Really a "secondary gill under the skin"? Unveiling "dorsal vessels" in freshwater slugs (Mollusca, Panpulmonata, Acochlidimorpha).

The freshwater slugs of the genus Acochlidium (Heterobranchia, Gastropoda, and Acochlidimorpha) are peculiar, one to two centimeter sized animals found only in small coastal rivers and streams of Southeast Asian and Western Pacific islands. When first described by Bücking, the author observed a branching "net of dendritic vessels connected to the heart," which he assumed to have replaced the original gastropod gill. In the present study, we compare the renopericardial systems of four Acochlidium species in microanatomical, histological and ultrastructural detail and identify where exactly the enigmatic, subepidermal "dorsal vessels" connect to the renopericardial system to examine if they can really function as a gill. Acochlidium have elaborate renopericardial systems compared to their ancestrally marine and also freshwater relatives. The primary site of ultrafiltration is the epicardium of the atrium with podocytes as usual for gastropods. The "dorsal vessels" in Acochlidium are extensions of the outer epithelium of the pericardial cavity and represent true vessels, that is, coelomatic channels, having an endothelium with podocytes. Hence, they considerably enlarge the site of ultrafiltration increasing the pericardial surface. "Dorsal vessels" in Acochlidium are therefore not homologous to externally similar morphological structures in Sacoglossa (marine panpulmonate slugs and snails). The multiplication of renopericardioducts in Acochlidium is a unique feature within Mollusca that enhances the negative pressure necessary for ultrafiltration in the thin, tube-like dorsal vessels and as a consequence the transport of primary urine from the pericardium to the kidney. The circulatory and excretory systems in Acochlidium are adaptations to a lifestyle in their freshwater environment in which snail bodies are hyposmotic and accrue considerable influx of surplus water into the body, which needs to be expelled.

Uncovering the shell game with barcodes: diversity of meiofaunal Caecidae snails (Truncatelloidea, Caenogastropoda) from Central America.

Caecidae is a species-rich family of microsnails with a worldwide distribution. Typical for many groups of gastropods, caecid taxonomy is largely based on overt shell characters. However, identification of species using shell characteristics is problematic due to their rather uniform, tubular shells, the presence of different growth stages, and a high degree of intraspecific variability. In the present study, a first integrative approach to caecid taxonomy is provided using light-microscopic investigation with microsculptural analyses and multi-marker barcoding, in conjunction with molecular species delineation analyses (ABGD, haplotype networks, GMYC, and bPTP). In total 132 specimens of Caecum and Meioceras collected during several sampling trips to Central America were analyzed and delineated into a minimum of 19 species to discuss putative synonyms, and supplement the original descriptions. Molecular phylogenetic analyses suggest Meioceras nitidum and M. cubitatum should be reclassified as Caecum, and the genus Meioceras might present a junior synonym of Caecum. Meiofaunal caecids morphologically resembling C. glabrum from the Northeast Atlantic are a complex of cryptic species with independent evolutionary origins, likely associated with multiple habitat shifts to the mesopsammic environment. Caecum invisibile Egger & Jörger, sp. nov. is formally described based on molecular diagnostic characters. This first integrative approach towards the taxonomy of Caecidae increases the known diversity, reveals the need for a reclassification of the genus Caecum and serves as a starting point for a barcoding library of the family, thereby enabling further reliable identifications of these taxonomically challenging microsnails in future studies.

The unique deep sea-land connection: interactive 3D visualization and molecular phylogeny of Bathyhedyle boucheti n. sp. (Bathyhedylidae n. fam.)-the first panpulmonate slug from bathyal zones.

The deep sea comprises vast unexplored areas and is expected to conceal significant undescribed invertebrate species diversity. Deep waters may act as a refuge for many relictual groups, including elusive and enigmatic higher taxa, but the evolutionary pathways by which colonization of the deep sea has occurred have scarcely been investigated. Sister group relationships between shallow water and deep sea taxa have been documented in several invertebrate groups, but are unknown between amphibious/terrestrial and deep-sea species. Here we describe in full and interactive 3D morphoanatomical detail the new sea slug species Bathyhedyle boucheti n. sp., dredged from the continental slope off Mozambique. Molecular and morphological analyses reveal that it represents a novel heterobranch gastropod lineage which we establish as the new family Bathyhedylidae. The family is robustly supported as sister to the recently discovered panpulmonate acochlidian family Aitengidae, which comprises amphibious species living along the sea shore as well as fully terrestrial species. This is the first marine-epibenthic representative among hedylopsacean Acochlidiida, the first record of an acochlidian from deep waters and the first documented panpulmonate deep-sea slug. Considering a marine mesopsammic ancestor, the external morphological features of Bathyhedyle n. gen. may be interpreted as independent adaptations to a benthic life style in the deep sea, including the large body size, broad foot and propodial tentacles. Alternatively, the common ancestor of Bathyhedylidae and Aitengidae may have been a macroscopic amphibious or even terrestrial species. We hypothesize that oophagy in the common ancestor of Aitengidae and Bathyhedylidae might explain the impressive ecological and evolutionary flexibility in habitat choice in the Acochlidiida.

Cryptic species in tropic sands--interactive 3D anatomy, molecular phylogeny and evolution of meiofaunal Pseudunelidae (Gastropoda, Acochlidia).

