Evolution of the Sacoglossa (Mollusca, Opisthobranchia) and the ecological associations with their food plants

Evolution in the opisthobranch order Sacoglossa has been closely linked to their specialized suctorial herbivorous habits. All shelled Sacoglossa (about 20% of the species) feed on one algal genus, Caulerpa. The non-shelled Sacoglossa have 'radiated' to other diets, mainly siphonalean or septate green algae (Class Ulvophyceae). Comparing the phylogeny of sacoglossan genera with the phylogeny of the Ulvophyceae indicates that co-speciation may have taken place at the basal node of the Sacoglossa, and that host switching has taken place several times in the two non-shelled clades. It is suggested that the most important evolutionary process has been speciation by 'resource-tracking'; the resource tracked is most probably cell wall composition of the algal prey. The fossil record of extant sacoglossan genera dates back to the Eocene and, based on the fossil record of siphonalean green algae, the Sacoglossa most likely appeared in the Cretaceous. It is hypothesized that the ancestral sacoglossan was epifaunal, suctorial and herbivorous, and the 'ancestral' food plant was not Caulerpa, but filamentous, calcified, now extinct, Udoteaceae.     

Molecular determination of kleptoplast origins from the sea slug Plakobranchus ocellatus (Sacoglossa,Gastropoda) reveals cryptic bryopsidalean (Chlorophyta) diversity in the Hawaiian Islands

The sacoglossan sea slug species complex Plakobranchus ocellatus is a common algivore throughout the tropical Pacific, including the Hawaiian Islands. Plakobranchus ocellatus is kleptoplastic—it sequesters and retains algal chloroplasts—a characteristic that can be exploited to molecularly characterize diminutive bryopsidalean algae that are typically difficult to locate, collect, and identify. Previous DNA barcode analyses of both P. ocellatus and its kleptoplasts have been conducted primarily in the western Pacific and have only minimally sampled the most eastern populations in the Hawaiian Islands. Using two chloroplast markers, rbcL and tufA, kleptoplast samples from an Oahu population of P. ocellatus were amplified and cloned to identify their algal sources. Plakobranchus ocellatus sequester chloroplasts from up to 11 bryopsidalean algal species, all but one being diminutive in thallus size. Notably, eight of the detected algal species were new records to the Hawaiian Islands. A sequestration preference study demonstrated that the O‘ahu population of P. ocellatus preferentially sequesters chloroplasts from diminutive, epilithic taxa. Using coxI barcoding of P. ocellatus, we showed the O‘ahu population to be part of a clade that includes sequences from the neighboring island Maui, Australia, and the Philippines. The use of P. ocellatus as a novel sampling tool allows the exploration of the green algal community diversity and composition at a fine scale.     

Phagocytosis of algal chloroplasts by digestive gland cells in the photosynthesis-capable slug Elysia timida (Mollusca, Opisthobranchia, Sacoglossa)

Parapodia of the sacoglossan slug Elysia timida were preserved by high-pressure cryofixation during feeding experiments and investigated with transmission electron microscopy. This slug has been known for its long-term retention of active chloroplasts and photosynthesis. We observed different stages of phagocytosis of chloroplast components from ingested algal food by slug digestive gland cells. Thylakoid stacks and stroma of chloroplasts were engulfed by the slug cells. In the slug cells thylakoids were surrounded by one membrane only. This membrane is interpreted as having been generated by the mollusk during phagocytosis. It is inferred to be eukaryotic in origin and unlikely, therefore, to be endowed with the translocons system ordinarily regulating import of algal gene-encoded plastid preproteins. Our structural findings suggest that chloroplast components in the slug cells are thylakoid stacks with chloroplast stroma only.     

The metabolism of photosynthetically fixed carbon by isolated chloroplasts from Codium fragile (Chlorophyta: Siphonales) and by Elysia viridis (Mollusca: Sacoglossa)

The metabolism of photosynthetically fixed ¹⁴C by isolated chloroplasts from Codium fragile is compared with that by Elysia viridis (which contains C. fragile chloroplasts). There are marked differences between the two in the formation and subsequent metabolism of both soluble and insoluble products. Less than 6% of the carbon fixed by the isolated chloroplasts during a 15 min pulse of ¹⁴C-bicarbonate in the light is released into the medium over the succeeding 24 h. During photosynthesis glycollate and glucose monophosphate are the only labelled compounds released; after the pulse very little glycollate is released and over 24 h only glucose monophosphate and an unidentified compound are found in the medium. In E. viridis photosynthetically fixed carbon can be recovered from compounds of all major classes found in animals. Soon after the pulse, hexoses are the most heavily labelled compounds, but two hours later amino acids are more heavily labelled than hexoses. The unidentified compound is not found in the animals. E. viridis can absorb and metabolize exogenous glycollate and glucose. Earlier authors' suggestions that glucose is the compound which moves from the chloroplast to the animal cell are discussed, and it is proposed that both glucose monophosphate and glucose are formed outside the chloroplast from triose phosphate exported from it.     

