ultrastructure of the loop of terebratulide brachiopods

Ultrastructure of the loop of terebratulide brachiopods SARAH MACKAY, DAVID I. MACKINNON AND ALWYN WILLIAMS

LETHAIA Mackay, S., MacKinnon, D.I. & Williams, A. 1994 03 21: Ultrastructure oftheloop ofterebratulide brachiopods. Lethaia, Vol. 26, pp. 367-378 Oslo. ISSN 0024-1 164.

The folded and twisted calcareous ribbon, forming both the ascending and descending lamellae of the loop of Waltonia inconspicua (Sowerby), is a two-layered structure consisting of a wedge of regularly stacked secondary layer fibres that overlie a thin layer of non-fibrous calcite (herein termed brachiotest). On one surface, that facing into the mantle cavity, secondary fibrous mosaic predominates, but smooth, finely banded brachiotest occurs as a narrow marginal lip upon which secondary layer fibres proliferate and progressively overlap. This growing edge of the ribbon is secreted by long, folded epithelial cells with digitate extensions to their apical plasmalemmas, which are distinguishable from the cuboidal epithelium-secreting fibres and their membranous sheaths. The other surface, facing the body cavity and the brachial coelom, consists entirely of roughened brachiotest exhibiting prominent banding that is aligned parallel to the growing loop edge. This surface is overlain by microfilamentar epithelium acting as a holdfast for the connective tissue frame ofthe lophophore. The other edge ofthe ribbon consists oftruncated sections ofboth secondary-layer fibres and brachiotest which bear signs of resorption consistent with the degen- erated state of the associated epithelium. Growth of the Waltonia loop is controlled by these localized processes of secretion and resorption of the fibrous and brachiotest layers and is typical of all terebratulides so far studied. The brachiotest is not homologous with the non-fibrous primary shell secreted at the valve margin. OBrachiopoda, Articulata, Terebratulida, ultrastructure, lophophore, loop.

Sarah Mackay, Department ofAnatomy, University of Glasgow, Glasgow G12 SQQ, Scotland; David I. MacKinnon, Department of Geology, University of Canterbury, Christchurch, New Zealand; Alyyn Williams, Palaeobiology Unit, 8 Lilybank Gardens, Department of Geology and Applied Geology, University of Glasgow, Glasgow G12 8QQ, Scotland; received 2nd September, 1992; revised version received 17th March, 1993.

The lophophore of the great majority of mature terebratu- tides is disposed as a plectolophe supported by a calcareous loop. This loop has continually undergone profound phylogenetic changes as well as intricate ontogenetic metamorpho- ses. A comprehension ofthese changes is fundamental to our understanding ofbrachiopod evolution as a whole. Unfortu- nately, however, comparative studies of the loop have generally been incomprehensible as well as ill-founded for two reasons. First, the terminology, created by Beecher (1895), Buckman (1910) and others (see Thomson 1927, pp. 170- 176), is based on the taxonomic names of those stocks pur- portingto characterize particular stages inloopdevelopment. Apart from the difficulties of determining precisely what they mean, such terms are inevitablythrown into confusion by any taxonomic revision of the groups on which they are based. Indeed, one frequently has to master the latest taxonomic details of the terebratulide classification in order to under- stand the description of the ontogeny of the loop of a single species. Steps have already been taken to replace this termi- nologywith one which is easier to use (Richardson 1975), and it is hoped that taxonomically based terms will have been

abandoned in the forthcoming revision of Part H (Brachio- poda) of the Treatise on Invertebrate Paleontology.

The second reason for the inadequacy of the standard comparative studies of loop development is that they were made with little regard for the mode of secretion of the loop or its anatomical relationship with the plectolophe. As far as we know, Thomson gave the prevailing view on the secretion of the loop when he noted that it ‘is the product of the inner lamina ofthe dorsal brachial lobe’ (1927, p. 91). His description of the brachiopod mantle (1927, p. 9) confirms that ‘inner lamina’ was used by him for the ciliated, inner epithe- liumwhichlinesthemantlecavityandisseparatedbyconnec- tive tissue from the outer epithelium which secretes both valves and all their calcareous apophyses, including the loop. In fact, the investment oftheloop by an extension ofthe outer epithelium and the role it plays in the secretion and resorption of the various calcareous components within the body cavity and the plectolophe seem not to have been described until 1956 (Williams, pp. 266-269). Yet, in subsequent studies of the terebratulide lophophore, few researchers, notably Atkins (1959,1961), Rudwick (1970) and Reynolds 8rMcCammon

368 Sarah Mackay, David I. MacKinnon Q Alym Williams LETHAIA 26 (1 993)

(1977), have taken account of the reported structural indi- viduality of the loop.

