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Research Article
Phylogenetic relationships within the Phyllidiidae (Opisthobranchia, Nudibranchia)
expand article infoBart E.M.W. Stoffels, Sancia E.T. van der Meij§, Bert W. Hoeksema|, Joris van Alphen|, Theo van Alen, María Angélica Meyers-Muñoz, Nicole J. De Voogd|, Yosephine Tuti#, Gerard van der Velde
‡ Radboud University Nijmegen (Nijmegen) and Naturalis Biodiversity Center, Leiden, Netherlands
§ Naturalis Biodiversity Center (Leiden, Netherlands) and Oxford University Museum of Natural History, Oxford, United Kingdom
| Naturalis Biodiversity Center, Leiden, Netherlands
¶ Radboud University Nijmegen, Nijmegen, Netherlands
# Research Centre for Oceanography, Indonesian Institute of Sciences, Jakarta, Indonesia
Open Access

Abstract

The Phyllidiidae (Gastropoda, Heterobranchia, Nudibranchia) is a family of colourful nudibranchs found on Indo-Pacific coral reefs. Despite the abundant and widespread occurrence of many species, their phylogenetic relationships are not well known. The present study is the first contribution to fill the gap in our knowledge on their phylogeny by combining morphological and molecular data. For that purpose 99 specimens belonging to 16 species were collected at two localities in Indonesia. They were photographed and used to make a phylogeny reconstruction based on newly obtained cytochrome oxidase subunit (COI) sequences as well as sequence data from GenBank. All mitochondrial 16S sequence data available from GenBank were used in a separate phylogeny reconstruction to obtain information for species we did not collect. COI data allowed the distinction of the genera and species, whereas the 16S data gave a mixed result with respect to the genera Phyllidia and Phyllidiella. Specimens which could be ascribed to species level based on their external morphology and colour patterns showed low variation in COI sequences, but there were two exceptions: three specimens identified as Phyllidia cf. babai represent two to three different species, while Phyllidiella pustulosa showed highly supported subclades. The barcoding marker COI also confirms that the species boundaries in morphologically highly variable species such as Phyllidia elegans, P. varicosa, and Phyllidiopsis krempfi, are correct as presently understood. In the COI as well as the 16S cladogram Phyllidiopsis cardinalis was located separately from all other Phyllidiidae, whereas Phyllidiopsis fissuratus was positioned alone from the Phyllidiella species by COI data only. Future studies on phyllidiid systematics should continue to combine morphological information with DNA sequences to obtain a clearer insight in their phylogeny.

Keywords

COI, Indonesia, mtDNA, nudibranch, phylogenetic relations, 16S

Introduction

Nudibranch gastropod molluscs have traditionally been classified with the Infraclass Opisthobranchia Milne Edwards, 1848, which consists of more than 6000 species (Yonow 2008). Although this taxon is not monophyletic and therefore is considered obsolete (Schrödl et al. 2011), taxonomic works still refer to “opisthobranchs” for practical reasons (e.g. Uribe et al. 2013) and Opisthobranchia is considered an “Informal Group” among the Heterobranchia (Wägele et al. 2014). These animals form, ecologically and morphologically, one of the most diverse groups of marine gastropods (Wägele et al. 2014). To avoid use of their misnomer, this well-known group of marine animals can also be referred to as sea slugs (Yonow 2015). Among these, the Nudibranchia Cuvier, 1817 form the largest order with an estimated number of more than 2000 species (Gosliner et al. 2008), although also estimates of nearly 3000 species are known (Vonnemann et al. 2005).

Much work has already been done to elucidate the phylogeny of the opisthobranchs by molecular analyses (e.g., Wollscheid and Wägele 1999, Grande et al. 2004a, 2004b, Vonnemann et al. 2005, Turner and Wilson 2008, Maeda et al. 2010, Pola and Gosliner 2010), but most of the phylogenetic relationships still remain unclear at family, genus, and species level, especially with regards to the nudibranchs. All nudibranch species and many other sea slugs are predators, which usually can be observed together with their prey (Behrens 2005, Pola and Gosliner 2010, van Alphen et al. 2011). Only rarely they are found together with potential predators such as sea anemones, mushroom corals, and pycnogonids (Piel 1991, Behrens 2005, van der Meij and Reijnen 2012, Mehrotra et al. 2015).

The present study aims to clarify the phylogenetic relationships within the Phyllidiidae Rafinesque, 1814, belonging to the Doridacea (Bouchet and Rocroi 2005). This family consists of more than 100 species divided over five genera: Ceratophyllidia Eliot, 1903, Phyllidia Cuvier, 1797, Phyllidiella Bergh, 1869, Phyllidiopsis Bergh, 1875, and Reticulidia Brunckhorst, 1990 (Bouchet 2015). The genera Fryeria JE Gray, 1853, and Reyfria Yonow, 1986, have been synonymised with Phyllidia (Valdés and Gosliner 1999).

Most nudibranchs of the family Phyllidiidae are commonly encountered on coral reefs, where they can easily be noticed because of their aposomatic colouration, which serves to deter possible predators from eating them (Ritson-Williams and Paul 2007). Nevertheless, only eight phyllidiid COI sequences can be found in GenBank, as well as two 18S sequences and 17 16S sequences. There are only a few published studies that incorporate even a single member of Phyllidiidae into a phylogenetic tree (e.g. Wollscheid-Lengeling et al. 2001) and even fewer deal with phylogenetic relationships among Phyllidiidae. Among the latter, most are using anatomical characters (Brunckhorst 1993, Valdés and Gosliner 1999, Valdés 2001, 2002) and only two are known to include a molecular and phylogenetic analysis (Valdés 2003, Cheney et al. 2014).

