Marine Heterobranchia ( Gastropoda , Mollusca ) in Bunaken National Park , North Sulawesi , Indonesia — A Follow-Up Diversity Study

Bunaken National Park has been surveyed for a fourth time in 14 years, in an attempt to establish the species composition of heterobranch sea slugs in a baseline study for monitoring programs and protection of this special park. These molluscs are potentially good indicators of the health of an ecosystem, as many are species-specific predators on a huge variety of marine benthic and sessile invertebrates from almost every taxonomic group. Additionally, they are known to contain bio-compounds of significance in the pharmaceutical industry. It is therefore of paramount importance not only to document the species composition from a zoogeographic point of view, but to assist in their protection for the future, both in terms of economics and aesthetics. These four surveys have documented more than 200 species, with an approximate 50% of each collection found only on that survey and not re-collected. Many species new to science have also been documented, highlighting the lack of knowledge in this field.


Introduction
Marine biodiversity studies in Indonesia are rare, and mainly documented in the Indonesian language [1].However, documentation on specific localities, which are threatened by manifold factors, from global climate change down to increased local impact by humans, is rare, despite a strong

Materials and Methods
Expeditions occurred from 15 October to 3 November 2016 and 4 to 12 September 2017, with 30 snorkelling and diving events (two during the night) in 2016 and ten (one during the night) in 2017.Underwater searching time in 2016 with two or three divers was approximately 60 min up to 120 min per dive; thus, person hours approximate 110 h for diving and 12 h for snorkelling.The diving took place between 2 and 30 m.During these dives, the substrate was searched by close observation and, especially in shallow waters, coral rubble was turned over.Only one or two divers collected in 2017, amounting to approximately 25 person hours; however, approximately 20 person hours were spent searching for animals in the intertidal flats.Additionally, rhizomes, roots of seagrasses and sediment were brought back into the lab with subsequent close observation for approximately 10 h in larger bowls.Nineteen sites were visited for collections in these two years, hours in larger bowls.Nineteen sites were visited for collections in these two years, focusing around Bunaken Island with 14 sites, and Mantehage (two sites that were not visited in 2003 and 2015; Figure 1).Manado Tua, Siladen, and the mainland were revisited, with an overlap of collecting sites on Manado Tua and Siladen; however, another site was visited on the mainland (for details see Figure 1 and Table 1).Table 1 lists all collection sites and their characteristics.Sea slugs were photo-documented under water on their natural substrate with an Olympus TG 4 camera when possible, and preliminarily identified using various literature sources [10,11].Additionally, photographic records were taken of each specimen, including its unique identifier number, in the lab with the same camera.The identifier contains information about the genus and species (first four letters), the year, and the area.Final identification followed by subsequent investigation of morphological details and consulting further literature (e.g., Yonow [12][13][14], Stoffels et.al. [15], Martynov and Korshunova [16], and Yonow and Jensen [17]).Validity of names was checked with the help of the World Register of Marine Species [18], and the systematics as well as genus affiliations suggested by this website were followed.Based on recent publications [19], several names have changed after the publication of Kaligis et al. [1].This is taken into consideration in Table 2.All animals were recorded with metadata that will be available in the internet portal of Diversity Workbench within the module Diversity Collection [20].Small animals less than 10-15 mm were usually preserved in 96% EtOH.Larger animals were preserved in formaldehyde/seawater and only a small piece of foot and/or body tissue was cut off and preserved in 96% alcohol for future molecular studies.Species numbers are analysed and visualised with Excel (Microsoft Office 14).

Oct 2016
Table 2. List of species and specimen identifiers for the three recent collection events, and including Kaligis et al. [1].Species that were recorded in Burghardt et al. [3] and recollected during the recent events are also indicated in the last column.Details of localities with abbreviations are specified in Table 1 or Kaligis et al. [1].* indicates species that are not documented in literature or identification books, thus representing at-the-moment-unique species of BNP.

Results
In total, 385 specimens comprising 112 marine heterobranch species were collected in 2016 and 108 specimens comprising 57 species in 2017 (see Figure 21A).Both collecting events were in October, the month when the rainy season usually starts.The overall species number recorded and collected from BNP during the dry season in 2015 attains 79 species.Collecting efforts in 2016 with 112 species was the highest, but training efforts as well as increased collecting time in the recent surveys affects the results: The number of species that were only found in 2016 (63, Figure 21A) is high, and represents nearly 30% of the overall species (215 species, see below) recorded from this area.This clearly shows that some species are very rare, and the area is still under-sampled.This can also be seen when looking at the results of 2017 (Figure 21A) with a shorter collecting period, but focusing on inter-reefal habitats (31 species, representing 15% of the overall species record).It is possible that the collection months had some bearing on the numbers collected; however, the El Niño situation in 2016 with an increased water temperature and the lack of a distinct dry season in 2017 with continuous rainfalls throughout the year (pers.obs.SR), does not allow statements about seasonal differences yet.
Table 2 lists all species collected in 2016 and 2017, as well as the previously published data from 2015 [1].The species recorded in 2003 [3] and recollected during these recent surveys are highlighted in a separate column.Any overlap with our recent survey is also highlighted in Figure 21A.Species that were ambiguous, and where re-identification was not possible (11 species in in Burghardt et al. [3], marked as +11 in Figure 21A), were not included in the species accumulation curve in Figure 21B.Identifiers are provided for unidentified and/or new species in Table 2 [1], and discussed in detail either when not mentioned in Kaligis et al. [1], when considered problematic in identification, or when new to science.The work has a focus on specific groups which were united in the past as Opisthobranchia, and which is still used in many identification books (see the review in Wägele et al. [21]).

