Homology and morphology of the neogastropod valve of Leiblein (Gastropoda: Caenogastropoda)
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- Golding, R.E. & Ponder, W.F. Zoomorphology (2010) 129: 81. doi:10.1007/s00435-009-0101-0
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The valve of Leiblein is a morphological synapomorphy defining Neogastropoda, but is also a significant adaptation in the evolution of carnivorous, predatory feeding and homologues have not been recognised amongst non-neogastropods to date. This study uses histology to examine the valve of Leiblein and associated oesophageal features of a buccinoidean (Euplica scripta—Columbellidae) and two muricoideans (Morula marginalba—Muricidae, and Columbarium pagodoides—Turbinellidae), and comparisons are made with the oesophagus of four non-neogastropods; the tonnoidean out-group Cabestana spengleri and three other littorinimorph caenogastropods. Several morphological details conflict with earlier descriptions of different neogastropod species, such as the presence of a diverticulum in the muricoidean valve and torsion in the buccinoidean valve. This supports recent findings that the valve of Leiblein is morphologically heterogeneous, but, unlike some other studies, does not dispute the homology of the valve in different neogastropod groups. Structure and histology support the homology of the valve of Leiblein in Muricoidea and Buccinoidea and its derivation from elements of the anterior oesophagus of higher littorinimorph caenogastropods (Tonnoidea). A thick, secretory, pseudostratified epithelium lining the ventral anterior oesophagus of C. spengleri is comparable with the tissue lining the glandular chamber of the valve of Leiblein. A modified theory regarding the evolutionary origin of the valve of Leiblein is proposed in which it is homologous to the anterior oesophagus of Tonnoidea.
The evolution of carnivory in Caenogastropoda is associated with a variety of morphological and physiological specialisations which facilitate prey detection, capture and digestion. These modifications include increased mobility, enhanced chemosensory abilities (enlarged osphradium), simplification of the midgut and the production of novel digestive enzymes through elaboration of components of the alimentary system, such as the oesophageal gland and salivary glands (Taylor et al. 1980; Taylor 1998; Andrews et al. 1999; Strong 2003). Although several groups of caenogastropods are carnivorous, the largest radiation has occurred within Neogastropoda, a hyper-diverse clade of predatory snails including cones, whelks and drills (Ponder 1974; Taylor et al. 1980; Kantor 1996, 2002). A morphological feature which distinguishes most neogastropods from other carnivorous caenogastropods is the valve of Leiblein; a structure which promotes the unidirectional passage of food through the oesophagus (Amaudrut 1898; Graham 1941; Fretter and Graham 1994).
The valve of Leiblein consists of a short, circular fold of tissue projecting posteriorly into an expanded cavity lined with a thick secretory epithelium (Graham 1941; Andrews and Thorogood 2005; Kantor and Fedosov 2009). Food passing through the circular fold into the cavity is coated with lubricating mucous secretions while strong, posteriorly directed ciliary currents around the apex of the fold prevent anterior regression of particles (Graham 1941). The hypothesised function of the valve may be to prevent regurgitation of food during feeding (Graham 1941), retain digestive secretions from the gland of Leiblein (Ponder 1974) or separate liquid from solid food for transport to the gland of Leiblein (Andrews and Thorogood 2005). Andrews and Thorogood (2005) described the detailed anatomy of the valve and gland of Leiblein of the muricid Nucella lapillus (Linnaeus, 1758). They discovered that a ‘siphon’ (=ventral groove or ‘scar’ dividing the oesophageal epithelium), bordered by the swollen dorsal folds, runs from the valve to the aperture of the gland of Leiblein and may be involved in the transportation of liquid and solutes to the absorptive gland of Leiblein (Andrews and Thorogood 2005).
