Ontogenetic changes in the spicule formation and their possible role in chromodorid opisthobranchs (Mollusca, Chromodorididae)

Abstract Many dorid nudibranchs have calcareous spicules in their integument. These structures were lost during their evolution in some dorid lineages, as usually stated for most genera of the family Chromodorididae. Nevertheless, in this article, the presence of calcareous spicules is reported upon for the first time in 12 southern European species belonging to the genera Felimare and Felimida: Felimare villafranca, Felimare picta, Felimare orsinii, Felimare fontandraui, Felimare bilineata, Felimare cantabrica, Felimare tricolor, Felimida luteorosea, Felimida purpurea, Felimida krohni, Felimida luteopunctata and Felimida britoi. The spicules are arranged in the notum, lateral region of the body, rhinophores, gills and feet, and their morphology is very variable, even in the same species. Moreover, changes in size, shape and arrangement between juvenile and adult stages can be observed. Potential biological roles of these structures are discussed.


Introduction
Many families of dorid nudibranchs are characterized by the presence of spicules in the body wall, while others do not have them or they have been observed only in some individuals of these families. The family Chromodorididae Bergh, 1891 belongs to the latter group in which, to date, these structures have only been recorded in the genera Cadlina Bergh, 1878 andCadlinella Thiele, 1931 (Rudman 1984) (although their inclusion in Chromodorididae is questionable, see below), and in some species belonging to other genera: thus, Gantès (1962) referred to the presence of spicules in the notum of juvenile specimens of Felimare villafranca (Risso, 1818) (referred to as Glossodoris gracilis); Thompson (1972) and Rudman (1999) observed these structures in post-larval specimens with direct development of Hypselodoris bennetti (Angas, 1864) and Felimare zebra (Heilprin, 1889) (referred to as Hypselodoris zebra), respectively; Schrö dl & Millen (2001) observed spicules in adults of Tyrinna delicata (Abraham, 1877) (referred to as Tyrinna nobilis Bergh, 1898), and Rudman (1984Rudman ( , 1999 in juveniles of Noumea haliclona (Burn, 1957) and Felimare zebra (the latter referred to as Hypselodoris zebra).
Despite spicules being present in all these genera and species, either along the entire life cycle or only during the juvenile stage, Rudman (1998), according to previous notes by Schmekel & Portmann (1982), pointed out that the absence of spicules is one of the characters to consider in the diagnosis of the family Chromodorididae. This affirmation could be a consequence of the lack of studies about the presence of spicules in the genera of this family and also to the fact that phylogenetic relationships of Chromodorididae, mainly at its basal level, were not resolved. Thus, some authors consider Cadlina and Cadlinella within Chromodorididae (Thiele 1931;Boss 1982;Rudman 1984;Gosliner & Johnson 1999;Wilson & Lee 2005), while others consider them to belong to Cadlinidae (Bergh 1891;Bertsch 1977;Thompson & Brown 1984;Vaught 1989), or include Cadlina within Cadlinidae and Cadlinella as a sister to the remaining chromodorid species (Johnson & Gosliner 2012). Schrö dl & Millen (2001) highlighted the problem, and molecular phylogenies support the hypothesis that Cadlina is not a member of Chromodorididae (Thollesson 1999;Grande et al. 2004;Turner & Wilson 2008). This view has been confirmed recently by Johnson (2011) after a comprehensive taxon sampling and outgroup selection.
Concerning the origin of the spicules, Ros (1976) suggested that they could be the remains of the shell or structures that have arisen secondarily as a response to the lack of hard structures. Nevertheless, Cattaneo-Vietti et al. (1993) considered the former hypothesis incorrect, because these structures are found not only in the notum but also in the foot, rhinophores and gills.