Animals Collected in 2016 (Figures 2-16)
3.1.1.Cephalaspidea and Runcinacea (16 species in eight genera belonging to five families, Figures 2A-4A) Haminoeidae usually have a short posterior foot, as can be seen in the probably undescribed Haminoea species (Figure 2A), shown in Haminoea sp. 2 in Gosliner et al. [11] (p. 30), or Haminoea spec (Figure 2C).Some members exhibit a typically long "tail."It is still unclear whether this group with long tails should have its own genus.One animal was retrieved (Figure 2B), which is similar to the Haminoeid depicted by Gosliner et al. [11] (p. 34) as Haminoeid sp. 2. This species is characterised by the white head appendages and a brownish flower-shaped pattern on a white body background.
Only during this study, two species of Phanerophthalmus were found with one animal each.Phanerophthalmus albocollaris (Figure 2D) is distinctive with white markings in the head region.However, the second species (Figure 2E) could not be assigned to any other described Phanerophthalmus species, although it resembles Phanerophthalmus sp. 3 in Gosliner et al. [11] (p. 33), a reddish to violet specimen with a white mark in the posterior part of the cephalic shield.The Bunaken specimen (Figure 2D) also bears some similarities to Phanerophthalmus luteus (Quoy and Gaimard, 1933) [17].It was also illustrated in Burghardt et al. [3] as Phanerophthalmus cf.smaragdinus.
The Aglajidae is a widespread taxon with its highest diversity probably in the Philippines [22].They are usually carnivorous, and many live in habitats with sand or sediment.Due to the focus on coral habitats, the species number present in our collection is limited to species characteristically known from coral habitats (Chelidonura amoena in Figure 2G, Chelidonura hirundinina [1], Odontoglaja guamensis in Figure 2H).One specimen (Figure 3A) looked similar to Tubulophilinopsis pilsbryi (Eliot, 1900), but with inverted coloration of that typical for T. pilsbryi.This specimen is tentatively assigned to this genus.
Gastropteridae are tiny-shelled slugs, some of them with striking colors.A small whitish Gastropteron was found with distinct white tubercles and patches (Figure 3B), similar to the one depicted by Gosliner et al. [11] (p. 56) as Gastropteron sp. 5. Kaligis et al. [1] illustrate five different Siphopteron species, including one new species.The new species (represented by two specimens) collected only at Siladen Island in 2015 was not found again at any other locality, and so a limited distribution is assumed.In contrast, S. brunneomarginatum (Figure 3D), Siphopteron ladrones (Figure 3E) and Siphopteron nigromarginatum (Figure 3F) were found again in various locations.
Runcinidae is a group of marine heterobranchs with small members of various colorations.They have not received much taxonomic attention in the past, and so identification is nearly impossible.Here a very small specimen (Figure 3G,H) is tentatively assigned to the genus Runcina.It has tiny pustules in the head area and a small pointed foot posteriorly that is a little bit longer than the notum.This animal is light brownish in color, and shows dots and spots even lighter in color.Another animal coming from the mainland was dark brown with no further color pattern (Figure 4A), and is clearly a different species.Gastropteridae are tiny-shelled slugs, some of them with striking colors.A small whitish Gastropteron was found with distinct white tubercles and patches (Figure 3B), similar to the one depicted by Gosliner et al. [11] (p. 56) as Gastropteron sp. 5. Kaligis et al. [1] illustrate five different Siphopteron species, including one new species.The new species (represented by two specimens) collected only at Siladen Island in 2015 was not found again at any other locality, and so a limited distribution is assumed.In contrast, S. brunneomarginatum (Figure 3D), Siphopteron ladrones (Figure 3E) and Siphopteron nigromarginatum (Figure 3F) were found again in various locations.
Runcinidae is a group of marine heterobranchs with small members of various colorations.They have not received much taxonomic attention in the past, and so identification is nearly impossible.Here a very small specimen (Figure 3G,H) is tentatively assigned to the genus Runcina.It has tiny pustules in the head area and a small pointed foot posteriorly that is a little bit longer than the notum.This animal is light brownish in color, and shows dots and spots even lighter in color.Another animal coming from the mainland was dark brown with no further color pattern (Figure 4A), and is clearly a different species.3.1.2.Anaspidea (two species in two genera belonging to one family, Figure 4B,C) Aplysia parvula is a circumtropical species which might represent a species complex [11,23].Our specimens (Figure 4B) showed the typical pinkish color with cream-colored dots, and darker parapodial and rhinophoral rims of the form associated with algae on coral reef flats and the intertidal [24].They were collected in coral rubble in less than 7 m depth.3.1.2.Anaspidea (two species in two genera belonging to one family, Figure 4B,C) Aplysia parvula is a circumtropical species which might represent a species complex [11,23].Our specimens (Figure 4B) showed the typical pinkish color with cream-colored dots, and darker parapodial and rhinophoral rims of the form associated with algae on coral reef flats and the intertidal [24].They were collected in coral rubble in less than 7 m depth.
3.1.3.Sacoglossa (16 species in four genera belonging to three families, Figures 4D-6D) Sixteen Costasiella species are described worldwide, and a further eight undescribed species are documented in Gosliner et al. [11].Jensen et al. [25] described and illustrated a specimen from Guam (preliminarily identified as Costasiella "kuroshimae") resembling our five specimens very closely, which were all collected together in Mantehage at Barracuda Point sitting close to a Flabellia alga in 14 m depth.The white head is typical for this species, which shows only a tinge of yellow in front and behind the eyes.The black markings behind the eyes render the impression of an "angry face" (Figure 4E).The green diamond-shaped patch, typical for C. kuroshimae, is absent.The rhinophores are rather transparent and do not show any black.Based on the phylogeny of Costasiella species from around the world, Jensen et al. [25] considered identification of C. kuroshimae as very difficult, because of the similarity of non-related specimens and a lack of a proper description of the original material of this species.
