could not be identified since a revision of the family Acanthogorgiidae is lacking.  Lorenz and Fehse 2009;Yamamoto 1973;Cumming 1997;Mase 1989; Crenavolva trailli Echinogorgia; Anthoplexaura (= Astrogorgia); Plexauroides (= Echinogorgia); Euplexaura; Subergorgia Goh et al. 1999;Mase 1989 Primovula rosewateri Subergorgia; Dendronephthya; Stereonephthya; Paratelesto Goh et al. 1999;Lorenz and Fehse 2009; this publication Primovula beckeri Acanthogorgia; Acabaria (= Melithaea); Unicella [sic] (= Eunicella); Lophogorgia (= Leptogorgia) Schiaparelli et al. 2005;Lorenz and Fehse 2009 Furthermore, examination of the ovulid species and their octocoral hosts revealed that in two instances individuals formerly identified as C. chiapponii and C. aureola would have cooccurred on the same host coral, in both cases Acanthogorgia sp.

Discussion
Based on the molecular data and morphological observations listed above, C. chiapponii is considered a junior synonym of C. aureola. The systematic account is therefore as follows:

Molecular data
The species Primovula rosewateri was previously placed in the genus Crenavolva by Cate (1973) but Fehse (2002a) moved it to Primovula, primarily based on the trian gular shape of the funiculum. The results of the molecular analyses (Fig. 3) support this decision. There is great genetic similarity between C. cf. rosewateri (= Primovula cf. rosewateri) obtained from GenBank, and C. aureola. However, the specimen from GenBank was collected from Balicasag Island, near Bohol, Philippines, which is the type locality of C. chiapponii. This location is approximately 85 km from Mactan Is land of Cebu, Philippines which is the type locality of C. aureola. It is not unlikely that the socalled C. cf. rosewateri from GenBank (AY161394 (16S), AY161627 (COI)) was misidentified and actually represents C. aureola. Moreover, the newly sequenced specimen of P. rosewateri from Malaysia convincingly clusters with Primovula beckeri. According to Lorenz and Fehse 2009, P. beckeri has an E African distribution and was originally described from South Africa. The specimen obtained from GenBank is from Sulawesi, Indonesia (Schiaparelli et al. 2005). It is therefore unlikely that this sequence represents P. beckeri but instead is the quite similar species from the central IndoPacific, P. rosewateri.

Host species and distribution records
The ranges of the presently discussed species all fit within the Coral Triangle (see Hoeksema 2007) and depend on the ranges of their host species. Species of the genus Acanthogorgia are not unique hosts for just Crenavolva spp. Reijnen (2010) already mentioned Acanthogorgia spp. as a host for Dentiovula eizoi Cate & Azuma, 1973(in Cate 1973 and D. colobica (Azuma & Cate, 1971). Acanthogorgia species and their ovulid associates are both known to occur from shallow to deep water in the Coral Triangle. In an overview of the Acanthogorgiidae by Stiasny (1947) the deep est record for an Acanthogorgia species is 4239 m, collected SE of Seram, Indonesia (Acalycigorgia densiflora = Acanthogorgia densiflora (Kükenthal & Gorzawsky, 1908). Nevertheless, Stiasny (1947) doubts the identification and compared it to congeneric species which are found in waters not exceeding 400 m depth. As a result Stiasny (1947) doubts the entire record. Therefore the deepest reliable record for an Acanthogorgia species in the Malayan Archipelago is 1254 m for Acanthogorgia multispina (Kükenthal & Gorzawsky, 1908). The deepest record for Crenavolva species is from approximately 1000 m, which is the deepest record for any ovulid species found to date (Lorenz and Fehse 2009). and Department of Fisheries Sabah. The MV Celebes Explorer accommodated the