The role of kleptoplasts in the survival rates of Elysia timida (Risso, 1818): (Sacoglossa: Opisthobranchia) during periods of food shortage

The importance of photosynthetic products derived from kleptoplasts in several sacoglossan species is being investigated in different fields, such as chemistry, biomolecular genetics and ecology. This study analyzes the effect of kleptoplasts on the survival rates of Elysia timida by evaluating the development of chlorophyll concentration, total length and survival rates of starved E. timida specimens kept in the light and in the dark. Although chlorophyll concentration values were similar in both cases, after 28 days specimens kept in the dark showed a greater size decrease and a lower survival rate (up to 30% lower) than those kept in the light. It is evident that kleptoplasts provide the mollusc with extra energy at the primary metabolism level to compensate for a shortage in food.     

Comparison of Alimentary Systems in Shelled and Non-shelled Sacoglossa (Mollusca,Opisthobranchia)

The Sacoglossa (= Ascoglossa) comprise a ‘complete’ evolutionary series from species with a large shell into which the animal can withdraw completely, through species with a reduced shell covering only the visceral mass, to shell-less (‘nudibranchiate’) forms some of which have lateral wing-like extensions, parapodia, others bearing leaf-like or cylindrical dorsal appendages, cerata. The Sacoglossa are all specialized suctorial feeders, and almost all are stenophagous herbivores. Hence many anatomical adaptations to a particular food occur in the alimentary system. However, a number of characters seem to reflect phylogenetic relationships as well. Among these are presence/absence, shape, and position of pharyngeal pouches, basic shape of radular teeth, branching pattern of digestive gland, and position of anus. The classificatory significance of these characters is discussed.     

Cylindrobulla and Ascobulla in the western Atlantic (Gastropoda, Opisthobranchia, Sacoglossa): Systematic review, description of a new species, and phylogenetic reanalysis

Two often-confused western Atlantic shelled opisthobranchs - Cylindrobulla beauii P. Fischer, 1857, and Ascobulla ulla (Marcus & Marcus, 1970) - are redescribed and compared, via a full literature review, scanning electron microscopy of shells and radulae, and serial histological sectioning of soft anatomy. A third sympatric species, C. gigas, from Florida, the Caribbean and Bermuda, is described as new, and also provides the first larval development data for the genus. The overlapping classificatory histories of the genera Cylindrobulla and Ascobulla are summarized and discussed with reference to the Cephalaspidea, Sacoglossa (= Ascoglossa), Anaspidea, and Diaphanoidea. Two of the most-recent studies (Jensen 1996u, Jensen 1996h; Mikkelsen 1996) have resulted in conflicting conclusions about the relationships and classification of Cylindrobulla. In the author's earlier phylogenetic analysis (Mikkelsen 1996), Cylindrobulla formed a monophyletic group with the shelled Sacoglossa with synapomorphies in digestive, reproductive, pallial, and nervous system characters. Jensen's two-part study (Jensen 1996a, Jensen 1996b) resulted in removal of Cylindrobulla to a new opisthobranch order, Cylindrobullacea. These two divergent cladistically produced classifications were tested by critically re-evaluating Jensen's dataset, especially with regard to Cylindrobulla, and re-running the analysis. Thirteen of Jensen's 52 characters were recoded for Cylindrobulla according to new morphological data and the new species described here. Other coding changes were required to update anatomical data for other taxa, add supplementary characters and taxa, more clearly define the outgroup, and to critically re-evaluate the cladistic criteria behind several characters. The resultant reanalysis produced a shorter tree using fewer a priori assumptions, but nevertheless preserved the basic tree topology, including three monophyletic sacoglossan clades, presented by Jensen (19966). Cylindrobulla, however, rejoined the shelled sacoglossans (Oxynoacea), supported by two synapomorphies. This analysis therefore reconfirmed Cylindrobulla's proper placement in the Sacoglossa, rather than in the traditional Cephalaspidea or segregated in its own monogeneric opisthobranch order. © 1998 The Norwegian Academy of Science and Letters     

The internal anatomy of Stiliger fuscatus (A. Gould, 1870) (Opisthobranchia: Sacoglossa)

The central nervous system of Stiliger fuscatus has two ganglia on the visceral loop. The superficial buccal musculature includes an ascus muscle; its contraction drives the lower limb of the radula in the action of piercing an algal wall. The reproductive system is described in functional terms and related to those of other species of the family Stiligeridae. The vagina does not open to the exterior and the penial style is of the penetrant type; fertilization is by hypodermic impregnation. The system could be called pseudo-diaulic and this term is explained in the additional notes.     