Eight species of Elysia were collected, but only few were observed on macro-algae.This is unfortunate as algal species are helpful in identification of sacoglossans since many are limited in their diet species.Most of them were collected from coral rubble or highly structured mini-habitats in the coral reef.Of the four undescribed Elysia species documented here, only one was found the previous year, identified as Elysia sp. 25 in Gosliner et al. [11] (p. 89) (see Figure 3g in Kaligis et al. [1]).This species has a characteristic black line in front of the parapodia and a peculiar crawling habit (Figure 5B).Two of the other new species of Elysia were collected in Alung Banua, on the southern coastline of Bunaken Island, which has a higher algal flora with tiny algal species, probably due to higher eutrophication.Elsp1_16Bu-1 (Figure 5C) is transparent (perhaps due to its small size of only 3 mm) with moderately branched green digestive gland shining through, whereas Elsp16Bu-1 was uniformly dark green with transparent rhinophores speckled with white dots and a few elevated white spots on the head, body, and parapodia (Figure 5D).Three specimens of another, probably undescribed, Elysia species were collected at a site in Mantehage.The rather white animals had a knobby appearance, and the outer sides of the parapodia were greenish (Figure 5E).
No new Thuridilla species were collected; however, one specimen of Thuridilla undula showed an aberrant habitus (Figure 6C): The parapodial rims were fused in the middle of the body.
3.1.4.Pleurobranchomorpha (one species in one genus belonging to one family, Figure 6E) During night dives, many specimens of Pleurobranchus forskalii were found, some of them feeding on tunicates of the family Didemniidae.We collected a smaller specimen of brownish color (Figure 6E), which was found under coral rubble during the day.It can be distinguished from Pleurobranchus peronii Cuvier, 1804 by its compound tubercles.3.1.4.Pleurobranchomorpha (one species in one genus belonging to one family, Figure 6E) During night dives, many specimens of Pleurobranchus forskalii were found, some of them feeding on tunicates of the family Didemniidae.We collected a smaller specimen of brownish color (Figure 6E), which was found under coral rubble during the day.It can be distinguished from Pleurobranchus peronii Cuvier, 1804 by its compound tubercles.The genus Kaloplocamus was represented with two different species, both new to science, possibly recorded by Gosliner et al. [11] (p. 116) as sp.8 (Figure 6F) and sp. 9 (Figure 6G); if confirmed, this extends the range of these two species further west.Kaloplocamus ramosus (Cantraine, 1835) exhibits bioluminescence [26], but the two species collected in this survey were not investigated to see if they also exhibited this peculiar phenomenon.
Kaligis et al. [1] listed Polycera japonica and Polycera risbeci, both from the northern shorelines of Bunaken.The former was found again in Mantehage (Figure 7A), but P. risbeci was not collected again.
We record here a third species of Polycera, which is very cryptic in coloration (Figure 7B).It is probably a new species, most similar to Polycera sp. 5 in Gosliner et al. [11] (p. 113).
Aegiridae were investigated by Fahey and Gosliner [27], who described a new species, Aegires malinus, typified by a reddish color, and apple-green rhinophores and gills.This species was collected only once in this study (Figure 7E), in addition to the bright lemon yellow Aegires citrinus (Figure 7D).
Several Gymnodoris species are present in the collection, including Gymnodoris tuberculosa (Figure 7F), furthermore an undescribed species very similar to Gymnodoris okinawae Baba, 1936, but with less distinct orange dots (Figure 7G; see also Gymnodoris sp. 2 in Gosliner et al. [11] (p. 152)), and one species which could not be assigned to any described or depicted species (Figure 7H).The genus is characterised by its rather uniform tapering shape.Distinguishing features are the arrangement of the gills; some have peculiar color characteristics [11].Nearly 60 undescribed species are depicted in Gosliner et al. [11], and so identification in surveys such as this must be preliminary until the genus is revised.
Some dorids mimic sponges and are therefore difficult to detect.A large cryptic species is Asteronotus mimeticus (Figure 8A,B) which shows various colorations, and sits on the underside of foliate sponges, such as Phyllospongia.One specimen tentatively assigned to Sclerodoris (Figure 8F) is depicted in Gosliner et al. [11] (p. 195) as Sclerodoris sp. 2.
Kaligis et al. [1] depicted a species under the name Chromodoris sp. with yellow rhinophores and gills, which is here identified as Chromodoris lochi; this yellow color morph is illustrated in Figure 9A.Several chromodorids show similarities in colors and patterns, and are therefore difficult to distinguish.The recent publication on color variation of some Chromodoris species mimicking other Chromodoris species adds to problems of correct identification [28].Kaligis et al. [1] recorded Chromodoris strigata, which is very similar to Chromodoris michaeli, but unfortunately, Gosliner and Behrens [29] (original description) did not compare their new species with C. strigata which was described earlier by Rudman [30].One main difference may be the more bluish color of C. michaeli compared to the more whitish C. strigata.Our specimen was not white but showed a tinge of blue (Figure 9B).The pattern of the black stripes is also very similar to the ones depicted in the original description of C. michaeli, and we therefore tentatively assign our animal to C. michaeli.Goniobranchus sp.(Figure 9H) appears similar to Gymnodoris species in its simple elongated body shape, and rather transparent coloration with tiny whitish dots on the dorsum.The notum has a narrow but distinct orange rim.