Phyllidiid slugs are characterized by their oval elongate and tough bodies, which generally possess hard notal tubercles on the dorsal side. Although their colouration is a main character used for their identification, many species cannot be identified based on colouration alone owing to their high intra-specific colour variation. Structure and pattern of the notal tubercles are important characters for identification. Other distinctive features of the Phyllidiidae are the retractile lamellate rhinophores, the compact digestive gland mass, and the triaulic reproductive system (Brunckhorst 1993). Another important character diagnosing the Phyllidiidae is the possession of numerous subdermal calcareous spicules of different microstructures (Chang et al. 2013). The Phyllidiidae have no jaws or radula and lack the dorsal, circumanal circlet of gills that is typical of other dorids (Brunckhorst 1993).

To study the phylogenetic relationships within the Phyllidiidae, a molecular analysis was performed based on DNA sequence data of the mitochondrial cytochrome oxidase I (COI) gene, combined with external morphological assessments of material collected in two areas in eastern Indonesia, the Raja Ampat islands (West Papua) and Ternate, off western Halmahera (Moluccas). Both locations are situated in the centre of maximum marine biodiversity, also known as the Coral Triangle (Hoeksema 2007). In earlier studies, high numbers of phyllidiid species were recorded from this area: 13 from the Bismarck Sea, Papua New Guinea (Domínguez et al. 2007), eleven from Ambon (Moluccas, Indonesia) (Yonow 2011), and eleven from the South China Sea (Sachidhanandam et al. 2000). Therefore, both of our areas were expected to show a high number of phyllidiid species that could be used for the present study.

Materials and methods

Sampling

Specimens were collected by SCUBA diving in West Papua by Gerard van der Velde in 2007, mostly in the coastal areas of Gam, Kri, Mansuar, and Batanta (Figures 12; see Hoeksema and van der Meij 2008). Additional specimens were mainly collected by Joris van Alphen and Nicole de Voogd, and also by Bert Hoeksema, Sancia van der Meij, and other expedition members (Hoeksema and van der Meij 2010) in 2009 off Halmahera (northern Moluccas), especially around Ternate (Figures 1, 3). A locality list of the sampling stations is provided in Table 1. Collected slugs were first photographed and subsequently preserved in 96% ethanol (West Papua 2007). Halmahera specimens were transferred into fresh 96% ethanol and labelled in order to prepare them for DNA analysis. These have been deposited in the mollusc collection of Naturalis Biodiversity Center, Leiden (coded as RMNH.Mol.), with the exception of some specimens that dried out after sequencing (Table 1; Figures 515; Suppl. material 1: COI sequences).

Figure 1.

Location of field areas: Halmahera (including Ternate) and West Papua (including Raja Ampat).

Figure 2.

Raja Ampat sites where Phyllidiidae were sampled in 2007.

Figure 3.

Halmahera and Ternate sites where Phyllidiidae were sampled in 2009.

Information on analysed Phyllidiidae species: RMNH.MOL catalogue number or field code number in case voucher specimen became lost; Genbank number if available; collection site, station number (RAJ = Raja Ampat, TER = Ternate, Halmahera), coordinates.