Aeolid nudibranchs (Gastropoda: Opisthobranchia) of the family Tergipedidae from New Zealand waters

Four species of the genus Cuthona Alder & Hancock are recorded for the first time. Two of these species are Cuthona beta (Baba & Abe, 1964) and C. alpha Baba & Hamatani, 1963: although the local specimens differ in some features, principally ceratal arrangement, length of the radula and colour, the differences are considered too slight to warrant separation. The other two are new, being distinguished by a combination of features: C. scintillans sp. nov. by the large size reached (24 mm), rhinophores, oral veil and number of ceratal rows (13), the rounded foot-angles, green diverticula and yellow surface pigmentation, and number of denticles on the largest radular teeth (9); C. reflexa sp. nov. by the simple colouration, short radula (30 teeth), terminal position of the cusp, a very short or no vagina and the renal opening above the anus.
The name Tergipedidae (= Cuthonidae) is given priority and its use reviewed. Three subfamilies are recognized viz. Cuthonellinae, Cuthoninae and Tergipedidinae, each founded on the division of the digestive gland. Thirteen genera are listed, but only seven are firmly established as being distinctive and belonging to the family; one of the remaining six, Guyvalvoria Vayssiere, 1906, is certainly valid, but, because the ceratal arrangement is only superficially known, its place in the family could not be determined.     

Aeolid nudibranchs (Gastropoda: Opisthobranchia) of the family Glaucidae from New Zealand waters

Two new species of aeolid, a new genus being created for one of them, are described along with another two species, one Phidiana militaris (Alder & Hancock) (= Learchis militaris ) a new record for New Zealand, the other, Glaucus atlanticus , found only once before. Jason mirabilis gen. et sp. nov. is distinguished by a ventral tongue-like process with a ridged chitinous covering which replaces the radula (still present but vestigial) and by the greatly subdivided rhinophoral lamellae. Babaina caprinsulensis sp. nov. differs from its Californian relative in having papillate rhinophores. The New Zealand specimens of Phidiana militaris differ slightly in colouration from those described for elsewhere in the Indo-Pacific.
The Glaucidae is enlarged to include the Babainidae, Facelinidae and Favorinidae, the main familial characteristics being the simple oral glands, sharp radular tooth and pugnacious behaviour. Possible evolutionary trends within the family based on the arrangement of the cerata and penial structure are traced out. The Babaininae, pleuroproctic with a notal ridge and irregular disposition of the cerata, is considered the most primitive subfamily and the Hervellinae, with cerata in single rows, the most advanced. The many facelinid genera previously recognized are reduced to two, Antionetta Schmekel and Phidiana Gray.     

Comparative immunohistochemistry of the cephalic sensory organs in Opisthobranchia (Mollusca,Gastropoda)

The structure and function of the central nervous systems of opisthobranch gastropods have been studied extensively. However, the organisation and function of the peripheral nervous system are poorly understood. The cephalic sensory organs (CSOs) are known to be chemosensory structures in the head region of opisthobranchs. In the present study, we used immunohistochemical methods and confocal laserscanning microscopy to comparatively examine the CSOs of different opisthobranchs, namely Acteon tornatilis, Aplysia punctata, Archidoris pseudoargus and Haminoea hydatis. We wanted to characterise sensory epithelia in order to infer the function of sensory structures and the organs they constitute. Immunoreactivity against the three antigens tyrosine hydroxylase, FMRFamide and serotonin was very similar in the CSOs of all investigated species. Tyrosine hydroxylase-like immunoreactivity was detected primarily in subepidermal sensory cell bodies, which were much more abundant in the anteriorly situated CSOs. This observation indicates that these cells and the respective organs may be involved in contact chemoreception and mechanoreception. The dominant features of FMRFamide-like immunoreactivity, especially in the posterior CSOs, were tightly knotted fibres which reveal glomerulus-like structures. This suggests an olfactory role for these organs. Serotonin-like immunoreactivity was detected in an extensive network of efferent fibres, but was not found within any peripheral cell bodies. Serotonin-like immunoreactivity was found in the same glomerulus-like structures as FMRFamide-like immunoreactivity, indicating a function of serotonin in the efferent control of olfactory inputs. Besides this functional implication, this study could also add some knowledge on the doubtful homology of the CSOs in opisthobranch gastropods.     