The taxon Hypselodoris has undergone changes in species affiliation in the last decade by new morphological and molecular analyses [31,32].Nearly nothing is known about color variation during ontogeny, rendering identification of small specimens more difficult.Two specimens were found in Bunaken Island which may be new to science (Figure 10D,E).Both animals were very small (5 mm and 6 mm respectively).They somewhat resemble Hypselodoris maculosa, which was also found several times, and the recently resurrected Hypselodoris decorata (Risbec, 1828); however, both specimens barely show the violet tinge on the foot and velum, and do not have the characteristic color banding of the rhinophores.The larger one also lacks the orange colored gills which are white in this specimen.We cannot exclude that both of them represent juveniles of described species.
Six phyllidiid species were collected.Only one specimen of Phyllidia elegans was found at Mikes Point (Figure 11B), whereas Kaligis et al. [1] recorded 13 specimens, mostly from Siladen.The animal is similar to Phyllidia alyta Yonow, 1996, with yellow rhinophores and longitudinal black stripes, including a black line on the foot sole.However, our animal shows black lines connecting the longitudinal stripes at the back, more typical for Phyllidia elegans.Another animal was tentatively assigned to Phyllidiella cf.annulata (Figure 11D) based on the distinctive black crossed pattern on the dorsum, the thin black line at the mantle margin and the many small tubercles covering the notum.more typical for Phyllidia elegans.Another animal was tentatively assigned to Phyllidiella cf.annulata (Figure 11D) based on the distinctive black crossed pattern on the dorsum, the thin black line at the mantle margin and the many small tubercles covering the notum.3.1.6.Nudibranchia, Cladobranchia (36 species in 20 genera belonging to 11 families, Figures 12A-16B) Four different species of Dermatobranchus were collected from various localities.Species of this genus often exhibit a rather whitish appearance with very unspecific dots or patches of darker color.Three of our specimens showed this coloration but probably belong to different species.Two of our specimens (Figure 11H) are very similar to Dermatobranchus kokonas Gosliner & Fahey, 2011, but have more brownish rhinophores with transparent tips.We tentatively assign our material to this species.Dermatobranchus piperoides Gosliner & Fahey, 2011 is distinguished by orange rhinophores with darker tips, but the single specimen we collected lacked the black tips, and assignment to D. piperoides is preliminary (Figure 12A).Another 3.1.6.Nudibranchia, Cladobranchia (36 species in 20 genera belonging to 11 families, Figures 12A-16B) Four different species of Dermatobranchus were collected from various localities.Species of this genus often exhibit a rather whitish appearance with very unspecific dots or patches of darker color.Three of our specimens showed this coloration but probably belong to different species.Two of our specimens (Figure 11H) are very similar to Dermatobranchus kokonas Gosliner & Fahey, 2011, but have more brownish rhinophores with transparent tips.We tentatively assign our material to this species.Dermatobranchus piperoides Gosliner & Fahey, 2011 is distinguished by orange rhinophores with darker tips, but the single specimen we collected lacked the black tips, and assignment to D. piperoides is preliminary (Figure 12A).Another specimen (Figure 12B) also looks very similar to D. piperoides in its notum coloration; however, this animal lacks the typical orange color on the rhinophores.This specimen depicted in Figure 12B cannot be assigned to any described Dermatobranchus species yet.Some Janolus species differ from the typical habitus of the genus in having densely papillate cerata.We assign our specimen tentatively to the species Janolus mirabilis based on the color, the moderately papillate cerata, and a large gap between the anterior and posterior groups of cerata (Figure 12C).
One species of Crosslandia was collected from floating Sargassum close to Siladen and close to Bunaken (Figure 12D).The few known species are recorded from algae or sea grass beds.Crosslandia daedali was recorded previously as living on Sargassum, and is described with a similar coloration as that of our eight specimens [33].These and subsequent records are all from the Pacific coast of America and south Atlantic coast of Brazil; however, there are recent records from areas around Singapore [34] that additionally confirms the presence of this species in the Indo-Pacific.
The genus Kabeiro (Dotidae) is usually found on leptothecate hydrozoans.It can be distinguished clearly from similarly looking aeolids (see below) by the presence of rhinophoral sheaths.Several specimens probably belonging to three different species were collected: Two undescribed species and three specimens of Kabeiro rubroreticulata (Figure 12F).The species depicted in Figure 12G was collected in several localities in the southern part of Bunaken Island, as well as Siladen Island.The species has characteristic tubercles arranged irregularly on the cerata.Another animal had more inflated cerata with a distinct terminal tubercle and just one row of smaller tubercles subapically around the cerata.This specimen was found in Siladen Island and certainly represents another species of Kabeiro (Figure 12H).
Three undescribed species of Tritonia were collected in different localities.One specimen similar to Tritonia sp. 10 in Gosliner et al. [11] (p. 321) was found on a partly overgrown hydroid colony (Figure 13B); however, Tritonia sp. 10 was described from a habitat with many Xeniid species.Specimens of a different species (Figure 13C) were observed swimming with a dorso-ventral movement, as mentioned by Gosliner et al. [11] for a very similarly colored animal (Tritonia sp. 9, p. 321).This species resembles Tritonia khaleesi Silva, Azevedo & Matthews-Cascon, 2014 from the tropical southern Atlantic in having very distinct whitish stripes on the dorsum [35], but our specimens showed a reticulate pattern in-between and underlying the broad white stripes.The third species (Figure 13D), only tentatively assigned to the genus Tritonia, was found on sponges.
The Eubranchus species illustrated in Figure 14B was found on similar leptothecate hydrozoans, as were the species of Kabeiro.This particular species is possibly the same as Eubranchus sp.22 of Gosliner et al. [11] (p. 341).
Bulbaeolidia alba (Figure 16B) is the only member of the Aeolidiidae in our collections.It can be easily distinguished by the overall white color and the red markings behind the rhinophores.