The valve of Leiblein occurs in all major groups of neogastropods, although in several groups it is greatly reduced or lost (Ponder 1974; Taylor and Morris 1988; Kantor 2002; Andrews and Thorogood 2005). Until a similar structure was recently identified in two species of Conidae (Kantor and Taylor 2002), the valve of Leiblein was not known from Conoidea. No homologue to the valve of Leiblein has been identified in the alimentary system of other caenogastropods. The unique structure and limited occurrence of the valve of Leiblein have resulted in it being considered one of the main synapomorphies of Neogastropoda (e.g., Taylor and Morris 1988), but its detailed structure has been examined in few taxa (Graham 1941; Kantor and Taylor 2002; Andrews and Thorogood 2005). The recent study by Kantor and Fedosov (2009) provided a wealth of morphological information, examining the valve of Leiblein in seven neogastropods from all major lineages.
Graham (1941) postulated that the valve of Leiblein is derived from the dorsal food channel of the mid-oesophagus. He proposed that the ventral section (oesophageal gland) of the mid-oesophagus (including the section in the vicinity of the valve of Leiblein) was stripped from the dorsal section to form the gland of Leiblein, allowing the remaining dorsal section of the oesophagus to be ‘pulled’ through the nerve ring. He postulated that the dorsal food channel of the mid-oesophagus, including the dorsal folds, was modified to form the valve and its glandular chamber. Evidence for this theory includes the presence of a ‘scar’ on the morphologically ventral surface of the valve of Leiblein, indicating the site of separation from the oesophageal gland, and the ‘lubricating’ secretions of the tissue lining the valve which Graham identified with the epithelia lining the dorsal food channel. Graham’s theory was further illustrated by Andrews and Thorogood (2005), who agreed that the valve of Leiblein was a modification of the dorsal section of the mid-oesophagus.
Ponder (1974) postulated that neogastropods are derived from ‘archaeogastropod’ or ‘lower caenogastropod’ stock, rather than higher caenogastropods and that the valve of Leiblein is a modification of ‘archaeogastropod’ oesophageal (buccal) pouches. These structures are glandular diverticula which lie immediately posterior to the buccal mass. However, this theory of the derivation of neogastropods is not supported by recent phylogenetic analyses which indicate that higher littorinimorph caenogastropods are the sister to neogastropods (Healy 1988; Ponder and Lindberg 1997; Riedel 2000; Strong 2003; Ponder et al. 2008). Ponder (1974) also suggested that the anterior oesophagus of neogastropods was an elongation of the posterior buccal cavity.
These hypotheses are incompatible, but there is limited evidence available to support either case. Perhaps due to the paucity of information, both Graham (1941) and Kantor and Fedosov (2009) concluded that the valve of Leiblein of Muricoidea and that of Buccinoidea are not homologous. However, the discussion is incomplete without comparing the homology of the tissues composing the valve of Leiblein to the oesophagus of other caenogastropods.
In order to address the questions raised earlier, we investigated the oesophagus of representative taxa from two neogastropod superfamilies, Muricoidea and Buccinoidea, a ranellid (Tonnoidea) and three other littorinimorph caenogastropods, to investigate the derivation and homology of the valve of Leiblein.
Materials and methods
Caenogastropod specimens examined in this study with classification to superfamily, family and species with authority
Strombus gibberulus (Linnaeus, 1758)
Shark Bay, Heron Island, QLD
Conuber melastomum (Swainson, 1822)
Careel Bay, Pittwater, NSW
R. Golding and M. Hill
E of Yamba, NSW, dredged at 70 m
K. J. Graham, FRV Kapala
Cabestana spengleri (Perry, 1811)
Mona Vale Beach, Sydney, NSW
R. Golding and P. Golding
Euplica scripta (Lamarck, 1822)
Bradley’s Head, Port Jackson, NSW
R. Golding, W. F. Ponder and D. Beechey
Morula marginalba (Blainville, 1832)
Mona Vale Beach, Sydney, NSW
R. Golding and P. Golding
Columbarium pagodoides (Watson, 1882)
Dredged off Sydney, NSW
The head and anterior alimentary system of one individual from each species was removed by dissection then dehydrated and saturated with Paraplast™ paraffin, using a Tissue-Tek® VIP processor. The specimens were then embedded in paraffin using a Tissue-Tek® TEC embedding station and serially sectioned on an American Optical microtome at between 5 and 7 μm, depending on specimen size. The sections were stained with Mayer’s Haematoxylin and Cason’s trichrome (acid fuchsin, aniline blue and orange G) and photographed using an Olympus DP70 digital camera mounted on an Olympus BX50 microscope with Olympus DPController image acquisition software. Additional specimens of C. spengleri were dissected under a Leica dissecting microscope with a camera lucida attached, to assist illustration of the oesophageal folds.