More recently, transmission and scanning electron mi- croscopy has helped to focus attention on the growth of the loop at the ultrastructural level. Williams (1968a, pp. 22-26), using the long axes of secondary fibres as growth vectors, produced maps of the loops of three terebratulide genera showing the direction of growth and the precise location of zones of fibre proliferation and loop resorption. This tech- nique has since been successfidly adopted for the study of spiriferide spiralia (MacKinnon 1974,1991; Samtleben 1972, 1975) and is currently being used by one of us (D.I.M.) for a comparative study ofloop dynamics. This latest terebratulide research, however, has been handicapped up to now by the absence of any ultrastructural studies of tissues associated with the loop. The deficiency has now been made good and affords an opportunity to present a more rational analysis of the growth of the terebratulide loop.

Materials and methods Preparation of soft parts Living specimens of Waltonia inconspicua ( Sowerby) were collected by Dr Mark James from Pudding Island, Otago Harbour. They were fixed in New Zealand by immersion for 2 hours in a freshly prepared solution of glutaraldehyde (2.5%) buffered with 0.2 M sodium cacodylate containing sodium chloride (1.9%) at 4°C. On receipt, specimens were givenabufferwashandthendecalcifiedin8%EDTAadjusted to pH 7.6. A wash in 0.2 M sucrose preceded the second fixation for 1 hr in 1% osmium tetroxide; these solutions were made up in cacodylate buffer containing sodium chloride. Material was then dehydrated through an ascending alcohol sequence and embedded in Spurr's resin; thin sections were cut on a Reichert Ultracut 'E' using a Diatome diamond knife. After staining with aqueous lead citrate and uranyl acetate, sections were examined on a Jeol JEM 100s transmission electron microscope. Additionally, semi-thin sections stained with toluidine blue were examined by light micros- copy to confirm the orientation of the specimens.

Preparation of hard parts Because of the delicate nature of the brachidia, special care was taken in the removal of soft tissue. Conjoined valves of Waltonia were placed in a petri dish containing a 1: 1 solution of water and domestic grade bleach (sodium hypochlorite) for approximately 10-15 minutes. During this stage the disintegration of pedicle and mantle tissue was monitored under a binocular microscope until it was apparent that the muscle system and lophophore had been largely digested. Using a scalpel blade the valves were then prised open and carefully disarticulated. Dorsal valves were replaced in the

bleach solution and further observed until all adherent tissue had disappeared. Each specimen was then washed, allowed to dry and mounted on an SEM stub using silver dag; coatings of carbon followed by gold were applied. Specimens were examined on a Cambridge 360 scanning electron microscope.

Micromorphology of the Waltonia loop SEM examination was carried out on a series of Wultonia specimens ranging in dorsal valve lengths from 0.7 mm to adult in excess of 12mm. Up to a shell length of about 1.5 mm, at which stage the lophophore is trocholophous, there are no skeletal outgrowths associated with the lophophore (Fig. 1A). Between 1.5 and 3 mm, coincident with a schizolophe, a high blade-like septal pillar extends from the valve floor, and rudiments of crura project from the anterior edges o€the inner socket ridges (Fig. 1C). By 3 mm length, the initial ascending loop elements are visible, firstly as an in- verted hood (Fig. ID) enveloping the posterior edge of the septal pillar (to accommodate the growing tips of a zygo- lophous lophophore) and then as a ring consisting of a pair of ascending lamellae united by a transverse band; at the same time, the descending elements, initially isolated and extending anteriorly from the crura and posteriorly from about midway down the septal pillar, fuse together to form a pair of descending lamellae. Further growth sees firstly the fusion of the anterior extremities of adjacent ascending and descending lamellae followed by their anterolateral separation from the septal pillar (Fig. 1E). As a result of continued peripheral expansion along the entire length of the ascending and descending lamellae the highly distinctive adult terebratellid loop form is generated (Fig. 1F).

Nothwithstanding the particular stage of loop development, four different kinds of skeletal fabric were identified on both ascending and descending parts of the loop. These were: orthodoxly stacked fibres; smooth, finely banded calcite; rough coarsely banded calcite; and truncated fibres.