RMNH.MOL or Field nr. Genbank accession number Species Locality Station Coordinates
336464 KX235918 Phyllidia babai Tanjung Ebamadu TER08 N0°45'23.4", E127°24'26.5"
336575 KX235920 Phyllidia cf. babai South Gam, shoal near mangroves RAJ37 S0°31'08.2", E130°38'28.0"
336614 KX235919 Phyllidia cf. babai Tanjung Ratemu (South of river) TER27 N0°54'44.5", E127°29'09.9"
336573 KX235921 Phyllidia coelestis Eastern entrance of passage RAJ44 S0°25'44.3", E130°33'56.8"
336574 KX235922 Phyllidia coelestis Wallace Lake RAJ13 S0°26'31.1", E130°41'08.0"
58 Phyllidia elegans Pulau Maka TER13 N0°54'42.7", E127°18'32.9"
137 Phyllidia elegans Pulau Pilongga, North TER34 N0°42'49.8", E127°28'45.4"
156 Phyllidia elegans Teluk Dodinga; Karang Ngeli West TER40 N0°46'25.3", E127°32'22.0"
336475 KX073972 Phyllidia elegans Tanjung Tabam TER12 N0°50'05.1", E127°23'10.0"
336478 KX073973 Phyllidia elegans Pulau Maka TER13 N0°54'42.7", E127°18'32.9"
336488 KX073974 Phyllidia elegans Tanjung Pasir Putih TER16 N0°51'50.4", E127°20'36.7"
336514 KX073975 Phyllidia elegans Dufadufa / Benteng Toloko TER24 N0°48'49.1", E127°23'21.6"
336515 KX073976 Phyllidia elegans Idem TER24 N0°48'49.1", E127°23'21.6"
336554 KX073985 Phyllidia elegans Passage RAJ43 S0°25'45.2", E130°33'37.3"
336555 KX073990 Phyllidia elegans Akber Reef RAJ14 S0°34'15.2", E130°39'33.7"
336556 KX073988 Phyllidia elegans Passage RAJ43 S0°25'45.2", E130°33'37.3"
336557 KX073987 Phyllidia elegans Idem RAJ43 S0°25'45.2", E130°33'37.3"
336558 KX073984 Phyllidia elegans Southwest Pulau Kri RAJ40 S0°33'58.1", E130°39'46.2"
336559 KX073991 Phyllidia elegans South Gam, shoal near mangroves RAJ37 S0°31'08.2", E130°38'28.0"
336560 KX073983 Phyllidia elegans Southwest Pulau Kri RAJ40 S0°33'58.1", E130°39'46.2"
336561 KX073986 Phyllidia elegans Passage RAJ43 S0°25'45.2", E130°33'37.3"
336562 KX073989 Phyllidia elegans Akber Reef RAJ14 S0°34'15.2", E130°39'33.7"
336628 KX073977 Phyllidia elegans Pulau Gura Ici, East TER32 S0°01'17.3", E127°14'17.2"
336629 KX073978 Phyllidia elegans Idem TER32 S0°01'17.3", E127°14'17.2"
336631 KX073979 Phyllidia elegans Pulau Pilongga, North TER34 N0°42'49.8", E127°28'45.4"
336632 KX073980 Phyllidia elegans Idem TER34 N0°42'49.8", E127°28'45.4"
336633 KX073981 Phyllidia elegans Idem TER34 N0°42'49.8", E127°28'45.4"
336649 KX073982 Phyllidia elegans Teluk Dodinga; Karang Ngeli West TER40 N0°46'25.3", E127°32'22.0"
336484 KX235923 Phyllidia exquisita Tanjung Ngafauda TER14 N0°54'38.3", E127°29'20.7"
336494 KX235924 Phyllidia ocellata Southwest of Tobala TER19 N0°44'56.6", E127°23'13.5"
336563 KX235926 Phyllidia ocellata Southeast Gam, Friwen Wonda RAJ11 S0°28'29.9", E130°41'54.8"
336564 KX235925 Phyllidia ocellata Idem RAJ11 S0°28'29.9", E130°41'54.8"
336565 KX235927 Phyllidia picta South Gam, Shoal near mangroves RAJ37 S0°31'08.2", E130°38'28.0"
336566 KX235929 Phyllidia picta Passage RAJ43 S0°25'45.2", E130°33'37.3"
336567 KX235928 Phyllidia picta North Batanta, West Telok Gegenlol RAJ29 S0°49'42.5", E130°42'42.0"
336619 KX235930 Phyllidia sp. Pulau Popaco, East TER28 S0°01'51.9", E127°14'01.8"
74 Phyllidia varicosa Tanjung Pasir Putih TER16 N0°51'50.4", E127°20'36.7"
336489 KX235931 Phyllidia varicosa Idem TER16 N0°51'50.4", E127°20'36.7"
336568 KX235942 Phyllidia varicosa Northeast Pulau Mansuar RAJ38 S0°34'05.0", E130°38'31.5"
336569 KX235941 Phyllidia varicosa Idem RAJ38 S0°34'05.0", E130°38'31.5"
336570 KX235943 Phyllidia varicosa North Batanta, West Telok Gegenlol RAJ29 S0°49'42.5", E130°42'42.0"
336571 KX235938 Phyllidia varicosa South Gam, Eastern entrance Besir Bay, Cape Besir RAJ25 S0°30'51.5", E130°34'11.5"
336572 KX235940 Phyllidia varicosa Idem RAJ25 S0°30'51.5", E130°34'11.5"
336604 KX235932 Phyllidia varicosa East side Ternate Harbour (outside) TER25 N0°46'55.3", E127°23'19.9"
336609 KX235933 Phyllidia varicosa Pasir Lamo (West side) TER26 N0°53'20.5", E127°27'34.2"
336612 KX235934 Phyllidia varicosa Idem TER26 N0°53'20.5", E127°27'34.2"
336617 KX235935 Phyllidia varicosa Tanjung Ratemu (South of river) TER27 N0°54'44.5", E127°29'09.9"
336621 KX235936 Phyllidia varicosa Pulau Popaco E TER28 S0°01'51.9", E127°14'01.8"
336637 KX235937 Phyllidia varicosa Teluk Dodinga East; North of Pulau Jere TER36 N0°50'47.8", E127°37'48.7"
336647 KX235939 Phyllidia varicosa Teluk Dodinga, Karang Galiasa Kecil West TER39 N0°51'09.1", E127°35'19.5"
336590 KX235944 Phyllidiopsis fissuratus Yenweres Bay RAJ46 S0°29'13.0", E130°40'23.6"