The dorsal food channel of the anterior oesophagus, bordered ventrally by the dorsal folds, is lined with a relatively low, densely ciliated, columnar epithelium composed of basophilic (dark purple-stained) cells with centrally positioned nuclei, interspersed with occasional mucous cells (Fig. 2a, b). The ventral channel is entirely covered with a much taller, pseudostratified epithelium (Fig. 2b; pse) composed of glandular cells which stain pale purple with Cason’s trichrome and contain basal nuclei. Small, wedge-shaped ciliary cells are positioned between the glandular cells, forming a second layer of nuclei near the surface. The epithelium lining the ventral channel is divided into three discrete sheets; two covering the ventral surfaces of the dorsal folds and a thick median sheet referred to here as the ventral glandular pad (Fig. 2b; vgp). The cells composing the glandular epithelium on the ventral surfaces of the dorsal folds show some histological variation, as the cell contents become progressively more transparent and the cells more inflated with cell contents posteriorly (Fig. 2c).
Posterior to the circumoesophageal nerve ring, the ventral glandular epithelium is reduced in height and confluent with the oesophageal gland (Fig. 2d; og). The oesophageal gland originates as a series of septate outpockets from the ventral wall of the mid-oesophagus, and these pockets are lined with a distinct epithelium composed of bright red-stained, protein-secreting cells (Fig. 2d). The dorsal folds adjacent to the oesophageal gland retain their asymmetry and a low epithelium typical of the anterior oesophagus.
The dorsal and ventral channels of the anterior oesophagus, and the interior surface of the circular fold, are lined with a relatively low epithelium containing mucous cells (Fig. 3a, e). Extremely long cilia are present on the interior surface of the circular fold, particularly around the posterior margin (Fig. 3c, g). The anterior dorsal and ventral surfaces of the glandular chamber and the outer surface of the circular fold are dominated by mucous cells (the mucous pad, Fig. 3b, f; mp). The remainder of the glandular epithelium within the chamber is composed of a pseudostratified epithelium (Fig. 3c, d; pse) of very tall, pale to dark purple-stained cells with basal nuclei, interspersed with sparsely ciliated cells to create an apparent double layer of nuclei (Fig. 3c, d, f–h). The posterior of the chamber is narrower, and the glandular epithelium tapers off to be replaced by low columnar cells similar to those found in the anterior oesophagus (Fig. 3h).
The epithelium lining the anterior oesophagus is composed of low, columnar cells with centrally positioned nuclei and sparse ciliation. The interior of the circular fold has a similar histology, but has longer and denser cilia (Fig. 5c). A ciliated, mucous epithelium is restricted to the dorsal wall of the glandular chamber, surrounding the fused surface of the circular fold (Fig. 5b, c). The remainder of the glandular chamber is composed of a tall, psueodostratified epithelium (Fig. 5b–d; pse) of pale purple-stained cells with basal nuclei, overlayed by a layer of densely ciliated cells.
Inadequate knowledge of the morphology of the valve of Leiblein has impeded interpretation of the functional and evolutionary significance of this structure. This study has described the morphology and histology of the anterior oesophagus for a small sample of neogastropods and non-neogastropod out-groups. The results have shown additional anatomical details of the valve of Leiblein in three neogastropod species and support for the homology of this structure within Neogastropoda. This study has also demonstrated similarities between the tissues present in the anterior oesophagus of a putative out-group to Neogastropoda, leading to a new hypothesis on the evolution of the valve of Leiblein.