Species Collected in 2017 (Figures 17-20)
In this section, we discuss the species that were not recorded previously, either in 2015 [1] or above.

Species Collected in 2017 (Figures 17-20)
In this section, we discuss the species that were not recorded previously, either in 2015 [1] or above.
3.2.1.Cephalaspidea (six species in five genera belonging to four families, Figure 17A,B) One cephalaspidean specimen was collected whose assignment even to a genus is difficult.According to the form of the head shield and the posterior body parts, it seems likely that this animal is an undescribed Philine (Figure 17A).

Species Collected in 2017 (Figures 17-20)
In this section, we discuss the species that were not recorded previously, either in 2015 [1] or above.
3.2.1.Cephalaspidea (six species in five genera belonging to four families, Figure 17A,B)  An undescribed Sagaminopteron species (Figure 17B) was collected from sponges on Siladen Island.Interestingly, the same species with same coloration was already photo-documented in 2016 from the same area, and it may be limited to a particular prey species found only at this locality.The species is similar to Sagaminopteron bilealbum Carlson & Hoff, 1973 with an overall white coloration and orange tips of the cephalic tentacles, but lacks the numerous orange dots, with only a few present along the rim of the parapodia.Sagaminopteron nigropunctatum is also similar to our specimen; however, this species is usually characterised by a covering of tiny black dots.We cannot exclude that this small animal with a size of only 4 mm is a juvenile of a described species.
3.2.2.Anaspidea (four species in four genera belonging to one family, Figure 17C,D) Collecting in the intertidal reef flats and upper sublittoral of the seagrass beds revealed two of the typical species for this area, Dolabella auricularia (Figure 17C) and Dolabrifera dolabrifera (Figure 17D).Although these species are large, they were not recorded in the previous years.
3.2.3.Sacoglossa (11 species in seven genera belonging to four families, Figures 17E-18C) Sieving sand containing sea grass roots and rhizomes revealed a tiny Cylindrobulla species with a shell length of 10 mm (Figure 17E).The cephalic shield was as long as the shell but could be retracted almost completely into the shell.
Several Cyerce specimens were found during this expedition in front of Panorama (see Table 2), tentatively assigned to Cyerce cf.bourbonica (Figure 17F).This species was also recorded in Kaligis et al. [1] from further places around Bunaken Island.
Costasiella kuroshimae (Figure 17 H), as well as an undescribed Costasiella species (Figure 18A) (as Costasiella sp. 8 [11] (p. 81)), was collected from the chlorophyte alga Avrainvillea.Costasiella sp. 8 is characterised by the black spots at the base of the rhinophores, whereas C. kuroshimae has black rhinophoral tips.One undescribed species with a typical black band behind the eyes and with a medium white spot in this band (Figure 18B) was collected from Flabellia and is depicted in Gosliner et al. [11] as Costasiella sp. 1 (p.79).
Plakobranchus ocellatus is a species complex, shown by Yonow and Jensen [17] and Meyers-Muñoz et al. [36]; the latter described a new Plakobranchus species from Indonesia that does not have ocellated spots but uniformly colored dots.Our specimen (Figure 18C) resembles best the variety they called P. ocellata variation F and Plakobranchus sp. 3 in Gosliner et al. [11] (p. 99) with large dark rings at the sides of the body and along the front of the head, filled with greenish to reddish color.It differs from the originally described specimen by van Hasselt (see Yonow and Jensen, Meyers-Muñoz et al. [17,36], which has smaller ringed dots next to the large rings, each with a dark center, on the parapodia.Our animal also had smaller rings on the parapodia, but these were darker in color with light-colored centers. 3.2.4.Pleurobranchomorpha (one species in one genus belonging to one family, Figure 18D) One specimen was collected under coral rubble that most resembles Berthellina citrina (Figure 18D).Other species can have white markings or dots on the notum, which are missing in our specimen.
3.2.5.Nudibranchia, Anthobranchia (24 species in 16 genera belonging to seven families, Figures 18E-20B) Two specimens of a Gymnodoris (Figure 18E) were collected in the coral rubble in front of Panorama.Both animals looked like Gymnodoris sp.46 [11] (p. 161), except for the frontal margin of the velum, which was not orange in our animal.
Two small reddish dorids, mimicking sponge surfaces (Figure 18F,G), were found in coral rubble.No assignment to any genus is possible for these two specimens until further morphological and molecular analyses.
A small dark brown dorid with white tips of the rhinophores and white gills was found in the seagrass beds in front of Panorama resort (Figure 19A).It also showed white tips of the tubercles surrounding the rhinophores, and is very similar to Discodorid sp. of Yonow [14] (p.154, Figure 15B); the same color pattern is illustrated for an undescribed Diaulula sp. 1 [11] (p. 197).This animal is probably associated with sponges living between the sea grass rhizomes.3.2.4.Pleurobranchomorpha (one species in one genus belonging to one family, Figure 18D) One specimen was collected under coral rubble that most resembles Berthellina citrina (Figure 18D).Other species can have white markings or dots on the notum, which are missing in our specimen.
3.2.5.Nudibranchia, Anthobranchia (24 species in 16 genera belonging to seven families, Figures 18E-20B) Two specimens of a Gymnodoris (Figure 18E) were collected in the coral rubble in front of Panorama.Both animals looked like Gymnodoris sp.46 [11] (p. 161), except for the frontal margin of the velum, which was not orange in our animal.Two specimens (Figure 19C,D) were collected which are simply assigned to the family Discodorididae pending further work.None of these species was illustrated in Tonozuka [37] covering Indonesian nudibranchs, or Yonow [14] describing dorids from Ambon or in Gosliner et al. [11].
A small white animal (Figure 19G) collected from a sponge on Siladen Island is similar to several Verconia species, especially Verconia decussata (Risbec, 1928) and Verconia simplex (Pease, 1871) (see Yonow [38] for generic placement discussion).However, our animal differed in the color pattern from all of these, by only exhibiting red rhinophores without any other red areas.This animal is small (4 mm) and might be a juvenile; therefore, assignment to any species is difficult.
Dendrodoris nigra and Dendrodoris fumata are known to exhibit a range of color patterns and to undergo ontogenetic color changes.Juvenile D. nigra usually have white dots irregularly distributed over the notum and are often pink to orange; however, the gills are numerous, 9-11, issuing from a small pocket.One of our specimens resembled the color of a juvenile D. nigra, but had only five gills.Furthermore, the texture appeared more rigid, and not soft as in typical Dendrodoris.We therefore do not assign this rather small specimen to a species (Figure 19H).
At a depth of approximately 25 m within a cave, a specimen of a new species of Phyllidiidae (Figure 20A,B) was found that completely lacks tubercles, thus resembling a species of Phyllidiopsis; however, the oral tentacles are not fused, which indicate it belongs to the genera Phyllidia or Phyllidiella.The coloration is also very distinctive, but this animal was not recorded from any other site and was only found once.An interesting note is that the area where this animal was found was visited several times and searched during both night and day dives, but revealed no more specimens.We tentatively assign this specimen to the genus Phyllidia.3.2.6.Nudibranchia, Cladobranchia (11 species in nine genera belonging to five families, Figure 20C-F) One Dermatobranchus species collected in Siladen was cream white with longitudinal lines, and the rim of the oral veil and foot in orange (Figure 20C).There are several creamy white Dermatobranchus species with longitudinal lines and darker dots in-between with similarly black rhinophores.Dermatobranchus sp. 8 depicted in Gosliner et al. [11] (p. 302) appears closest to our animal but lacks the orange foot rim.The rhinophores and orange margins are very similar to those in D. albus, which lacks the dark spots present in our species.
In 2015 several new Noumeaella species were collected, mainly from sponges, but also in-between Caulerpa racemosa [1].One new Noumeaella species was observed for the first time in BNP living underneath foliaceous sponges (Figure 20D).The two collected specimens had the characteristic cream color with a distinct dark spot at the base of each ceras.Gosliner et al. [11] (p. 368) illustrate a similarly colored form from Indonesia.
A small facelinid species was collected by divers from a locality in the south of Bunaken, which has a very distinct color pattern with orange undulating lines running across the body (Figure 20F).The animal is whitish to transparent with many opaque white patches and dots of various sizes covering all parts of the body, including all tentacles.3.2.6.Nudibranchia, Cladobranchia (11 species in nine genera belonging to five families, Figure 20C-F) One Dermatobranchus species collected in Siladen was cream white with longitudinal lines, and the rim of the oral veil and foot in orange (Figure 20C).There are several creamy white Dermatobranchus species with longitudinal lines and darker dots in-between with similarly black rhinophores.Dermatobranchus sp. 8 depicted in Gosliner et al. [11] (p. 302) appears closest to our animal but lacks the orange foot rim.The rhinophores and orange margins are very similar to those in D. albus, which lacks the dark spots present in our species.