336589 KX235945 Phyllidiella rudmani Southeast Gam, Friwen Wonda RAJ11 S0°28'29.9", E130°41'54.8"
336434 KX235946 Phyllidiella nigra Off Danau Laguna TER02 N0°45'29.7", E127°20'59.2"
336471 KX235947 Phyllidiella nigra Maitara Northwest TER10 N0°44'32.0", E127°21'50.9"
336472 KX235948 Phyllidiella nigra Idem TER10 N0°44'32.0", E127°21'50.9"
336501 KX235949 Phyllidiella nigra Sulamadaha I TER22 N0°52'03.6", E127°19'33.1"
336505 KX235950 Phyllidiella nigra Sulamadaha II TER23 N0°52'02.0", E127°19'45.8"
336576 KX235952 Phyllidiella nigra South Gam, Eastern entrance Besir Bay, Pulau Bun RAJ26 S0°30'59.3", E130°33'48.7"
336577 KX235951 Phyllidiella nigra South Gam, Southeast Besir Bay RAJ32 S0°30'45.2", E130°35'00.1"
75F Phyllidiella pustulosa North Batanta, West Telok Gegenlol RAJ29 S0°49'42.5", E130°42'42.0"
336436 KX235953 Phyllidiella pustulosa Off Danau Laguna TER02 N0°45'29.7", E127°20'59.2"
336460 KX235954 Phyllidiella pustulosa Desa Tahua TER07 N0°45'09.1", E127°23'31.3"
336461 KX235955 Phyllidiella pustulosa Idem TER07 N0°45'09.1", E127°23'31.3"
336470 KX235956 Phyllidiella pustulosa Northwest side of Maitara TER10 N0°44'32.0", E127°21'50.9"
336474 KX235957 Phyllidiella pustulosa Tanjung Tabam TER12 N0°50'05.1", E127°23'10.0"
336495 KX235958 Phyllidiella pustulosa Tanjung Ratemu (South of river) TER21 N0°54'24.7", E127°29'17.7"
336508 KX235959 Phyllidiella pustulosa Dufadufa / Benteng Toloko TER24 N0°48'49.1", E127°23'21.6"
336510 KX235960 Phyllidiella pustulosa Idem TER24 N0°48'49.1", E127°23'21.6"
336578 KX235965 Phyllidiella pustulosa South Gam, Southeast Besir Bay RAJ32 S0°30'45.2", E130°35'00.1"
336579 KX235971 Phyllidiella pustulosa South Gam, Besir Bay RAJ35 S0°48'58.3", E130°59'16.6"
336580 KX235967 Phyllidiella pustulosa Southwest Pulau Kri RAJ40 S0°33'58.1", E130°39'46.2"
336581 KX235963 Phyllidiella pustulosa South Gam, Besir Bay RAJ35 S0°48'58.3", E130°59'16.6"
336582 KX235968 Phyllidiella pustulosa Southwest Pulau Kri RAJ40 S0°33'58.1", E130°39'46.2"
336583 KX235964 Phyllidiella pustulosa South Gam, East entrance Besir Bay, Cape Besir RAJ25 S0°30'51.5", E130°34'11.5"
336584 KX235961 Phyllidiella pustulosa West Pulau Yeben Kecil RAJ48 S0°29'20.6", E130°30'04.9"
336585 KX235969 Phyllidiella pustulosa Southeast Gam, Desa Besir RAJ41 S0°27'48.1", E130°41'14.6"
336586 KX235966 Phyllidiella pustulosa Idem RAJ41 S0°27'48.1", E130°41'14.6"
336587 KX235962 Phyllidiella pustulosa South Gam, Eastern entrance Besir Bay, Cape Besir RAJ25 S0°30'51.5", E130°34'11.5"
336588 KX235970 Phyllidiella pustulosa West Pulau Yeben Kecil RAJ48 S0°29'20.6", E130°30'04.9"
336453 KX235972 Phyllidiopsis krempfi Kampung Cina / Tapak 2 TER06 N0°47'15.0", E127°23'25.0"
336462 KX235973 Phyllidiopsis krempfi Tanjung Ebamadu TER08 N0°45'23.4", E127°24'26.5"
336466 KX235974 Phyllidiopsis krempfi Idem TER08 N0°45'23.4", E127°24'26.5"
336469 KX235975 Phyllidiopsis krempfi West Maitara TER09 N0°43'47.6", E127°21'44.7"
336512 KX235976 Phyllidiopsis krempfi Dufadufa / Benteng Toloko TER24 N0°48'49.1", E127°23'21.6"
336594 KX235979 Phyllidiopsis krempfi Southwest Pulau Kri, Kuburan RAJ15 S0°33'42.8", E130°39'40.4"
336595 KX235984 Phyllidiopsis krempfi Southwest Pulau Kri RAJ40 S0°33'58.1", E130°39'46.2"
336596 KX235983 Phyllidiopsis krempfi Northwest Pulau Mansuar, Lalosi reef RAJ49 S0°32'53.5", E130°29'51.1"
336597 KX235978 Phyllidiopsis krempfi Southwest Pulau Kri, Kuburan RAJ15 S0°33'42.8", E130°39'40.4"
336598 KX235980 Phyllidiopsis krempfi North Batanta, North Pulau Yarifi RAJ28 S0°46'46.7", E130°42'42.7"
336599 KX235982 Phyllidiopsis krempfi East Kri, Sorido Wall RAJ12 S0°33'13.2", E130°41'16.9"
336600 KX235981 Phyllidiopsis krempfi Northeast Mansuar RAJ38 S0°34'05.0", E130°38'31.5"
336650 KX235977 Phyllidiopsis krempfi Teluk Dodinga; West Karang Ngeli TER40 N0°46'25.3", E127°32'22.0"
336451 KX235985 Phyllidiopsis shireenae Kampung Cina / Tapak 2 TER06 N0°47'15.0", E127°23'25.0"
336652 KX235986 Phyllidiopsis shireenae Teluk Dodinga; East Karang Luelue TER41 N0°46'32.8", E127°33'43.4"
336591 KX235987 Phyllidiopsis xishaensis Southeast Gam, Pulau Kerupiar, Mike’s Point RAJ05 S0°30'57.1", E130°40'22.1"
336592 KX235988 Phyllidiopsis xishaensis East Pulau Kri, Cape Kri RAJ07 S0°33'22.2", E130°41'28.7"
336593 KX235989 Phyllidiopsis xishaensis Eastern entrance of passage RAJ44 S0°25'44.3", E130°33'56.8"
336640 KX235990 Reticulidia fungia East Teluk Dodinga; North of Pulau Jere TER36 N0°50'47.8", E127°37'48.7"
336455 KX235991 Reticulidia halgerda Kampung Cina / Tapak 2 TER06 N0°47'15.0", E127°23'25.0"