Secondary-layer fibres Except for a narrow marginal zone of smooth, finely banded calcite, the entire lateral to dorsal facing surface of each descending lamella and the medially facing surfaces of the ascending lamellae and transverse band were occupied by orthodoxly stacked secondary-layer fibres (Figs. 2,3E, F, 4A, B). The fibres were normally regularly disposed with well- developed terminal faces in alternate rows. The typical terminal face, llke that characterizing fibres lining the floors of valves, consisted of a granular surface defined by the convex edge of a fibre and an acutely angular boundary of about 70" marking the limit ofthe superficially smooth proximal axis of the fibre, which frequentlybore chevron-like banding parallel with the distal angular boundary. At resolutions approaching

LETHAL4 26 (1993) The loop of terebratulides 369

Fig. 1. SEM micrographsofdorsalvalveinteriors of Wultonia inconspicua (Sowerby) showingvariousstagesofloop development. 0A.Valve 0.7 mminlength showing absence of any brachial supporting structure, ~ 9 5 . DB. Detail of anterior margin of specimen in Fig. lA, showing peripheral rim of primary layer (PL) forming seeding substrate for secondarylayer fibres (FL) whose terminal faces enlarge away from the shell margin, ~ 2 4 0 . DC. Juvenile valve showing medial septa1 pillar (SP) and incipient crura (CR) extending fromanterior edgesofinner socket ridges, ~ 1 8 . UD. Development ofhood (HD) on septalpillar; descending lamellae (DL) incomplete, x 14. OE. Ascending (AL) and descending lamellae beginning to separate anterolaterally from the median septum, x8. OF. Typical young adult with descending branches braced by lateral connecting bands arising from median septum, X5.

370 Sarah Mackay, David I. MacKinnon &Alwyn Williams LETHAL4 26 (1993)

growing edge

descending lamella

Fig. 2. Diagram of a young adult dorsal valve interior of Waltonia inconspicua (Sowerby) showing the distribution of secondary fibres (growth vectors arrowed) and rough coarsely banded calcite (with growth banding) on loop surfaces; growth stage comparable with that of specimens illustrated in Figs. IE, 3, and 4.

100 nm, fracture sections of fibres showed no differentiated fine structure other than cleavage planes.

Using the long axes of the terminal faces as growth vectors for the shell (and its secreting cells), the generation of new skeletal and outer epithelial constituents could be deter- mined. Apart from complications arising from some localized whorls of terminal faces, vectors of growth ran towards the anteroventral margins of the descending and ascending lamellae (Fig. 2). The terminal faces of mature fibres averaged 15 pn in width (in 50 estimates) and varied significantly in size only in rarely occurring small fibres budding off mature ones.

However, towards the junction with the smooth, finely banded surfaces, the fibres became smaller and at the boundary averaged no more than 6 pn (in 25 estimates). Indeed, many were little more than anteriorly directed lobate protu- berances contained within smooth depressions also with outwardly convex boundaries.

Smoothly banded calcite

Forming a rounded rim to the secondary mosaic, around the entire periphery of both ascending and descending lamellae and the transverse band, was a narrow strip of smooth, finely banded calcite, on average no more than 5 pn wide (Figs. 3E, F, 4A, B). Aseries of fine, radially disposed striations, made up of impersistent ridges and about 100 nm thick, extended around the outer edge (Fig. 3F). The surface was also sporadically indented with elliptical pits about 150 nm in diameter

but was devoid of larger features and generally had a homo- geneous texturelike that ofthe axes offibres. Smooth surfaces were underlain by calcite which, while displaying characteristic cleavage, also bore inclined tension cracks up to 2 pm long and 500 nm in maximum width (Fig. 3F).

Rough, coarsely banded calcite

This fabric was the most widespread of all, covering the ventral to medial surface of the descending lamellae, the dorsal to lateral surface of the ascending lamellae and the posteroventral surface of the transverse band where it was bounded by the smooth rounded surface along one margin and by the truncating flat surface along the other (Figs. 2,3A, B, 4C-E). The surface had a rough microtopography and was corrugated by coarse growth bands parallel with the smooth rounded margins. In detail, microtopographic variation it- cluded low, rounded, smooth or granular masses, up to 20 pn in size, and flatter laminar surfaces pierced by inliers

Fzg. 3. SEM micrographs of Waltonia inconspicua (Sowerby) showing various skeletal fabrics associated with a broken ascending lamella. OA. General view of a dorsal-valve interior showing descending lamellae fused toahighseptalpillarthatextendsposteriorlyasalowseptum.Thering-1ike ascending lamella, comparable to that shown in Fig. 2, has been broken off, ~ 1 5 . OB. More detailed view of the anteroventral edge of the septal pillar showing the location of the remnant ascending lamellar edges, ~ 5 5 . OC. Moredetailedview ofFig. 3Bshowingresorbededges (RE) ofthe ascending lamellae located above the anteroventral edge of the septal pillar, x450.