A Comparison of the Bunaken NP Studies
In 2015 several new Noumeaella species were collected, mainly from sponges, but also in-between Caulerpa racemosa [1].One new Noumeaella species was observed for the first time in BNP living underneath foliaceous sponges (Figure 20D).The two collected specimens had the characteristic cream color with a distinct dark spot at the base of each ceras.Gosliner et al. [11] (p. 368) illustrate a similarly colored form from Indonesia.
A small facelinid species was collected by divers from a locality in the south of Bunaken, which has a very distinct color pattern with orange undulating lines running across the body (Figure 20F).The animal is whitish to transparent with many opaque white patches and dots of various sizes covering all parts of the body, including all tentacles.

A Comparison of the Bunaken NP Studies
In the following section, we compare the three years' collections from 2015 [1] to 2017 with the previous study of Burghardt et al. [3] based on a collection in 2003.We also try to re-identify the unidentified animals of this earlier study in light of further collections and recent documentation.In this study [3] 86 species are listed, 32 of which were not found again in the recent collections (Figure 21A).Many species are only listed as undescribed species, and therefore at least 11 species are difficult to compare with our recent material due to lack of photo-documentation.They are not included in the species accumulation curve in Figure 21B., Burghard et al. [3], and this study; Ambon [12][13][14]17]; Indonesia with emphasis on Bali [37]; Vietnam [16]; and Papua New Guinea [39].Burghard et al. [3], and this study; Ambon [12][13][14]17]; Indonesia with emphasis on Bali [37]; Vietnam [16]; and Papua New Guinea [39].
Liloa mongii (listed as Haminoea curta in Burghardt et al. [3]) and Limulatys species are tiny cephalaspideans living in silty habitats.Our recent studies did not cover any silty areas, which might be the reason this species was not collected again.No figures were provided of the other haminoeid species, and therefore no comparison with our recently sampled undescribed Haminoea species is possible.
Phanerophthalmus cf.smaragdinus (Rüppell and Leuckart, 1830), now accepted as Phanerophthalmus olivaceus (Ehrenberg, 1828) and depicted in Figure 23 of Burghardt et al. [3], is similar to an animal we collected in 2016.We tentatively identify our specimen as P. cf. albocollaris as well as the animal depicted in Burghardt et al. [3] (Figure 23).
Burghardt et al. [3] (Figure 25) illustrated a specimen as Phyllaplysia sp. 1.Despite searching through seagrasses, where this genus is often found, we were not able to find another specimen of this or any other species of the genus again.
One Cyerce specimen documented in Burghardt et al. [3] (Figure 28) as Cyerce sp. 1 is very similar to the specimens we found during our current expeditions in various sites around BNP (see Table 2).They are all tentatively assigned to Cyerce cf.bourbonica (Figure 17F).Cyerce elegans Bergh, 1870 (see in Burghardt et al. [3] (Figure 29), Gosliner et.al. [11], and Yonow and Jensen [17]) is characterised by its transparency and pale body color; it was not collected again.
Thuridilla bayeri and Thuridilla gracilis were mentioned by Burghardt et al. [3] as separate species, but photographs were not provided.The subsequent synonymy of T. bayeri Er.Marcus, 1965 with T. gracilis and Thuridilla ratna Er.Marcus, 1965 has been teased out by Yonow and Jensen [17] who advocate individual descriptions until the precise identities of this complex are firmly established.Burghardt et al. [3] also collected an undescribed Thuridilla species, but did not provide any photo-documentation of this animal.We did not find any unusual or undescribed Thuridilla species in our recent collections.
Burghardt et al. [3] listed Pleurobranchus peronii Cuvier, 1804 as well as an unidentified Pleurobranchus species.P. peronii may be mistaken for Pleurobranchus forskalii, which is the only Pleurobranchus species we recollected several times.The compound tubercles and a more heterogeneous coloration are typical for P. forskalii, in addition to the distinctive white markings, whereas P. peronii only has single tubercles and is more uniformly colored, be it in many shades of brown, orange, or red.Both species can show a wide variety of colors and patterns, especially the juveniles.
Burghardt et al. [3] listed two Gymnodoris species, Gymnodoris citrina Bergh, 1877, and an unidentified animal; neither is illustrated.Gymnodoris species are notoriously difficult to identify because of extensive color variations which are similar between species; there has been no review of this group.We were unable to assign any of the recently collected specimens to G. citrina.
Burghardt et al. [3] mentioned the dorid Tambja morosa Bergh, 1877, which usually inhabits steep slopes and drop-offs, as present on the southern coastline of Bunaken Island.It feeds on arborescent bryozoans, which have also been observed in BNP, but this species was not collected again in our recent survey.Interestingly, all recently recorded Polyceridae species ( [1], this study) were low in specimen numbers, even supposedly common ones such as Nembrotha kubaryana and Nembrotha cristata (e.g., [13]).
Goniobranchus decorus (Pease, 1860) (as Chromodoris decora in Burghardt et al. [3]) shows similar color patterns to some species of Thorunna and Hypselodoris.Usually they can be found under and among coral rubble as in this study, e.g., Hypselodoris maculosa and Thorunna furtiva.Rudman [40] discussed the Chromodoris decora color group, including at least 14 similarly colored species belonging to the above-named genera.While G. decorus is nominally a common species and appears to have somewhat variable color patterns, it is recognisable, but was not found again in studies subsequent to the collecting period in 2003 [3].
The same problem is observed for Goniobranchus tinctorius (Rüppell and Leuckart, 1830) (listed as Chromodoris tinctoria in Burghardt et al. [3], but not depicted), which is very similar in color pattern to Goniobranchus reticulatus (recorded in Kaligis et al. [1]) and Chromodoris inopinata Bergh, 1905.Yonow [13] has shown that the two species are distinct, although Rudman [41] had previously considered C. reticulata and C. inopinata as synonymous.Collections from the nearby island of Ambon included red-reticulated specimens that were clearly identified as Chromodoris inopinata [13].Several Goniobranchus species with reticulate red pattern on a white background are depicted in Gosliner et al. [11].
Burghardt et al. [3] listed Verconia varians (Pease, 1871) (as Noumea varians) without including a picture.We also found a species of Verconia, but our animal is not Verconia varians, which is very distinct in body form and coloration; therefore V. varians is recorded only from the collection in 2003 [3].
Dendrodoris is a genus with members living in shallow water with a high variety in color morphs and similar color patterns.Burghardt et al. [3] recorded (but not illustrated) Dendrodoris elongata; we recently found only Dendrodoris albobrunnea [1], which is very similar to D. elongata.The differences were discussed in Yonow and Jensen [17].
Burghardt et al. [3] listed Phyllidiella nigra (van Hasselt, 1824), which is very similar to P. pustulosa, but can be differentiated upon close examination (e.g., Yonow [13] pp. 937-938).In our recent collections, P. pustulosa dominated in specimen numbers, and none of the recent specimens could be assigned to P. nigra.Identification of these species is difficult [15], and molecular analyses are warranted for future studies.
Two Janolus species are figured in Burghardt et al. [3] (Figures 40 and 41).Janolus mirabilis, which is characterised by the white pattern and the distinct gap between anterior and posterior cerata, was recollected (this study, Figure 12C).The other Janolus species (as Janolus sp. 1 in Burghardt et al. [3], Figure 41) resembles Janolus mirabilis (both were of similar size of 6 mm).We found a third form that differs from both J. mirabilis and J. sp. 1 of the 2003 collection [3], exhibiting much denser cerata with more acute papillae.
Phyllodesmium jakobsenae Burghardt and Wägele, 2004 (see Burghardt et al. [3]: Figure 44) is a typical xeniid feeder that was first described from the southern shorelines of Bunaken Island in shallow water, resulting from the 2003 collecting period [3,42].Although Xeniidae are still present in this area, this species was not found again in the recent sampling periods despite extended snorkelling in this area.
Burghardt et al. [3] (Figure 45) illustrated an undescribed Phyllodesmium species that was subsequently described as Phyllodesmium rudmani by Burghardt and Gosliner (2006) [43].We were not able to find this species either in the recent expeditions to BNP.
Phestilla species are closely associated with hard corals, such as Porites and Goniopora, and are easily overlooked despite large sizes of up to 60 mm.However, a thorough search of these corals, especially in the southern shorelines of Bunaken Island, did not reveal either of the two recorded species in Burghardt et al. [3], P. lugubris (Bergh, 1870), and P. minor Rudman, 1981.
Pseudovermis and Acochlidiimorpha are interstitial forms rarely recorded, unless specifically sampled for.Their absence in the recent sediment collections from the sea grass area might be attributed to the rough sieving, which destroys delicate animals more quickly than the shelled ones, or to not sampling the appropriate sediment habitats.