Morphological study

Collected specimens were identified according to their external morphology using Brunckhorst (1993), Yonow et al. (2002), and Yonow (2011). In addition, field guides showing in situ photographs were used (Gosliner et al. 2008). All individuals except for three could be identified to species level. All specimens were photographed alive or in the preserved state (Figures 515); these photos can be linked to the phylogeny reconstruction of the Phyllidiidae based on COI gene sequence data (Figure 4).

Figure 4.

Phylogeny reconstruction of the Phyllidiidae based on COI gene sequence data of 109 specimens (including outgroups). Topology derived from Bayesian inference 50% majority rule, significance values are posterior probabilities / bootstrap values. Numbers refer to GenBank accession numbers / RMNH.Moll catalogue numbers.

Figure 5.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidia elegans. Order of specimens (a–h) according to Figure 4 (f, h dorsal and ventral sides). Numbers refer to RMNH. Moll catalogue numbers.

Figure 6.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidia elegans. Order of specimens (a–i) according to Figure 4 (d dorsal and ventral sides). Numbers refer to RMNH.Moll catalogue numbers and locality codes (137 and 156, dried-out).

Figure 7.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidia elegans (a–f), Phyllidia sp. (g dorsal and ventral sides), P. exquisita (h), P. coelestis (i). Order of specimens (a–i) according to Figure 4. Numbers refer to RMNH.Moll catalogue numbers or locality code (058, dried-out).

Figure 8.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidia coelestis (a), P. varicosa (b–i). Order of specimens (a–i) according to Figure 4 (d dorsal and ventral sides). Numbers refer to RMNH.Moll catalogue numbers.

Figure 9.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidia varicosa (a–f), P. ocellata (g–i). Order of specimens (a–i) according to Figure 4 (c dorsal and ventral sides). Numbers refer to RMNH.Moll catalogue numbers or locality code (074, dried-out).

Figure 10.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidia picta (a–c), Phyllidia babai (d), Phyllidia cf. babai (e–f), Reticulidia fungia (g), Reticulidia halgerda (h), Phyllidiopsis fissuratus (i). Order of specimens (a–i) according to Figure 4 (e dorsal and ventral sides). Numbers refer to RMNH.Moll catalogue numbers.

Figure 11.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidiella rudmani (a), Phyllidiella nigra (b–h), Phyllidiella pustulosa (i–j). Order of specimens (a–j) according to Figure 4. Numbers refer to RMNH.Moll catalogue numbers.

Figure 12.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidiella pustulosa. Order of specimens (a–j) according to Figure 4. Numbers refer to RMNH.Moll catalogue numbers or locality code (75F, dried-out).

Figure 13.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidiella pustulosa (a–h), Phyllidiopsis xishaensis (i–j). Order of specimens (a–j) according to Figure 4. Numbers refer to RMNH.Moll catalogue numbers.

Figure 14.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidiopsis xishaensis (a), Phyllidiopsis shireenae (b–c), Phyllidiopsis krempfi (d–i). Order of specimens (a–i) according to Figure 4 (c dorsal and ventral sides). Numbers refer to RMNH.Moll catalogue numbers.

Figure 15.

External morphology and colouration of Phyllidiidae specimens used for COI phylogeny reconstruction: Phyllidiopsis krempfi. Order of specimens (a–g) according to Figure 4 (f, g dorsal and ventral sides). Numbers refer to RMNH.Moll catalogue numbers.

DNA extraction

For each species encountered in the field surveys one or more individuals were chosen for DNA analysis as well as from the morphologically distinct unidentified specimens, resulting in a total of 99 samples (Table 1). DNA was extracted from tissue of small foot fragments with the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s protocol. DNA was eluted in DEPC treated water. The quality of the extracted DNA was tested by agarose gel (0.7%) electrophoresis.

PCR amplification, purification, and sequencing

Extracted DNA was used for Polymerase Chain Reaction (PCR) to amplify fragments of the mitochondrial gene COI (cytochrome c oxidase subunit 1). The primers used for the amplification of the COI gene were: LCO1490 (5’GGT CAA CAA ATC ATA AAG ATA TTG G 3’) and HCO2198 (5’TAA ACT TCA GGG TGA CCA AAA AAT CA 3’) (Folmer et al. 1994). Thermal cycling conditions used for the amplification of the COI gene were: initial denaturing at 94 °C for 3 min followed by 38 amplification cycles of denaturation at 94 °C for 15 sec, primer annealing at 50 °C for 30 sec, and elongation at 72 °C for 1 min. A final elongation step at 72 °C for 5 min was performed. After checking by agarose (1%) electrophoresis if the PCR resulted the unique PCR fragments of the expected size (approximately 658 bp), the fragments were purified using the GeneJET PCR Purification Kit (Thermo Scientific, Landsmeer, NL). Purified PCR products were sequenced with corresponding primers.

Sequence alignment and phylogenetic analyses

The quality of the sequences was checked using Chromas Lite (Technelysium Pty Ltd.). Subsequently the sequences were edited in MEGA 6 (Tamura et al. 2013) and analysed by BLAST searches (http://www.ncbi.nlm.nih.gov). COI sequences of Dendrodoris citrina (Cheeseman, 1881) and Doriopsilla areolata Bergh, 1880 were collected from GenBank and used as outgroups. Additional COI sequences of Phyllidia coelestis Bergh, 1905, Phyllidia elegans Bergh, 1869, Phyllidia ocellata Cuvier, 1804, Phyllidia picta Pruvot-Fol, 1957, Phyllidia varicosa Lamarck, 1801, Phyllidiella lizae Brunckhorst, 1993, Phyllidiella pustulosa (Cuvier, 1804), Phyllidiopsis cardinalis Bergh, 1875 were obtained from GenBank (Table 2).

Mitochondrial COI sequences of Phyllidiidae (and outgroups) obtained from GenBank.

Species Accession number Reference Collection locality
Dendrodoris citrina GQ292043 Shields et al. (2009 unpubl.) Ross Sea, Antarctica?
Doriopsilla areolata AJ223262 Thollesson (2000) Cadiz, Andalusia, Spain
Phyllidia coelestis KJ001305 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidia elegans AJ223276 Thollesson (2000) Tab I., Papua New Guinea
Phyllidia ocellata KJ001307 Cheney et al. (2014) Mooloolaba, Queensland, Australia
Phyllidia picta KJ001304 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidia varicosa KJ001306 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidiella lizae KJ001309 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidiella pustulosa KJ001310 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidiopsis cardinalis KJ001308 Cheney et al. (2014) Mooloolaba, Queensland, Australia