LETHAIA 26 ( 1993) The loop of terebratulides 371

OD. More detailed view of Fig. 3C showing stacked fibres (FL) overlying thin brachiotest (BT) on resorbed edge with exposed fibre trails of median-facing surface to left, x2080. OE. Growing edge (bottom) and broken edge (to right) of ascending lamella showing narrow strip of smoothly banded calcite (BC) forming a substrate for overlapping secondary layer fibres, Xl160 . OF. More detailed view of edge illustrated in Fig. 3E, showing fine radially disposed striations (ST) at the leading (lower) edge; tension cracks (TC) visible on fractured edge, ~4720.

372 Sarah Mackay, David I. MacKinnon QAlwyn Williams LETHAIA 26 (1993)

Fig. 4. SEM micrographs of Waltonia inconspicua (Sowerby) (the same specimen illustrated in Fig. 3A) showing various skeletal fabrics associated with a descending lamella ofthe loop. OA. General view ofleft hand crus (CR), slightly damaged crural process (CP) and proximal edge ofdescending lamella (DL), x190. OB. More detailed view of Fig.4A showing growing edge of descending branch with narrow margin of smoothly banded calcite (BC) overlapped by secondary layer fibres (FL), x1800. UC. General view of ventral surface of descending lamella showing rough, coarsely banded calcite (RC) with growth


microfilamentar cells I

connective tissue

Fig. 5. Stylized block section showing the differentiation of the outer euithelium enveloping a segment of the two-layered descending lamella of the loop of Waltonia inconspicua (Sowerby).

with rhombohedral boundaries (Fig. 4E). The junction with the truncated surface was sporadically seamedwith rhombohedral etch pits and revealed that the rough, coarsely banded surface was underlain by a layer of calcite, about 2 pm thick, which was occasionally stratified.

Truncated fibres Along the medially facing edges of both ascending and descending lamellae, fibres and smoothly banded calcite were truncated by flattish surfaces. These surfaces were made up principally of transverse, oblique or even longitudinal sections of fibres (Figs. 3C, D, 4D, F) dependent on the disposi- tion of the surfaces of truncation to the fibre axes. As noted above, the adjacent irregular margin of the brachiotest was extensively pitted (Fig. 4F).

banding runningparallel togrowing edge (GE) andtruncatedbyresorptive edge (RE), x93. OD. More detailed view of Fig. 4C showing banding on rough, coarsely banded calcite, x430. OE. Microtopography of rough, coarsely banded surface showing irregular deposits of calcite, X1830. OF. More detailed view of Fig. 4C showing resorptive edge of descending lamella with etched, truncated secondary layer fibres (FL) overlying a thin layer of brachiotest (BT), the edge of which is highly pitted, x3120.

All four fabrics have been confirmed in dorsal valves as small as 3 mm in length.

Epithelium associated with the Waltonia loop The precise location of the loop components in decalcified sections was seldom difficult to determine, as the secondary fibres were each ensheathed within proteinaceous membranes which interconnected and usuallyretained thecharac- teristic outlines of orthodoxly stacked fibres. Within a section of valve and mantle, such a network was successively underlain by outer epithelium, connective tissue and inner epithelium which lined the mantle cavity containing the lophophore (Fig. 5 ) . Similar successions, intimately associatedwith sections of the lophophore, must also bound the mantle cavity and afford a means of orientating strips of epithelium when seen at high magnifications (Fig. 6A).

For this study, a montage was made ofa transverse section of the complete outer epithelial envelope around a mid- section ofthe left-descending lamella of a decalcified loop and was supplemented by detailed surveys of key parts of the

374 Sarah Mackay, David I. MacKinnon QAluyn Williams LETHAIA 26 (1993)

epithelium and adjacent tissues. In the montage, the outer epithelium delineated a suboval area, nearly450 p long and just over 2 0 O p wide, and was differentiated into four histologically distinctive arcs. Along the lateral arc facing the mantle cavity (Figs. 5,6B), the outer epithelium was indistin- guishable from that lining the valve floor and indeed was also succeeded by a thin layer of connective tissue and ciliated inner epithelium bordering the mantle cavity. It consisted of a monolayer of cells which were ostensibly cuboidal, being about 6 pm high and up to 10 pn or so wide, but were usually complexly interdigitatedwith one another. The large nucleus was medially situated although frequently displaced, rounded mitochondria with open rosettes of short cristae were quite common, as was rough endoplasmic reticulum. Large vacuoles also occurred, containing membrane-bound electron-dense proteinaceous bodies and smaller vesicles, no more than 50 p in size, which were either empty or filled with unstained materials.