A new poecilogonous species of sea slug (Opisthobranchia: Sacoglossa) from California: comparison with the planktotrophic congener Alderia modesta (Loven, 1844)

Cryptic species are increasingly recognized as commonplace amongmarine gastropods, especially in taxa such as shell-less opisthobranchsthat lack many discrete taxonomic characters. Most cases ofpoecilogony, the presence of variable larval development withina single species, have historically turned out to representcryptic species, with each possessing a single canalized typeof development. One well-characterized example of poecilogonywas attributed to the sacoglossan opisthobranch Alderia modesta;in southern California, slugs resembling this member of a monotypicgenus produce both long-lived, planktotrophic and short-lived,lecithotrophic larvae. Paradoxically, however, A. modesta isexclusively planktotrophic everywhere else in the northern Pacificand Atlantic Oceans. A recently completed molecular study foundthat slugs from poecilogonous populations south of Bodega Harbor,California, comprise an evolutionarily distinct lineage separatefrom northern, strictly planktotrophic slugs. We now describethe southern species as A. willowi n. sp., based on differencesin morphology of the dorsum and radula, characteristics of theegg mass, larval development mode and nuclear and mitochondrialgenetic markers. A DNA barcode is provided, based on 27 fixeddifferences in the cytochrome c oxidase subunit I gene thatcan reliably differentiate Pacific specimens of Alderia species.Genetic and morphological data are concordant with developmentalevidence, confirming that A. willowi is a true case of poecilogony.An improved understanding of the ecological differences betweenthese sister taxa may shed light on the selective pressuresthat drove the evolution of lecithotrophy in the southern species. (Received 1 November 2005; accepted 20 September 2006)     

Behavioral thermoregulation of Bulla gouldiana (Gastropoda: Opisthobranchia: Cephalaspidea)

1. 1. The preferred temperature of Bulla gouldiana is 26.7–28.7°C.

2. 2. In constant scotophase, photophase, and light and dark photoperiod the organisms do not have a diel cycle of thermoregulation.

3. 3. It takes the animal 6–16 h to reach the preferred temperature.

4. 4. The lowest and highest temperatures visited were 11 and 33°C.

5. 5. Spawning of the species occurred in the thermal gradient between 27 and 28.5°C.

     

Molecular confirmation of the occurrence of Elysia cf. tomentosa (Mollusca: Heterobranchia) in the Persian Gulf

The molluscan fauna of the Persian Gulf has recently been relatively well documented, yet there are few records of heterobranch sea slugs (opisthobranchs) from the Arabian parts and no report from the Iranian waters. Here we report for the first time the occurrence of one of these molluscs in the northern Persian Gulf (Bandar Abbas, Iran). Sacoglossan specimens were collected in association with the seaweed, Caulerpa sertularioides. Since morphological attributes were not adequately reliable for species identification, molecular approaches were carried out. Maximum-likelihood and Bayesian Inference analysis of partial DNA sequences of the mitochondrial cytochrome c oxidase subunit I (COI) locus were used for DNA barcoding of large-bodied specimens of Elysia. All Persian Gulf specimens were genetically confirmed as Elysia cf. tomentosa sp. 5, one of at least five morphologically similar but genetically distinct species in the taxonomically challenging and unresolved E. tomentosa complex. This species has previously been recorded only from Australia and Thailand and our finding adds another distant point to the geographic distribution of this species.     

Towards a phylogeny and evolution of Acochlidia (Mollusca: Gastropoda: Opisthobranchia)

The Acochlidia are unique among opisthobranch gastropods in combining extremely high morphological and ecological diversity with modest species diversity. The phylogeny of acochlidians has never been addressed by cladistic means, as their evolution has remained unknown. This study gives a first overview on more than 150 biological and morphological characters that are potentially useful for phylogenetic analysis. Based on 107 characters, a parsimony analysis (PAUP) was performed for all 27 valid acochlidian species together with 11 (plus two) outgroup taxa. The resulting strict consensus tree shows a moderate overall resolution, with at least some bootstrap support for most resolved nodes. The Acochlidia are clearly monophyletic, and originate from an unresolved basal opisthobranch level. The Acochlidia split into the Hedylopsacea (Tantulum (Hedylopsis (Pseudunela (Strubellia (‘Acochlidium’, ‘Palliohedyle’))))) and Microhedylacea (Asperspina (Pontohedyle, ‘Parhedyle’, ‘Microhedyle’, (Ganitus, Paraganitus))). The formerly enigmatic Ganitidae, resembling sacoglossan opisthobranchs by having dagger‐like rachidian radular teeth, are likely to be highly derived microhedylids. The paraphyly of some of the traditionally recognized family level taxa induced a preliminary reclassification. From the phylogenetic hypothesis obtained, we conclude that the acochlidian ancestor was marine mesopsammic. The colonization of limnic systems occurred twice, independently: first in the Caribbean (with the development of the small interstitial Tantulum elegans), and second in the Indo‐Pacific, with a radiation of large‐sized benthic acochlidian species. The evolution of extraordinary reproductive features, such as hypodermic impregnation by a complex copulative aparatus in hedylopsaceans, cutaneous insemination via spermatophores in microhedylaceans, and gonochorism in Microhedylidae s.l. (including Ganitidae), is discussed. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 124–154.