The newly obtained COI sequences and the sequences from GenBank were aligned using the Guidance server (Clustal W; Penn et al. 2010), resulting in an alignment score of 1.000. There were no unreliable columns. Prior to the model-based phylogenetic analysis, the best-fit model of nucleotide substitution was identified by means of the Akaike Information Criterion (AIC) calculated with jModeltest (Posada 2008), resulting in TVM+I+G as the most suitable model. Phylogenetic reconstructions were carried out with Bayesian inference in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the most complex GTR+I+G model of nucleotide substitution. Bayesian inference coupled with Markov Chain Monte Carlo techniques (MCMC; six chains) were run for 5,000,000 generations with a sample tree saved every 1000 generations. The burnin was set to 25%. Likelihood scores stabilized at 0.007476. Consensus trees were visualized in FigTree v.1.3.1 (Rambaut 2009). A maximum likelihood analysis (GTR+I+G; 1000 bootstraps) was carried out with Phyml 3.1 (Guindon et al. 2010) using the Seaview platform (Gouy et al. 2010).

Initial phylogenetic analyses showed high intraspecific variation on the COI region between specimens identified as Phyllidiella pustulosa. Tests to estimate the average evolutionary divergence over sequence pairs between and within groups were carried out in MEGA 6.06. Phyllidia elegans, P. varicosa, Phyllidiella nigra (van Hasselt, 1824), P. pustulosa, and Phyllidiopsis krempfi Pruvot-Fol, 1957 were used as representatives for each of the species groups, because of the larger number of available sequences for these species. The Phyllidiella pustulosa sequence from GenBank (KJ001310) was excluded from this analysis: based on its position in the phylogeny reconstruction the identification of this specimen as P. pustulosa is doubtful. The web version of ABGD (Automatic Barcode Gap Discovery, Puillandre et al. 2012) was used to estimate the genetic distance corresponding to the difference between a speciation process versus intra-specific variation in Phyllidiella pustulosa. Runs were performed using the default range of priors (pmin = 0.001, pmax = 0.10) using the JC69 Jukes-Cantor measure of distance. The analysis involved 20 nucleotide sequences with a total of 588 positions in the final dataset.

All available mitochondrial 16S sequences of Phyllidiidae on GenBank (Tholesson 2000, Wolfscheid-Lengeling et al. 2001, Valdés 2003, Cheney et al. 2014, Shields et al. unpublished) were used for a phylogeny reconstruction based on this marker, which allowed us to study the phylogenetic position of 17 phyllidiid species including two species (Phyllidia rueppelii (Bergh, 1869) and Phyllidiopsis sphingis Brunckhorst, 1993) for which no COI data were available. Doriopsilla albopunctata (JG Cooper, 1863) was used as outgroup (Table 3). The sequences were aligned using the Guidance server (ClustalW; Penn et al. 2010), resulting in an alignment score of 0.996281. All unreliable columns (confidence score below 0.93) were removed. Prior to the model-based phylogenetic analysis, the best-fit model of nucleotide substitution was identified by means of the Akaike Information Criterion (AIC) calculated with jModeltest (Posada 2008), resulting in TVM+I+G. Because of the unavailability of TVM in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), we used the most complex GTR+I+G model of nucleotide substitution. Bayesian inferences coupled with MCMC techniques (six chains) were run for 3,000,000 generations, with a sample tree saved every 1000 generations and the burnin set to 25%. Likelihood scores stabilized at a value of 0.005654. Consensus trees were visualized in FigTree v.1.3.1 (Rambaut 2009). A maximum likelihood analysis (GTR+I+G; 1000 bootstraps) was carried out with Phyml 3.1 (Guindon et al. 2010) using the Seaview platform (Gouy et al. 2010).

16S sequences of Phyllidiidae obtained from GenBank.

Species Accession number Reference Collection locality
Doropsilla albopunctata AF430354 Valdés (2003) Baja California, Mexico
Phyllidia coelestis AF430361 Valdés (2003) Lifou I., New Caledonia
Phyllidia coelestis KJ018917 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidia elegans AF430362 Valdés (2003) Lifou I., New Caledonia
Phyllidia elegans AJ225201 Thollesson (2000) Tab I., Papua New Guinea
Phyllidia ocellata AF430363 Valdés (2003) Lifou I., New Caledonia
Phyllidia picta KJ018916 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidia rueppelii AF430358 Valdés (2003) Hurghada, Egypt
Phyllidiella lizae AF430365 Valdés (2003) Lifou I., New Caledonia
Phyllidiella lizae KJ018918 Cheney et al. (2014) Lizard I., Queensland Australia
Phyllidiella pustulosa AF249232 Wollscheid-Lengeling et al. (2001) Great Barrier Reef, Australia
Phyllidiella pustulosa AF430366 Valdés (2003) Lifou I., New Caledonia
Phyllidia varicosa AF430364 Valdés (2003) Lifou I., New Caledonia
Phyllidiopsis cardinalis AF430367 Valdés (2003) Lifou I., New Caledonia
Phyllidiopsis sphingis AF430368 Valdés (2003) Lifou I., New Caledonia
Phyllidiopsis xishaensis* AF430369 Valdés (2003) Lifou I., New Caledonia
Reticulidia fungia AF430370 Valdés (2003) Lifou I., New Caledonia
Reticulidia halgerda AF430371 Valdés (2003) Lifou I., New Caledonia

Results and discussion

Position of genera

The reconstruction based on COI (Figure 4) is derived from the Bayesian inference 50% majority rule consensus. This topology is congruent with the one resulting from the maximum likelihood analysis. Three large groupings can be discerned (indicated as A, B, and C in Figure 4), albeit with low support for the higher taxonomic levels. The support values in the distal branches are high. The genera Phyllidia, Phyllidiella, Phyllidiopsis, and Reticulidia are retrieved in distinct clades, with Reticulidia as a sister clade to Phyllidia. Phyllidiopsis fissuratus Brunckhorst, 1993 formed a separate lineage basal to Phyllidiella species (albeit without support). Phyllidiopsis cardinalis does not cluster with its congeners, but instead forms a separate lineage in the Phyllidiidae.