For the purposes of this study, however, the most distinctive feature of typical outer epithelium is that each cell was attached by hemidesmosomes to a proteinaceous membrane which was part ofthe interconnecting system of sheets investing the axes of all secondary fibres. Since they survived decalcification more or less in their original position relative to the fibres, they were good indicators of the extent and alignment of the secondary shell, although not necessarily of the original thickness of the biomineralized components. In the montage, the network of proteinaceous membranes, markingthe site ofthe secondaryshell component oftheloop, occupied a triangular area adjoining the lateral epithelial arc. The base of this triangular network (about 80 p wide) lay along the dorsomedial arc of epithelium facing toward the body cavity and the apex at the ventrolateral margin.

Dorsomedially towards the base of the triangular network of interconnecting membranes, very large vacuoles with sparsely distributed contents began to dominate the epithelial milieu. This development heralded profound changes in the outer epithelium adjoining the base of the membranous network marking the former site of the secondary fibres. Membrane-bound vesicles with electron-dense contents undergoing digestion still occurred along with rough endoplasmic reticulum; but the latter showed signs of disintegrating into electron-dense particles, about 150 nm in size, which were interspersed with bits of membranes seemingly derived from the break-up of the apical secreting plasmalemma (Fig. 6C). Varying lengths of membranes also sporadically occurred in folded bundles and in vesicles, up to 2.5 p in diameter, where they were mixed up with a variety of electron-dense particles.

This breakdown in outer epithelial constituents was re- flected in the discontinuity ofthe membranous networkat the interface with the secreting plasmalemmas (Fig. 6D). Here membranes were broken into interrupted traces, and the electron-dense particles associated with the disintegration of rough endoplasmic reticulum were found scattered well in-

side the zone of secondary fibres, mainly attached to the membranes inwardly for about 80 pm, towards the medial arc of outer epithelium. The cells continued to show signs of degeneration accompanied by an increase in subcircular or oval, electron-dense bodies up to 1 pm in size. Extracellularly there was no sign of interconnected membranes. This ventrolateral arc of resorptive outer epithelium was underlain by a thinlayer ofconnective tissueinwardlylinedby coelomiccells facing the body cavity.

Ventrolaterally towards the other end ofthe lateral arc, the outer epithelium abounded with mitochondria and rough endoplasmic reticulum. Finely granular electron-dense particles, about 40-70 p\ in size, were common, especially aggre- gated in clusters along the rough endoplasmic reticulum or in vacuoles. Vesicles, in the process of exocytosis, were seen at those parts of the apical plasmalemmas which were attached by fibrillar hemidesmosomes to the extracellular membranes.

This normal kind of outer epithelium suddenly gave way to a narrow zone of 5 or 6 elongated cells as much as 18 pn long but isoclinally foldedwithin one another about an axial plane parallel with the basal lamina so that they were seldom more than 1 to 2 p thick (Fig. 6E). The cells contained rounded mitochondriawith a dense matrix and swollen cristae, abundant rough endoplasmic reticulum associated with fine electron-dense granules, and many membrane-boundvesicles of electron-dense material in various stages of depletion, which tended to accumulate apically immediately below finger-like protrusions of the secretory plasmalemma, up to 1 pn or so long. The digitate protrusions lay immediately beneath an electron-dense membrane which closelycontoured the apical surfacesofthecellswithagap ofabout 20 nm. Themembrane was very much like those investing secondary fibres which were otherwise absent from this part of the montage as they were from the rest of a zone, up to 150 p thick, separating the medial arc of outer epithelium from the triangular network of membranes of the secondary shell.

The fourth kind of outer epithelium, that forming the medial arc, was subtended between the ventrolateral arc of folded cells and the dorsomedial arc of resorptive epithelium. At both junctions, the passage from one type of epithelium to another was sudden. The innermost folded cell, for example, was succeeded by a columnar cell, up to 13 pn high and 7.5 pm wide, which, with its convoluted basal lamina accom- modating closely spaced bundles of microfilaments extending to the apical plasmalemma, had the characteristic organi-

Fig. 6. TEM micrographs of a transverse section through the tissue associated with the left hand descending lamella of the loop of Waltonia inconspicua (Sowerby). OA. Outer epithelum (OE) associated with membranous sheaths of fibres and supported by connective tissue (CT). Inner epithelium (IE) lines mantle cavity, asterisk is in coelomic cavity, ~3,500. OB. Arrowheads indicate nuclei of typical outer epithelium. Note proteinaceous membranes investing secondary fibres (arrows), ~5,000.