Regarding the function of spicules in 'dorids', a defensive role against predators has been proposed (Thompson 1960;Ros 1976Ros , 1977Thompson & Brown 1976;García et al. 1986;Cattaneo-Vietti et al. 1993;Wägele & Willan 2000). According to Todd (1981), it is likely that spicules decrease the energetic value of these nudibranchs, making them less attractive to predators. Rudman (1984) suggested that the presence of spicules in the notum could make the nudibranch resemble the sponge upon which it preys, providing a camouflage against predators. Another proposed role is structural (Ros 1976), maintaining the stiffness and shape of the body (Vayssière 1901;Cattaneo-Vietti et al. 1993, 1995 and supporting the role of cariophyllidia tubercles (Foale & Willan 1987). Penney (2006) carried out a morphological and functional study of the spicules of Cadlina luteomarginata McFarland, 1966, conducting experiments on their effectiveness as defensive structures by exposing specimens to attack by crabs and sea anemones. He concluded that spicules per se do not deter predators and proposed that their main role should be related to the support of the body.
Due to the lack of data about the presence/absence of spicules in the genera of the family Chromodorididae, and considering the utility that some authors give to these structures from a phylogenetic point of view (Valdés 2002), we made a study of some southern European species of the genera Felimare and Felimida according to the updated taxonomy of Johnson & Gosliner (2012), from which we have recorded the presence of spicules in all studied species. Moreover, we have studied the ontogenetic shifts that occur regarding their size, form and position, that may have different roles. Felimida luteorosea (Rapp, 1827): 1 spec. of 18 mm; 1 spec. of 22 mm. Felimida purpurea (Risso in Guérin, 1831): 1 spec. of 1 mm; 1 spec. of 5 mm; 1 spec. of 13 mm; 1 spec. of 20 mm. Felimida krohni (Vérany, 1846): 1 spec. of 5 mm; 1 spec. of 11 mm; 1 spec. of 22 mm. Felimida luteopunctata: 1 spec. of 6 mm (preserved); 1 spec. of 13 mm (preserved). Felimida britoi (Ortea & Pérez, 1983): 1 spec. of 5 mm; 1 spec. of 20 mm.

Most
The specimens were photographed and measured in vivo (except those of Felimida luteopunctata, which were not collected directly by the authors); later they were frozen in seawater, being defrosted by the addition of ethanol (98%). Once defrosted, the rhinophores and gills were removed, and the remaining body was dissected laterally and longitudinally to remove the internal organs. The body wall was divided into three parts: notum, lateral part of the body and the foot. Each part was mounted between a slide and a coverslide; a saturated solution of KOH was added for 5 min. Later they were photographed with a Sony P93 digital camera mounted on a light microscope, and the spicules were drawn with a camera lucida.
The terms used to define the relative abundance of the spicules, except in the interphase notumÁ hyponotum in which the amount is indicated, are as follows: very numerous, more than 100 per mm 2 ; numerous, between 50 and 100 per mm 2 ; scarce, between 10 and 50 per mm 2 ; very scarce, fewer than 10 per mm 2 .
The normality of the variables 'number of spicules' and 'length of the specimen' were tested with the Shapiro Wilk's test for Felimare and Felimida specimens. In both cases data were normally distributed for the length of the animal and nonnormally distributed for the number of spicules (W 00.84335, p B0.05 in Felimare and W 0 0.66055, p B0.05 in Felimida). Therefore, the Spearman's non-parametric correlation was used in all cases.

Results
Spicules in the rhinophores, gills, notum, lateral region of the body and foot were observed in all the species. These structures, independently of their abundance, were distributed in a scattered manner, not constituting a continuous rigid structure. The number of the spicules varied depending on the length of the specimens and the region of the body.
Spicules were grouped into 12 categories according to their shape: Type A: thick spicules, with both tips pointed or slightly blunt, and usually slightly curved in their central part ( Figure 1A).
Type B: thin spicules, straight and with both tips pointed ( Figure 1B).
Type C: thick spicules with rounded tips and with tubercles on one of them ( Figure 1C).  Type D: straight thick spicules with both tips rounded ( Figure 1D).
Type E: curved spicules with both tips rounded ( Figure 1E).
Type F: straight very thick spicules with both tips rounded and enlarged ( Figure 1F).