Discussion
Setiawan et al. [7] described declining conditions of reef fish and coral cover within BNP compared to 2006 and 2007, and related this to an increased number of local and foreign visitors, in addition to an increased number of permanent residents.Towoliu [44] identified diving and snorkelling activities as a major source of the decline in living coral coverage by comparing different sites around Bunaken Island.Whereas undisturbed areas had a live coral coverage of nearly 55% in 3 m depths, areas with snorkelers and divers showed coverage of only 17% coral cover at this depth.Although BNP is protected under regulations within the Regional Spatial Development Plan formed in 1990s, the existing tourist area was already twice the size in 2015 of the originally proposed area [45].Despite the lack of wastewater management, investigation into water pollution did not yet show a decreased water quality around Bunaken Island [46].Local people benefit from tourism and increase their income [47]; nevertheless, without regulations, these benefits will only be short-term, and allowing development beyond prescribed regulations is short-sighted.With an increase in diving tourism in the whole of the BNP [48] and an increase in general anthropogenic influences by locals [49] and tourists, demands on management strategies for dive sites are paramount within BNP.Only monitoring programs on characteristic and economically important but diverse taxa can provide the necessary data to recognise damages and threats to specific habitats, and will allow taking counteractions, preventative measures, or providing alternatives to a future sustainable use and maintenance of this unique region.A number of studies exist in which marine heterobranchs have been used as indicators for anthropogenic influences, such as the study on cadmium levels in the Mediterranean Sea [50] or the molluscan algal epifauna as a measure of habitat quality along the coast of Spain [51].Another study shows that Retusa obtusa dominated in the most enclosed stations inside a harbor where sediments contained very high values of organic matter, lipids, and heavy metals, and thus could serve as a harbor sediment indicator in conjunction with three bivalves [52].Shifts in nudibranch species composition has been used as an indicator for climate change in Australian waters [53].Thus, surveying such a diverse group, which relies heavily on a healthy diverse habitat, including the water column for their larval distribution, and the adults with a high affiliation to specific prey items, will certainly provide information about environmental changes when properly monitored.With this study, including already published information from the same region, we create the baseline for monitoring habitats around the BNP in the future.
The total species number of marine heterobranchs in Bunaken National Park, North Sulawesi, attains 226 species, when including the 11 uncertainties in species re-identification of Burghardt et al. [3] (Figure 21A).The groups that were absent in our most recent collections, the Acochlidiimorpha, Hedylopsidae, and Thecosomata increase the overall number of species.However, photo-documentation of non-collected species is not included in this number.The Venn diagram presented in Figure 21A shows the overlap of species in the three recent studies compared to the previous study in 2006 [3]; only 32 species of 215 species were collected twice.Of the 172 species recorded in the most recent three studies, only 23 species were common to all three.Pairwise comparison revealed an overlap of 44 species between 2015 [1] and 2016, 27 species between 2015 [1] and 2017, and 28 species between 2016 and 2017.
The present study revealed that out of the 69 undescribed species, representing 30% of the recorded species from BNP, 34 species have not been listed in diversity or taxonomic studies before, and they are presented here for the very first time (Figure 21B).The richness of this particular study area, also when comparing overall species diversity to other studies in Indonesia or other areas nearby [1,2] may be an indication that protection of a small area with a very diverse habitat structure is successful and worthwhile (Figure 21C).
The high species number found only once in all four surveys (162 species: Figure 21A) also suggests that the small area is worth protecting.While many marine heterobranchs, and especially nudibranchs, are ephemeral, the occurrence of even one specimen in a habitat presumes a richness of other marine invertebrate species, since many are species-specific with regards to food items.It also indicates that species diversity is not fully documented and still needs more sampling.The number of confirmed heterobranch species from BNP is higher than in the geographically close area of Ambon (summarised from [12][13][14]17]), but similar to the general study from Indonesia and Bali [37] (Figure 21C).While interesting, and providing a good base for future studies, the reader must bear in mind that collecting time, effort, and sampled habitats varied in all the studies compared here; thus, direct comparisons are clearly not possible.
Looking at species abundance, one of the most commonly recorded species is Phyllidiella pustulosa (nearly 60), three Chromodoris species, C. annae (>80), C. dianae (>70), and C. willani (40), as well as Phyllodesmium briareum (about 60), where most of the species were collected from a large Briareum colony (see also Table 2).Pungus et al. [54] began a survey on nudibranchs in Lembeh Strait, North Sulawesi, counting the most common species in a certain area.They also documented P. pustulosa and C. annae as the most frequently recorded species in that area.However, other species mentioned in their study, such as Tambja gabrielae, were not found in BNP.Aunurohim et al. [55] and Sari and Aunurohim [56] recorded a similar species composition with C. annae and P. pustulosa as the most dominant nudibranchs in Situbondo, East Java, and Phyllidiidae the dominant nudibranch family.The recently completed studies on the marine heterobranchs from Ambon [12][13][14]17] revealed Dendrodoris fumata as the most commonly recorded species, followed by Phyllidiella pustulosa, Chromodoris magnifica, and Phyllidia varicosa.Species composition and diversity of these enigmatic marine heterobranchs certainly differ in these regions of Indonesia; however, more studies are needed to understand influence of habitat structures caused by geological origins, currents, availability of food (and many others), as well as the influence of a changing environment (seasonal or long term).Another factor that hinders an accurate comparison at the moment is certainly the differences in collection efforts, experience of collectors, and habitat selection of the researchers.We therefore can merely describe the differences between the four study periods, without providing reasons.
Eight species were collected in 2003 [3], but not again in 2015 [1] to 2017.These comprise taxa from various habitats: Cyerce elegans from algal communities, Phyllodesmium jakobsenae from reef flats feeding on Xenia species, Verconia varians feeding on sponges, Tambja morosa from coral structures feeding on bryozoans, Embletonia gracilis and Bornella stellifera from coral structures feeding on hydrozoans, and finally the two Phestilla species, P. lugubris and P. minor, closely associated with certain hexacoral species and feeding on them.However, there is one example that might indicate a shift or deterioration of the habitat: Phyllodesmium jakobsenae was collected by snorkelling from xeniid colonies in the inter-reefal area in southern Bunaken Island, close to an area with several resorts, a small market, and a jetty.The type locality of this species faces a high increase in tourist numbers and boat traffic, including the building of a new resort as well as renovating an older one in the last five years.This high disturbance of the habitat may have contributed to the absence of P. jakobsenae from the recent collections, despite a particular search for this species.To address the increase of direct anthropogenic activities as one factor of species extinction, future collecting and monitoring of this area is desperately needed, clearly shown by studies on other metazoan taxa or organisms [7,44].
Based on the overall results from this special area, we consider the number of 215 species (plus another 11 uncertainties) a good baseline for future monitoring projects, and this baseline will help in understanding shifts of species composition, irrespective of causation factors.Our results also indicate that the diversity of marine heterobranchs is high in BNP, compared to the other results published on Indonesia [12][13][14]17,37].Studies from Papua New Guinea [39] and the Philippine Islands [11], which were based on many more collection events and collecting times, indicate with much higher species numbers, what can be expected in the long run.Ongoing studies in nearby areas (Bangka Archipelago, Lembeh Strait, and Sangihe Island) will also help to better understand the richness in this hotspot of species diversity, as well as will help to understand differences in habitat structures resulting in a different species composition of these areas.