Morphological variation of the valve of Leiblein
Previous studies have demonstrated the morphological heterogeneity of the valve of Leiblein across different neogastropod groups (Graham 1941; Kantor and Fedosov 2009). There are several inconsistencies between previous descriptions of the valve of Leiblein of Buccinoidea and Muricoidea and those presented here, which may be due to either more detailed examination or to differences between species. The valve of Euplica scripta (Buccinoidea) was found to be twisted by torsion, unlike the buccinoidean valves described by Graham (1941) and Kantor and Fedosov (2009). In addition, the blind-ending diverticulum detected in the right ventrolateral surface of the glandular chamber of the muricoidean valve has not been described in previous studies. This curious structure has no obvious function but is perhaps related to the operation of the siphon (i.e., the ventral groove) which connects the valve to the gland of Leiblein in Nucella sp. (Andrews and Thorogood 2005).
Morphological variation in the structure of the valve of Leiblein has been interpreted by some authors as evidence of independent derivation (Graham 1941; Kantor and Fedosov 2009), in opposition to the more widely held view that the valve of Leiblein is a highly significant morphological synapomorphy supporting the monophyly of Neogastropoda (e.g., Taylor and Morris 1988; Strong 2003). The valve of Leiblein present in some conoidean and cancellarioidean taxa is not well characterised and is of uncertain homology to the valve in Buccinoidea and Muricoidea (Kantor and Fedosov 2009) and, as they were not examined in this study, are not the focus of the following discussion.
Graham (1941) theorised that the buccinoidean and muricoidean valves are not homologous because only the latter are positioned on the site of torsion. Based on the findings of the present study, we dispute this reasoning, because at least one buccinoidean species (E. scripta) displays torsion through the valve of Leiblein. Kantor and Fedosov (2009) also consider buccinoidean and muricoidean valves to be putatively analogous, in part because the ventral groove between the dorsal folds which is present in Muricoidea does not extend through the glandular chamber of the buccinoidean valve. However, Graham (1941) illustrated the valve of Leiblein of Buccinum undatum (Buccinidae) with a ventral groove originating in the posterior part of the glandular chamber of the valve, which he interpreted as the remainder of the dorsal folds. The buccinoidean examined in this study does not have a ventral groove, but the glandular chamber is clearly marked by a pair of ventrolateral depressions which may be the vestigial remains of the tissue separating the dorsal folds from the oesophageal wall. This feature requires further examination of other taxa to determine its significance.
The gross structure of each valve is similar, with a narrow anterior aperture leading to a posteriorly directed circular fold, surrounded by a glandular chamber.
The valve is positioned immediately anterior to the nerve ring.
The epithelium lining the circular fold is relatively low and dark-staining.
The glandular epithelium is predominantly pseudostratified, with dense ciliation.
The glandular epithelium is differentiated near the junction with the circular fold, where inflated cells with pale-stained contents (probably mucous cells) are present.
The glandular epithelium is marked ventrally by a longitudinal groove (Muricoidea) or pair of indentations (Buccinoidea).
These similarities strongly support the homology of the valve of Leiblein. The homology of this structure with non-neogastropods is discussed in the following paragraphs.
Evolutionary derivation of the neogastropod valve of Leiblein
The evolutionary origin of the valve has not been investigated in any recent comparative morphological studies, nor are there any previous reports of valve of Leiblein-like structures in the oesophagus of any non-neogastropod taxa. This study presents evidence of a putatively homologous structure in the anterior oesophagus of the ranellid, Cabestana spengleri.
There is considerable evidence that the circular fold in the valve of Leiblein is a modification of (and homologous to) the dorsal folds, as proposed by Graham (1941) and supported by Andrews and Thorogood (2005). In Muricoidea, the circular fold is divided ventrally, and the base of each side of the circular fold is fused to the corresponding dorsal fold (Fig. 3b). The presence of the dorsal folds through the glandular chamber is also confirmed by the fact that the left fold is larger than the right. This asymmetry is also present on the anterior and mid-oesophageal dorsal folds of C. spengleri (Fig. 2b), Lamellaria sp. (Fig. 1b) and some other caenogastropods (Andrews and Thorogood 2005). Further evidence is provided by histology, with both the dorsal food channel (bordered by the dorsal folds) and the circular fold lined with a relatively low, dark-staining, ciliated epithelium.