Type G: straight very thick spicules with many tubercles and with rounded tips ( Figure 1G).
Type H: slightly curved and very thick spicules with rounded tips ( Figure 1H).
Type I: straight thin spicules with many tubercles ( Figure 1I). Type J: more or less spherical spicules with many thick tubercles ( Figure 1J).
Type L does not occur in Tables IÁXII. V-shaped spicules ( Figure 1L).
A shift could be observed in the number of spicules of the notum, but also in the shape and arrangement, from juveniles to adults (Figures 2  and 3). Thus, two small examined specimens of Felimare villafranca (750 mm) and Felimida purpurea (1 mm) had a total amount of 72 and 52 spicules of Type A, respectively. These were arranged mainly on the edge of the notum, with one of the tips usually oriented towards the outside ( Figure 3AÁC). Neither of the two specimens had developed mantle dermal formations (MDFs). In slightly larger specimens of F. villafranca (2.5 mm) and F. purpurea (5 mm), the number of spicules did not vary significantly (80 and 50, respectively), but an increase in the size could be detected ( Figure 4) and the MDFs could be observed. Moreover, these larger spicules were placed in the interphase notumÁhyponotum. In the remaining specimens, the number of spicules in the interphase decreased as the size of the animal increased. Thus, in specimens of F. villafranca longer than 20 mm the number of spicules in this interphase was lower than 10 ( Figure 5) and they were not observed in specimens of F. purpurea longer than 13 mm. This decrease occurred in all studied Felimare species in such a way that there was a statistically significant negative correlation (r s 0 (0.90, p B0.05; n 019) between the size of the animal and the number of spicules in the interphase notumÁhyponotum ( Figure 6). The same situation was observed in specimens of Felimida ( Figure 7), with the correlation also being    In adult specimens of Felimare villafranca, Felimare tricolor, Felimare bilineata and Felimare cantabrica the spicules of the interphase notumÁhyponotum were substituted with smaller spicules of Type F ( Figure  8A) distributed throughout the entire notum and lateral region of the body; the spicules of the rhinophores were thicker at the base and with a more or less uniform transversal section and rounded tips ( Figure 8B), and thinner and fusiform at the apex ( Figure 8C). Finally, the spicules of the inner region of the body wall of specimens larger than 1 mm of the species of Felimida were generally spherical-shaped with many tubercles ( Figure 8D,E).
In all studied species, except in Felimida luteorosea, small spicules of Type L could be observed sparsely distributed in the body wall, rhinophores and gills. Their height ranged between 5 and 15 mm ( Figure 1L).
Detailed information about spicule shape, distribution, size (minimum and maximum, mean and standard deviation, and 'n' 0 the number of spicules), and abundance in the studied species are provided in Tables IÁXII (see also Supplementary  material).

Discussion
Calcareous spicules are present in some family-taxa of dorid nudibranchs, but are absent in others, and according to Schmekel & Portmann (1982), Rudman (1984Rudman ( , 1998 and Valdés (2002) the genera Hypselodoris Stimpson, 1855 and Chromodoris Alder & Hancock, 1855 (many of the species previously considered to be in those genera, including all the species studied by us, have recently been transferred to the genera Felimare and Felimida) lack these structures. However, our results reject this view. It is likely that the idea of a lack of spicules results from only superficial studies of the external adult morphology of these groups, as spicules have been observed in the rhinophores, gills, notum, lateral part of the body and foot of the examined specimens in both genera.
The shape, arrangement and high number of spicules from the edge of the notum and the interphase notumÁhyponotum in the early juvenile stages,  which lack chemical defences or are not yet fully developed, lead us to propose the hypothesis of a possible deterrent role which would be in agreement with the defensive role suggested for these structures by Thompson (1960), Ros (1976Ros ( , 1977, Thompson & Brown (1976), García et al. (1986), Cattaneo-Vietti et al. (1993) and Wägele & Willan (2000). The decrease in the number of spicules in the interphase Table III. Spicules shape distribution, size (minimum and maximum, mean and standard deviation, 'n' 0 number of spicules) and the abundance in three specimens of Felimare orsinii.   notumÁhyponotum as the animals grow, when the efficiency of chemical defences increases, would support this hypothesis.