OC. Zone ofresorption: phagocytosis of membranous material (arrows), X15,OOO. OD. Zone of resorption: large vacuole in epithelial cell (arrowhead) and disintegration of membranes around fibres (arrows). Outer epithelium is separated by connective tissue from coelomic epithelium (CE), ~3,500. OE. Zone of elongated isoclinally folded outer epithelial cells. Note rough endoplasmic reticulum (arrow), vesicle (V) containing electron-dense material and apical electron-dense membrane (arrowhead), ~30,000. OF. Outer epithelial cells with microfilaments (arrow) and apical granular membrane (arrowhead), ~30,000. Inset shows hemidesmosomal plaque (arrow) attaching filaments to the collagenous tissue (asterisk) below the basal lamina, X30,OOO.

376 Sarah Mackay, David I. MacKinnon &Alwyn Williams LETHAIA 26 (1993)

zation of the entire arc (Fig. 6F). The microfilaments were connected by hemidesmosomal plaques to the connective layer underlying the basal layer and to a granular, proteinaceous membrane, about 7 nm thick, immediately distal ofthe secretory plasmalemma. Rough endoplasmic reticulum was more abundant than in other kinds of outer epithelium, and aggregates of fine granular particles and variously sized vesicles were common, as were microtubules and lysosomes.

The microfilamentar cells were uniformly columnar for about 150 pm along the ventrolateral part of the medial arc, but in mid-region, cells were very low in patches, 20 pn or so across, so that they shared an undulating boundarywith that part of the loop free of interconnected membranes. The microfilamentar outer epithelium was everywhere succeeded medially by connective tissue and a layer of sporadically distributed coelomic cells lining the body cavity.

Conclusions Secretion of the WALTONIA loop

A correlation of the micromorphology of the descending lamella of the Waltoniu loop with its investing soft tissues shows that changes in the fine structure of the lamella are compatible with those in the enveloping outer epithelium. The four surfaces identified on the lamella coincide with four distinctive types of epithelium. Two of them bound an outer, wedge-shaped layer of fibrous calcite, the other two an inner, relatively thin layer of non-fibrous calcite. The balance main- tained between these two layers during the growth of the lamella can be related to inferred changes in the secretory regimes of the enveloping epithelium.

The wedge of fibres with their membranous sheaths is secreted in the same way as the secondary layer of the terebratulide shell. The chevron banding along the axes of fibres confirms that they grow anteroventrally away from the base of the wedge which coincides with the dorsomedial surface truncating fully developed fibres. At the other end, however, the wedge thins out as a band of incipient fibres along the junction with smooth, non-fibrous calcite. This is evidently the site of fibre proliferation contributing to the ventral expansion of the lamella.

There is also a striking difference between the epithelial zones, associated with the truncation of mature fibres at one end ofthe wedge and the generation of new ones at the other. Along the zone of fibre truncation, the degenerate condition of the outer epithelium, with the many signs of membrane digestion (and presumably the concomitant solution of calcite), points to active resorption of the biomineral components of the lamella. In contrast, dong the zone of fibre proliferation, the associated epithelium is characterized by an abundance of mitochondria, rough endoplasmic reticulum, vesicles and electron-dense granules, all indicative of high rates of secretion.

Unlike the fibrous wedge, the layer of non-fibrous calcite is a novel structural feature of the biomineral successions of articulate brachiopods; and serves as both a substrate for the seeding of fibres and a biomineral frame for the adhesion of the connective-tissue support of the lophophore. The forward growth of its ventrolateral edge is controlled by the folded cells which also secrete a proteinaceous membrane immediately distal of the digitate protrusions of their apical plasmalemmas. This growth is counter-balanced by resorption of the layer by resorptive cells occupying its truncated, etch-pitted, dorsomedial surface.