Histological examination of the anterior oesophagus of C. spengleri shows that a very thick epithelium lining the ventral channel closely resembles the lining of the glandular chamber of the valve of Leiblein. Both tissues have a strikingly similar histology—a thick, pseudostratified epithelium (Brown 1969; Kantor and Fedosov 2009) with tall, columnar cells, bearing low ciliation. In both groups, the epithelium is ventral in the anterior oesophagus, although the glandular chamber of the valve in muricoids is divided by a narrow strip of non-glandular tissue between the dorsal folds. This groove is possibly inconsistent with the homology inferred here (also see in the following paragraphs), but may be a specialisation associated with the siphon (i.e., ventral groove) which connects the valve to the gland of Leiblein. A reduction in the epithelium in this region in some taxa would perhaps be advantageous to the passage of liquid and solutes (Andrews and Thorogood 2005).
As outlined in the Introduction, two opposing theories have been put forward to explain the evolutionary origin of the valve of Leiblein. Graham’s (1941) theory of oesophageal modification proposed that the valve of Leiblein is homologous to the mid-oesophagus of other caenogastropods. He suggested that the ventral tissue of the mid-oesophagus was stripped entirely away from the mid-oesophagus (including the valve) to form the gland of Leiblein, leaving only a ventral groove (‘scar’) in the ventral midline of the oesophagus. The results presented here do not support this interpretation, because the epithelium lining the glandular section of the valve of Leiblein is totally dissimilar to the tissue lining the dorsal section (dorsal food channel) of the mid-oesophagus of other caenogastropods. The alternative theory, put forward by Ponder (1974), has the valve of Leiblein derived from the oesophageal pouches in the posterior region of the buccal mass of an archaeogastropod or lower caenogastropod ancestor. As discussed earlier, this is unlikely given current phylogenetic interpretations of Gastropoda (for example, Ponder and Lindberg 1997; Aktipis et al. 2008).
Instead, we propose a modification of Ponder’s (1974) hypothesis. The histological similarities between the glandular layers in the valve of Leiblein and the anterior oesophagus of the ranellid C. spengleri suggest that the valve is homologous to the anterior oesophagus of closely related caenogastropods. There is good evidence for a close relationship between Tonnoidea and Neogastropoda (Riedel 2000; Colgan et al. 2007; Ponder et al. 2008), supporting the theory that the epithelial layers in these structures are corresponding and homologous.
This theory is supported by the weak or absent dorsal folds of the anterior section of the neogastropod oesophagus. In other caenogastropods, the dorsal folds are prominent in the anterior oesophagus, but they are absent or reduced in the buccal region of the gut (Strong 2003). In addition, evidence for the buccal origin of the anterior part of the oesophagus in neogastropods comes from ontogenetic studies. Previous investigations of the ontogenetic development of the caenogastropod digestive system suggest that part of the foregut of predatory species with planktotrophic larvae is formed prior to metamorphosis from either a ventral outpocketing of the larval buccal mass or, in the most extreme cases, an isolated parallel anterior oesophagus and buccal mass (Ball et al. 1997; Page and Pedersen 1998; Page 2000, 2005). Page (2000, 2005) described the foregut ontogeny of a nassariid and showed that the post-metamorphic buccal mass develops separately from the larval foregut. In addition, the anterior oesophagus forms from an elaboration of this parallel buccal capsule (Page 2005) supporting the buccal origin of that structure.
This research was supported by a Chadwick Biodiversity Fellowship from the Australian Museum, Australian Biological Resources Study PhD Research Scholarship, funding from an Australian Museum Postgraduate Award and University of Sydney Postgraduate Research Support Scheme awarded to REG. The constructive comments of three anonymous reviewers greatly improved the manuscript. Professor Maria Byrne of the Department of Anatomy and Histology at the University of Sydney provided guidance and laboratory space and is thanked for her generous support. Alison Miller and Ian Loch at the Australian Museum are thanked for their kind assistance.