The 16S phylogeny reconstruction is also derived from the Bayesian inference 50% majority rule consensus of the trees remaining after the burnin. There are low support values in the basal part of the tree and high support values in the distal phylogenetic branches (Figure 17). The Bayesian inference topology is congruent with the topology resulting from the maximum likelihood analysis. The outgroup Doriopsilla albopunctata is separated by a long branch. Within the overall clade four main groupings can be distinguished: Phyllidiella, Phyllidiopsis, and Reticulidia, and a mixed clade of Phyllidiella and Phyllidia. Based on this analysis only the genus Reticulidia is monophyletic. Phyllidiopsis cardinalis does not cluster with any of the other analysed taxa, and holds a separate position in the phylogeny reconstruction. The latter is in accordance with the COI reconstruction (Figure 4).

The arrangement of the four phyllidiid genera based on the molecular data (Figures 4, 16a) is similar to that of Brunckhorst (1993) that was based on morphological and anatomical data (Figure 16b). The only exception is the position of the genus Fryeria. Brunckhorst (1993) distinguished Fryeria from Phyllidia based on the position of the anus and other anatomical features. Phyllidia picta (with its synonyms Fryeria picta (Pruvot-Fol, 1957), Fryeria menindie Brunckhorst, 1993, Phyllidia menindie (Brunckhorst, 1993)) was included in our analyses which, according to Brunckhorst, should belong to the genus Fryeria. Valdés and Gosliner (1999) synonymized both genera, which was later followed by Valdés (2003) and Cheney et al. (2014). The present reconstruction based on COI (Figure 16a) reconfirms the inclusion of Fryeria in the genus Phyllidia.

Figure 16.

a Cladogram based on COI gene sequence data showing topology of four genera of Phyllidiidae b Cladogram according to Brunckhorst (1993) based on morphological data showing topology of six genera of Phyllidiidae c Cladogram based on 16S mtDNA sequence data showing topology of four genera of Phyllidiidae (Valdés 2003) d Cladogram based on morphological data (Valdés 2002) showing topology of five genera of Phyllidiidae.

Figure 17.

Phylogeny reconstruction of the Phyllidiidae based on 16S mtDNA of 17 specimens of 14 species (including outgroup). Topology derived from Bayesian inference 50% majority rule, significance values are posterior probabilities/bootstrap values. Numbers refer to GenBank accession numbers. *Re-identification according to Yonow (pers. comm.)

The cladogram of the genera based on 16S mtDNA sequence data collected by Valdés (2003) (Figure 16c) is roughly similar to the cladogram based on COI, except for the different positions of Phyllidiopsis and Phyllidiella. The cladogram based on morphological and anatomical data as shown by Valdés (2002; Figure 16d) is different from the other proposed classifications (Figures 16a–c). Brunckhorst (1993) considered Ceratophyllidia a sister group to all the other genera (Figure 6b). Valdés (2002; Figure 16d) distinguished two larger groupings within the Phyllidiidae; Ceratophyllidia and Phyllidiopsis as one group and Phyllidia, Phyllidiella, and Reticulidia as the other group. Phyllidia and Phyllidiella in turn formed a sister group of Reticulidia (Figure 16d). The cladogram by Brunckhorst (1993) and our cladogram based on COI (Figure 4) both show that Phyllidiella is a sister clade of Reticulidia and Phyllidia. In contrast, Phyllidiella is not a sister group of Phyllidia but to all the other genera grouped together in the cladogram of Valdés (2003).

Unfortunately no Ceratophyllidia specimens were available to complete our analysis at genus level. Up to this point the phylogenetic position of the genus Ceratophyllidia remains unclear, and additional molecular analyses are necessary to establish its position.

Species level analysis

Species level analysis was mainly based on COI (Figure 4). Four nominal species were sequenced in the genus Phyllidiella. Phyllidiella nigra formed a highly supported clade. In the clade containing P. pustulosa much variation is visible indicating larger genetic differences among individuals. The ABGD analysis shows that four Molecular Operational Taxonomic Units (MOTUs) are present in Phyllidiella pustulosa, suggesting the presence of cryptic species or, alternatively, high intraspecific variation. The P. pustulosa of Cheney et al. (2014) falls in between the group consisting of P. nigra and P. pustulosa on one side and P. rudmani Brunckhorst, 1993 on the other and probably represents another species. Our specimen of P. rudmani clustered with the specimen identified as P. lizae in Cheney et al. (2014). Phyllidiella rudmani and P. lizae resemble each other (Brunckhorst 1993) and hence it is possible that the species identified as P. lizae in Cheney et al. (2014) is in fact P. rudmani.

Specimens of seven nominal Phyllidia species were sequenced. Sequences of 25 individuals of Phyllidia elegans (including one from GenBank) formed a highly supported clade, just like the clades containing P. ocellata, P. picta, and P. varicosa. Phyllidia coelestis was also retrieved as a highly supported clade. An individual identified as P. picta by Cheney et al. (2014) was part of this group suggesting that it should probably be identified as P. coelestis. Brunckhorst (1993) already noticed the close similarity between the two species but still confused them (Yonow 1996), and hence identification errors are likely to occur. Individuals identified as Phyllidia babai Brunckhorst, 1993 and P. cf. babai were retrieved in two different clades. Specimens 336464 and 336614 differ in 75 base pairs, 336464 and 336575 by 68 base pairs and 336614 and 336575 by 32 base pairs. Differences based on COI suggest that they represent two, or possibly three, different species. The genus Reticulidia was retrieved as a sister group of Phyllidia.

