Sponges (Porifera) display a wide range of reproduction strategies, both sexual and asexual. Asexual reproduction in sponges occurs as a variety of mechanisms, including budding, fragmentation and gemmulation . In general, asexual reproduction seems to be part of the ground pattern of all Metazoa . However, primary agametic-asexual reproduction mechanisms such as fission or budding should be clearly distinguished from secondary asexual reproduction mechanisms such as parthenogenesis. Apart from in the poriferans, primary asexual reproduction is present in all non-bilaterian clades: Placozoa , Cnidaria  and Ctenophora , and even in most bilaterian clades , e.g. fission in Platyhelminthes. In addition, looking at Ediacaran fossils we find mainly colonial organisms, which also indicate that the ability to bud is fundamental to the metazoan condition . Even though it has been suggested that much genomic repatterning occurred during Cambrian explosion , it seems likely that the regulation of agametic asexual reproduction in all these metazoans can be put down to a common early root, i.e. a set of genes which regulate pattern formation . In the cnidarian model systems Hydra sp., Nematostella vectensis STEPHENSON, 1935 and Podocoryna carnea M. SARS, 1846, asexual reproduction follows strict temporal patterns which are genetically regulated . Regulation of cnidarian budding involves developmental pathways, such as the Wnt-pathway , typically found in larval development. In contrast, asexual development in sponges is usually thought to be less specific and is interpreted as a consequence of the general ability of this group to reorganize constantly . The high level of mobility of all cell types within the mesohyle of sponges (excluding the epithelial-like pinacocytes) is referred to as 'constant morphogenesis' . Within the broader metazoan context the question arises as to whether poriferan asexual reproduction follows similar temporal patterns to those in cnidarians. If this is the case, asexual reproduction in sponges is most likely strictly regulated by developmental pathways.
In order to address this question it is useful to distinguish the processes in poriferans which are generally regarded as asexual reproduction. Fragmentation of specimens represents the most primitive mode. Ecologically and in the context of population dynamics, fragmentation maximizes dispersal and thus represents an important reproduction strategy which might exceed sexual reproduction rates in many species [1, 13]. However, the main prerequisites for this mode of reproduction are the general morphological plasticity of sponges and their ability to reorganize. The literature does not point towards the involvement of highly regulated processes . This is further supported by the fact that fragmentation has been shown to be linked to specific sexual reproduction events in some cases .
Gemmulation and the structure of gemmules in freshwater sponges has been studied intensively  and is an important part of the life history of several species. However, gemmules cannot be regarded as asexual reproduction bodies in the strict sense. In most cases, they serve as dormant structures that can be produced to overcome unfavorable environmental conditions. Gemmules are formed inside the sponge tissue in high numbers and remain embedded in the skeleton framework when the mother sponge decays. Although gemmules might be dispersed (which in fact represents asexual multiplication), they mainly repopulate the skeletal structures of the former mother sponge . Gemmule formation and hatching thus represent what is presumably a highly derived case of sponge morphogenesis restricted to freshwater sponges (suborder Spongillina) and a few other families.
While both fragmentation and gemmulation cover a variety of purposes in the life history of some sponges, budding only fulfils one function. Bud formation, release and subsequent morphogenesis are exclusively processes of asexual reproduction. Although budding is only obligate in the life histories of the two families Polymastiidae and Tethyidae, it occurs on an irregular basis in most, if not all demosponge families [1, 17].
Maas , and Connes  reported some fundamental principles of budding in Mediterranean Tethya species. During the early stages the stalked buds consist of homogeneous mesenchymal cell masses arranged around a stalk (sclera bundle) rising from the mother sponge. Subsequently, cells migrate into the bud and the mineral skeleton develops. Finally, the bud breaks off, is dispersed by currents and develops into a new sponge. Generally, as long as it is connected to the mother sponge, neither canals nor functional choanocyte chambers form. This means that the adult functions of water pumping and particle uptake are not present at this stage. In some species, however, buds do gain canal system functionality during early development and while still attached to the mother sponge. Buds of Mycale contarenii (MARTENS, 1824) are fully functional juvenile sponges which display a notable level of organization and in which all cell types differentiated . The same applies to Radiospongilla cerebellata (BOWERBANK, 1863), the only freshwater sponge to display budding . All cases of sponge budding are characterized by the formation of cell aggregates that indicate mesenchymal morphogenesis. In addition, Ereskovsky and co-workers recently reported epithelial budding in the homoscleromorph sponges of the genus Oscarella [17, 22]. This mode of budding is more similar to budding in cnidarians than the mesenchymal budding of other sponges. However, both mechanisms result in functional clonal juveniles immediately or soon after the release of the buds.
In the present study, we investigated on the basis of the tropical sponge Tethya wilhelma SARÀ, SARÀ, NICKEL & BRÜMMER, 2001 (Demospongiae, Hadromerida) whether sponge budding represents a spatiotemporal sequence of morphological events. Adult specimens of T. wilhelma typically display a spherical body shape with a distinct outer cortex and an inner choanosome core (see Additional file 1). The cortex is rich in lacuna, while the inner choanosome is characterized by a higher cell density. The architecture of the silica skeleton is strikingly well organized. The most prominent structures of the skeleton are megasclere bundles radiating from the centre, which distally sometimes form small forked fans. Megasters are mainly found in the cortex-choanosome boundary, embedded in strong collagen layers within the mesohyle and forming a megaster sphere around the choanosome. The same applies to micrasters, which form a tylaster layer that is connected to the exopinacoderm . These highly organized parts of the skeleton are made up of biological compound materials (i.e. particle enhanced elastomeres with silica spicules embedded into a collagen/cell matrix) which form functional skeletal superstructures [23, 24]. This particular morphological pattern, which is typical of most species of the genus Tethya, accounts to the high contractibility of these species [25–27].
Under laboratory conditions T. wilhelma seems to reproduces exclusively asexually by budding, but nothing is yet known about reproduction in the wild. Budding occurs all year round, and its frequency and the amount of buds produced vary between individuals. Specific factors influencing budding might be water temperature, salinity or nutrient availability . There seems to be a trend in T. wilhelma to intensify budding under changing environmental conditions (unpublished observations). Budding usually starts with the occurrence of tubercles on the surface, which may produce longer filaments that are able to grow into stalked buds [29–31]. It typically takes 48 hours from the moment a developing bud becomes visible on the surface until it is detached from the mother sponge. In the mean time a number of cells and skeletal elements are transported into the bud along the connecting megasclere bundle to form a highly organized almost spherical bud resembling adult morphology [see [23, 24]].
We hypothesized that the conspicuous level of organization in T. wilhelma develops step by step during bud formation and maturation. We used typical morphological characters like skeleton, canal system and choanoderm as markers to investigate the spatiotemporal patterning of budding. We used synchrotron radiation-based x-ray microtomography (SR-μCT) on complete buds and analyzed the resulting 3D images [23, 24, 32, 33]. In contrast to previous descriptive studies on sponge budding (e.g. [34, 35, 20, 36, 17, 18, 21]) SR-μCT additionally allows for volumetric analyses. Hence, for the first time, our study addresses morphological changes during bud development in a quantitative manner.