Figure 1 .
Figure 1.Location of study area: (A) Indonesia and Sulawesi with dashed lines indicating the close-up area in (B); (B) Diving sites (orange dots only in 2015, purple only in 2016 and 2017, and red in both studies) in Bunaken National Park and the islands sampled.

Figure 1 .
Figure 1.Location of study area: (A) Indonesia and Sulawesi with dashed lines indicating the close-up area in (B); (B) Diving sites (orange dots only in 2015, purple only in 2016 and 2017, and red in both studies) in Bunaken National Park and the islands sampled.

Diversity 2018 , 1 Figure 21 .
Figure 21.Summary of species numbers and overview in Bunaken National Park (BNP): (A) Comparison of collection efforts of the years 2003 with 2015, 2016, and 2017.(B) Species accumulation curves of overall species numbers (blue line), of undescribed species (red line), and of species that are not documented in literature or identification books, thus representing at-the-moment-unique species of BNP, North Sulawesi (green line).The species number for the collection event 2003 does not contain the 11 species that could not be reassessed.(C) Comparison of species numbers from geographic areas close by North Sulawesi.Data based on BNP: Kaligis et al.[1], Burghard et al.[3], and this study; Ambon[12][13][14]17]; Indonesia with emphasis on Bali[37]; Vietnam[16]; and Papua New Guinea[39].

Figure 21 .
Figure 21.Summary of species numbers and overview in Bunaken National Park (BNP): (A) Comparison of collection efforts of the years 2003 with 2015, 2016, and 2017.(B) Species accumulation curves of overall species numbers (blue line), of undescribed species (red line), and of species that are not documented in literature or identification books, thus representing at-the-moment-unique species of BNP, North Sulawesi (green line).The species number for the collection event 2003 does not contain the 11 species that could not be reassessed.(C) Comparison of species numbers from geographic areas close by North Sulawesi.Data based on BNP: Kaligis et al. [1],Burghard et al.[3], and this study; Ambon[12][13][14]17]; Indonesia with emphasis on Bali[37]; Vietnam[16]; and Papua New Guinea[39].

Table 1 .
Information about collection sites and dates of collections in 2016 and 2017.
•46 57.5" E Wall-like coral reef structure with terraces and many tiny caves; with hard and soft corals, gorgonians, black corals, colonies of Parazoanthus, hydrozoans, and ascidians.
[11]meaella cf.sp.6 in Gosliner et al.[11]: 368 and in the Figure legends.Species authorities are provided in Table 2 and authorities will only be mentioned in the text for those species not listed in Table 2. Figures 2-16 depict all species collected in 2016, and Figures 17-20 those collected additionally in 2017.Species and genera are compared to previous results