Material of four nominal species in the genus Phyllidiopsis was sequenced, with additional data of one species from GenBank (P. cardinalis). Phyllidiopsis fissuratus clusters basal to Phyllidiella, without support. Phyllidiopsis shireenae Brunckhorst, 1990 and P. xishaensis (Lin, 1983) cluster as sister species, in highly supported clades. Phyllidiopsis krempfi also formed a clear group. Phyllidiopsis cardinalis does not cluster with any of the phyllidiid genera based on either the 16S or the COI analysis. This result suggests that P. cardinalis should be separated from the other Phyllidiopsis species, but further morphological analyses are needed to confirm this outcome. Brunckhorst (1993) noted that P. cardinalis is the type species of the genus Phyllidiopsis, and that it has a unique and complex coloration totally different from that of any other known phyllidiid species, as well as a different anatomy, especially in the foregut. Valdés (2003) states “Additionally, the genus Phyllidiopsis is not monophyletic when molecular characters are used, because Phyllidiopsis cardinalis is at the base of the Phyllidiidae clade, and not nested with the other members of Phyllidiopsis”. Surprisingly, in the analysis of Cheney et al. (2014), based on a concatenated dataset of 16S and COI mtDNA, P. cardinalis was retrieved in a highly supported clade with several species of Phyllidiella and Phyllidia.

Variation within Phyllidiella pustulosa

Phyllidiella pustulosa is the only species in the COI cladogram (Figure 4) in which highly supported subclades can be discerned. To estimate the average evolutionary divergence within Phyllidiella pustulosa the base differences were compared per site for all grouped sequences of the species Phyllidia elegans (n = 24), P. varicosa (n = 15), Phyllidiella nigra (n = 7), P. pustulosa (n = 20), and Phyllidiopsis krempfi (n = 13) (Tables 45).

Estimates of average evolutionary divergence (p-distance) over sequence pairs within groups, in percentages.

Species Distance (%)
Phyllidia elegans 0.7
Phyllidia varicosa 0.7
Phyllidiella nigra 0.6
Phyllidiella pustulosa 3.9
Phyllidiopsis krempfi 1.2

Estimates of average evolutionary divergence (p-distance) over sequence pairs between groups, in percentages.

Distance (%)
Species Phyllidia elegans Phyllidia varicosa Phyllidiella nigra Phyllidiella pustulosa Phyllidiopsis krempfi
Phyllidia elegans
Phyllidia varicosa 12.1
Phyllidiella nigra 15.8 15.5
Phyllidiella pustulosa 18.3 18.9 10.5
Phyllidiopsis krempfi 15.8 16.4 14.6 17.2

The genetic variation on the barcoding marker COI is much higher within Phyllidiella pustulosa (3.9%) than within the other four species, which showed genetic variations between 0.6 and 1.2% (Table 4). The interspecific genetic variation (involving three different genera) ranges between 10.5 and 18.9% (Table 5). The congeners Phyllidiella nigra and P. pustulosa differ by 10.5%, and the congeners Phyllidia elegans and P. varicosa differ by 12.1%. The observed levels of genetic variation within Phyllidiella pustulosa (Table 4) and between the five species (Table 5) call for additional studies on possible cryptic speciation in P. pustulosa.

Conclusions

The barcoding marker COI works well to separate the different species in the Phyllidiidae, and confirms that the species boundaries in highly variable species, such as Phyllidia elegans, P. varicosa, and Phyllidiopsis krempfi, are correct as presently understood. However, a multi-locus approach, preferably including nuclear markers, is needed to improve the resolution for the higher taxonomic levels. With the exception of a few species that are difficult to place (Phyllidiopsis fissuratus, Phyllidiopsis cardinalis) the studied genera (Phyllidia, Phyllidiella, Phyllidiopsis, and Reticulidia) were retrieved as separate genera within the family. Additional representatives of Ceratophyllidia are needed to indicate the position of this genus within the Phyllidiidae. The observed groupings within Phyllidiella pustulosa suggest that multiple (cryptic) species could be present in this species, for which further analyses are needed including morphological data and multiple markers. Chang and Willan (2015) indicated that at least nine clades could be recognized in Phyllidiella pustulosa that could be separated slightly according to morphological characters. We recommend that future studies combine DNA sequences with morphological characters, which can easily be done by adding pictures of the specimens to avoid increasing confusion in the identification of specimens.

Acknowledgements

The expeditions were part of the research programme “Ekspedisi Widya Nusantara (E-Win)” of PPO-LIPI. The research permit applications were sponsored by Prof. Dr. Suharsono of PPO-LIPI. LIPI granted research permit 6559/SU/KS/2007 for the fieldwork in the Raja Ampat Islands, West Papua. We want to thank Max Ammer and staff of Papua Diving at Kri Eco Resort and Raja Ampat Research and Conservation Center (RARCC) for logistic support at Kri Island, Raja Ampat. Dr. Mark Erdmann (Conservation International, Sorong, West Papua) provided useful advice and encouragement. The Indonesian State Ministry of Research and Technology (RISTEK) granted research permit 0248/FRP/SM/X/09 for the fieldwork in Ternate and Halmahera. We want to thank Mr. Fasmi Ahmad and staff of the LIPI field station at Ternate for logistic support. We are also grateful to Mr. Samar and Mr. Dodi of Universitas Khairun at Ternate for their participation and field assistance. Financial support was given by Adessium Foundation, the van Tienhoven Stichting, the Schure-Beijerinck-Popping Fund (KNAW), The Groningen University Fund, the Leiden University Fund, the Jan Joost ter Pelkwijk Fund (Naturalis), and the Alida Buitendijk Fund (Naturalis). We thank Erik-Jan Bosch (Naturalis) for making the maps. We thank Richard C. Willan and one anonymous reviewer, as well as the editor Nathalie Yonow for critical and constructive remarks, which helped to improve the paper.

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