The microfilamentar epithelium, associated with the medial surface of the non-fibrous lenticle, not only acts as a holdfast between the lamella and the lophophore but also thickens thislayer oftheloop by further differential secretion. The interface between the fibrous and non-fibrous layers is easily traced throughout the lamella from its outcrop just within the ventral edge of the loop to the dorsomedially located edge of resorption. It involves the sporadic secretion of stratified laminae, normally in phase with stepped growth banding, which may represent brief periods of rhythmic sedimentation. On the other hand, rounded, convex masses scattered over the surface, are more likely to reflect the microtopography of the secreting plasmalemmas. In particular, the extent ofoverlap among such masses and the variation in surface texture suggest the presence of extracellular membranes during secretion. An interrupted membrane is usually found immediately distal of the apical plasmalemma of the microfilamentar cells. It is ultrastructurally comparable with the membranes investing fibres and that occurring distally of the digitate protrusions of the folded cells. Such membranes must have been permeable to the passage of Ca2+ and HCO; ions and any intracrystalline organic compounds within the calciticloop; and at least contributed to the compaction ofthe thickeningbiomineral succession. This would account for the comparably smooth textures of the axes of fibres as well as the ventral edges and convex surfaces of the non-fibrous layer, all of which are covered by proteinaceous membranes.

Although an exhaustive correlative study of hard and soft parts of the Wultonia loop has only been made of one section through the calcareous ribbon, rangom surveys of other parts of the biomineral skeleton and decalcified soft tissue confirm that the relationships inferred from the section are typical of the lophophore support as a whole.

Comparison of loop and shell structure

The secretory regime determining the succession of the in- tegument of Wuttoniu has been known for some time (Wil- iams 1968b, p. 276). It gives rise to a complex periostracum (Williams 1968b, p. 274) with an electron-dense basal layer, 100nm or so thick, serving as a seeding sheet for a non- fibrous primary layer which in turn becomes the substrate for the first formed fibreswiththeir membranous sheaths, marking the onset of the secondary layer. The basal layer of the

LETHAIA 26 (1993) The loop of terebratulides 377

periostracum and most, if not all, of the succeeding primary layer are secreted by the elongate vesicular cells forming the outer mantle lobe at the valve margin (Williams & Mackay 1978, p. 194).

The secondary fibrous layer is secreted by cuboidal cells constituting the outer epithelium of the mantle proper. This spread of outer epithelium, demarcated by the outer mantle lobe, is responsible for the differential thickening of the fibrous secondary shell. In morphological terms, such thickening includes the secretion and growth of internal features arising from the floors and hingelines of the valves, like septa and articulatory devices as well as lophophore supports. Accordingly, the occurrence of a non-fibrous layer of calcite on the inside of the ribbon of the Wultonia loop, appears at first sight to be anomalous.

A non-fibrous succession, however, may signal nothing more than a cessation in the secretion of membranous sheaths by outer epthelium undergoing changes in function. A sharp retraction of the mantle, for example, activates new forms ofsecretion by outer epithelium. In terebratulides these can include the deposition, on the shell surface evacuated by retreat, of a proteinaceous membrane followed by pads of non-fibrous calcite, up to 8 pn thick, before the secretion of fibres with their enveloping membranes is reinstated (Will- iams 1971, p. 64).

Lenses of prismatic calcite can also occur within the secondary shell of spiriferides, which is otherwise fibrous (Wil- liams 1968a, p. 32); andevenformsacontinuous tertiarylayer in a number of spiriferide stocks (MacKinnon 1974, p. 255) and in many terebratulids like Gryphusand Liothyrellu (Mac- Kinnon & Williams 1974, p. 179). With regard to the terebratulids, it has been shown that individual prisms are extensions of fibres and owe their distinctiveness to the absence of investing membranes.

A more relevant comparison, however, can be drawn between the non-fibrous layer of the Wultonia loop and the patches of modified shell underlying muscle bases. These muscle scars consist of lenticular masses of secondary shell (myotest of Krans 1965, p. 9 9 , which have been so altered by resorption and differential secretion that their surfaces may be etched with trails of depressions and pits tracing the outlines of the epithelial cells at the muscle bases (Williams 1968a, p. 21) even though the identity (and therefore the membranous sheaths) of fibres may be lost (MacKinnon 1977, p. 10). Comparable modifications have been found on the medial non-fibrous surface of the terebratulide Mugel- laniufluvescens (Lamarck) (Williams 1968a, p. 30).

The examples just cited confirm that the Waltonia loop is by no means unique in being composed of juxtaposed layers of fibrous and non-fibrous calcite. The important question is, however, whether both layers are an integral part of the secondary succession or whether the non-fibrous layer is the homologue of the primary shell.