BACKGROUND: Towards realistic estimations of the diversity of marine animals, tiny meiofaunal species usually are underrepresented. Since the biological species concept is hardly applicable on exotic and elusive animals, it is even more important to apply a morphospecies concept on the best level of information possible, using accurate and efficient methodology such as 3D modelling from histological sections. Molecular approaches such as sequence analyses may reveal further, cryptic species. This is the first case study on meiofaunal gastropods to test diversity estimations from traditional taxonomy against results from modern microanatomical methodology and molecular systematics. RESULTS: The examined meiofaunal Pseudunela specimens from several Indo-Pacific islands cannot be distinguished by external features. Their 3D microanatomy shows differences in the organ systems and allows for taxonomic separation in some cases. Additional molecular analyses based on partial mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA markers revealed considerable genetic structure that is largely congruent with anatomical or geographical patterns. Two new species (Pseudunela viatoris and P. marteli spp. nov.) are formally described integrating morphological and genetic analyses. Phylogenetic analysis using partial 16S rRNA, COI and the nuclear 18S rRNA markers shows a clade of Pseudunelidae species as the sister group to limnic Acochlidiidae. Within Pseudunela, two subtypes of complex excretory systems occur. A complex kidney already evolved in the ancestor of Hedylopsacea. Several habitat shifts occurred during hedylopsacean evolution. CONCLUSIONS: Cryptic species occur in tropical meiofaunal Pseudunela gastropods, and likely in other meiofaunal groups with poor dispersal abilities, boosting current diversity estimations. Only a combined 3D microanatomical and molecular approach revealed actual species diversity within Pseudunela reliably. Such integrative methods are recommended for all taxonomic approaches and biodiversity surveys on soft-bodied and small-sized invertebrates. With increasing taxon sampling and details studied, the evolution of acochlidian panpulmonates is even more complex than expected.

Tiny but complex - interactive 3D visualization of the interstitial acochlidian gastropod Pseudunela cornuta (Challis, 1970).

BACKGROUND: Mesopsammic acochlidians are small, and organ complexity may be strongly reduced (regressive evolution by progenesis), especially in microhedylacean species. The marine interstitial hedylopsacean Pseudunela cornuta (Challis, 1970), however, was suggested as having a complex reproductive system resembling that of much larger, limnic and benthic species. The present study aims to reconstruct the detailed anatomy and true complexity of P. cornuta from serial, semithin histological sections by using modern computer-based 3D visualization with Amira software, and to explain it in an evolutionary context. RESULTS: Our results demonstrate considerable discordance with the original species description, which was based solely on paraffin sections. Here, we show that the nervous system of P. cornuta has paired rhinophoral, optic and gastro-oesophageal ganglia, three distinct ganglia on the visceral nerve cord, and a putative osphradial ganglion, while anterior accessory ganglia are absent. The presence of an anal genital cloaca is clearly rejected and the anus, nephropore and gonopore open separately to the exterior; the circulatory and excretory systems are well-differentiated, including a two-chambered heart and a complex kidney with a long, looped nephroduct; the special androdiaulic reproductive system shows two allosperm receptacles, three nidamental glands, a cavity with unknown function, as well as highly complex anterior copulatory organs with two separate glandular and impregnatory systems including a penial stylet that measures approximately a third of the whole length of the preserved specimen. CONCLUSION: In spite of its small body size, the interstitial hermaphroditic P. cornuta shows high complexity regarding all major organ systems; the excretory system is as differentiated as in species of the sister clade, the limnic and much larger Acochlidiidae, and the reproductive system is by far the most elaborated one ever observed in a mesopsammic gastropod, though functionally not yet fully understood. Such organ complexity as shown herein by interactive 3D visualization is not plesiomorphically maintained from a larger, benthic ancestor, but newly evolved within small marine hedylopsacean ancestors of P. cornuta. The common picture of general organ regression within mesopsammic acochlidians thus is valid for microhedylacean species only.

Computer-based three-dimensional reconstruction of the anatomy of Microhedyle remanei (Marcus, 1953), an interstitial acochlidian gastropod from Bermuda.

Phylogenetic analysis of the highly enigmatic and aberrant acochlidian opisthobranch gastropods is mainly hindered by the lack of reliable anatomical data. Due to their small size, marine interstitial acochlidian species are not suitable for anatomical investigations by dissecting. The tinyness and complexity of, e.g., opisthobranch central nervous and reproductive systems make data derived from older paraffin-based histology and interpretation by hand-based graphical reconstruction questionable. In a former study, a hermaphroditic member of the basal acochlidian genus Hedylopsis ("Hedylopsacea") has been examined in detail. The present study uses the gonochoric Microhedyle remanei (Microhedylidae) as a model organism of the other traditional major acochlidian subgroup, the "Microhedylacea." More than 20 specimens with up to 2 mm body length were extracted from coarse subtidal sand near Castle Roads, Bermuda Islands. Their central nervous, digestive, circulatory, excretory, and genital systems were reconstructed three-dimensionally from serial semithin histological sections using AMIRA software. The radula was analyzed by SEM. Our specimens closely resemble the original description of M. remanei (Marcus, 1953; as Unela) from Brazil; apparent differences regarding the number of visceral loop ganglia or details of male genitalia are assumed to be due to oversights by the former author or because of different ontogenetic stages. Microhedyle remanei differs from all congeners due to the lack (vs. presence) of eyes; further distinguishing features are discussed. In contrast to members of the hedylopsacean Hedylopsidae, Acochlidiidae, and Tantulidae, M. remanei shows a nervous system with numerous precerebral accessory "ganglia" that are not differentiated into cortex and medulla. While all Microhedylidae previously were thought to lack a heart, M. remanei shows a two-chambered heart, as is usual for opisthobranchs. The oocytes of M. remanei are yolky and large in relation to body size and suggest intracapsular larval development. A comparative microanatomical discussion and the distribution of characters within acochlidian taxa indicate that the current classification of the Acochlidia does not reflect phylogenetic relationships. Detailed structural investigations on further poorly known species are required; the computer-based 3D reconstruction of semithin serial sections with AMIRA is shown to be an ideal tool for efficient analysis and presentation of the microanatomy of small specimens.