At first sight, the latter relationship appears to be feasible. The boundary between the fibrous and non-fibrous layers

along the ventral edge of the loop is marked by a narrow zone ofimmature fibres invarious stages ofdevelopment (Fig. 2A), as is so at the primarylsecondary shell junction just within the valve margin (Fig. 1B). Moreover, the vesicular cells, secreting theperiostracumandmuchoftheprimarylayer, resemblethe folded cells, controlling the secretion oftheventral edge ofthe loop, in having digitate apical plasmalemmas and in the occurrence ofvesicles, albeit on a much more abundant scale. Basic differences in structure and function, however, refute any homology. Away from its interface with the fibrous secondary shell, the primary shell is bounded by the periostracum (secreted bythevesicular cells). The equivalent surface of the Wultonia loop ribbon is occupied by microflamentar epithelium and folded cells; and, although an extraceuular membrane usually intervenes between that surface and the epithelium, it cannot be the homologue of the periostracum as biomineral secretion takes place through, not on, it.

Furthermore, in juvenile shells up to about 1.5 mm in length (Fig. lA), the sites of future crura and septal pillar consist entirely of orthodoxly stacked secondary-layer fibres secreted by normal cuboidal outer epithelum. The novel skeletal fabrics associatedwith theloop describedin this paper are thus demonstrably not in continuity with any part of the primary layer but originate, llke myotest fabric underlying muscle bases, as localized modifications ofthe secondary shell succession.

In summary, we conclude that, since the non-fibrous constituent of the Wultoniu loop is a variant of the secondary shell, it owes its distinctiveness to the special physio-chemical relationships between its secreting outer epithelium and the tissues of the lophophore, which it envelopes. The layer certainly has much in common with the myotest; but it is uniquely fashioned by a balance between secretion and resorption, both of which functions contribute to its morphological singularity. We, therefore, propose that the term ‘brachiotest’ be used to identify this deposit.

Structure of the terebratulide loop

The loop is a complex feature of the terebratulide shell, which develops in many different ways by differeptial accretion and resorption of its component parts. Nevertheless, when visu- alized as a single, twisted and folded ribbon subtended between a pair of crura extending from the hinge line, its structure and growth can be simply categorized (Fig. 2). The ventral (or ventrolateral) edge of the ribbon undergoes expansion by secretion; while the dorsal (or dorsomedial) edge is subject to attrition by resorption. In addition, both sides of the ribbon thicken by biomineral accretion. The ‘outer’ side, facing the mantle cavity, thickens by the forward growth of fibres which proliferate along the ventral edge. The ‘inner’ side, facing the body cavity and the coelom of the brachia, consists of differentially thickening brachiotest which also expands along the ventral edge where it acts as a substrate for the proliferating fibres.

378 Sarah Mackay, David I. MacKinnon e+Aluyn Williams LETHAIA 26 (1993)

The ultrastructural differences between the outer and inner surfaces of the loop were first described by Williams (1968, pp. 22-26) for Terebratulina caput-serpentis (Linne), Magel- lank flavescens (Lamarck) and Laqueus californicus (Koch). But the structural and micromorphological relationship between the fibrous layer and the brachiotest was unknown although the dorsal and ventral edges of the loop were cor- rectly identified as zones of growth and resorption respec- tively.

Since then, little work has been done on the dynamics of loop development at the ultrastructural level. Recently, however, one ofus (D.I.M.) hasbeen studyingthe micromorphology of the terebratulide loop with the aim of rationalizing our views on its ontogeny and phylogeny. So far, all the fine structures characterizing the development of the WaZtonia loop have been found in every Recent species examined. They include:

Neothyris lenticularis (Deshayes) Magasella sanguinea (Leach) Magellania javescens (Lamark) Magellania venosa (Solander) Laqueus californicus (Koch) Laqueus rubellus (Sowerby) Pictothyris picta (Dillwyn) Jolonica nipponica (Yabe & Hatai)

Macandrevia cranium (Muller) Emomiosa gerda (Cooper) Anakinetica cummgi (Davidson) Pirothyris vercoi (Blochmann) Mergelia truncata (Linnk) Megathyris detruncata (Gmelin) Argyrotheca cordata (Risso)

Similar fabrics have also been positively identified on loop fragments of fossil species including Miocene Magadina browni Thomson, Oligocene Waiparia elliptica Thomson and Rhizotkyris kokoamuensisBowen & Campbell, and Cre- taceous Magas pumilus Sowerby.

We are therefore confident that the ultrastructural features just described have always been typical of the terebratulide loop and will help to determine the various modes of growth which have led to the phylogenetic diversification of such a distinctive lophophore support.

Acknowledgements. -This w0rkwascarriedoutbyS.M. andA.W. withthe aid of facilities provided by NERC grant GR91408 and by D.I.M. during study leave as an Honorary Research Fellow at the University of Glasgow. We are indebted to the University and the Council for their support.

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