One of the regions of the body which is more exposed to predators is the notum, particularly in species in which the edge of the notum is flap-like or has a straight, prominent edge. Thus, most of the studied species of Felimare have many small spicules in both regions. Based on species of sponges and anthozoans, Koehl (1982) conducted a study on the mechanical design of spicules and their role in strengthening connective tissue in animals. The Table VII. Spicule shape, distribution, size (minimum and maximum, mean and standard deviation, 'n' 0 number of spicules) and abundance in three specimens of Felimare tricolor.   Figure 8D) author concluded that there is a direct relation between the number and dimension of spicules and stiffness. A higher number of smaller spicules increases the stiffness of the body. This could account for the small size of the spicules on the notum and the lateral region of the body in adult specimens of Felimare villafranca, Felimare tricolor, Felimare bilineata and Felimare cantabrica. In Felimida luteorosea, Felimida purpurea and Felimida krohni a thin layer formed by many small spicules, spherical or slightly elongated and with many tubercles, arranged at the inner side of the body wall ( Figure  8D,E), could decrease the eventual injuries caused by a predator. The greater capacity of contraction, typical of the species of this family ( Figure 9AÁD) also help in this regard. Thus, a high number of small spicules could constitute a stronger inner armature when the animal is attacked and compresses its body. The studied chromodorids, as well as many other dorids, when attacked quickly retract the gills, partially or completely, while the rhinophores, which are the main sensorial organs that receive information from the environment, are held outside ( Figure  10A). Thus, these detach from the body and become one of the more vulnerable regions of the animal, mainly with fish that conduct exploratory attacks.

Region of the body
Fish are one of the most important groups of predators of nudibranchs according to Todd (1981) and Penney (2006). The arrangement of the spicules in the rhinophores, more pointed in the apical region ( Figure 8C) and higher in number compared with those of the gills ( Figure 10B), may play a possible deterrent role against these attacks or, as the above authors point out, represent an indirect system to decrease the energetic value due to the great number of spicules and, consequently, be rejected by the fish as food.
A second role attributed to the spicules is structural (Ros 1976;Cattaneo-Vietti et al. 1993;Foale & Willan 1987), but their scattered arrangement in the studied species of the genera Felimare and Felimida lead us to propose that their importance in this role is less than in those dorids in which bundles are accumulated. In any case, their arrangement and the higher number in some regions of the body, as in the anterior part and the edge of the notum of the Felimida species, seem to significantly contribute to the stiffness of the animal as well. In this region, the spicules are mostly arranged forming a small angle with respect to the edge or perpendicular to it, and their size increases towards the inside ( Figure 10C).
According to Penney (2006), the role of the spicules of the rhinophores and gills is to maintain Table X. Spicule shape, distribution, size (minimum and maximum, mean and standard deviation, 'n' 0 number of spicules) and abundance in three specimens of Felimida krohni.  the separation of the lamellae, so that these perform their physiological role more efficiently. Their distribution should be both in the axis and in the lamellae, as in fact occurs in Cadlina luteomarginata (Penney, 2006). However, in the studied species most of the spicules are located in the axis of the  rhinophores ( Figure 8B,C) and they are almost absent in the lamellae. Concerning the gills, in general, the number of spicules is low and they are arranged mainly at the base, and in a lower region along the rachis ( Figure 10B). Moreover, these organs perform perfectly in other dorid species which lack spicules. Finally, Jö rger et al. (2008) noted for a species of the family Parhedylidae Thiele, 1931 that the aggregation of needle-like spicules between the oral tentacles might give the head additional stability while the animal is moving and digging between sand granules in the interstitial environment, although they also pointed out that the function of these accumulations required further investigations. Nevertheless, the species studied by us, as with other Chromodorididae, are not burrowing.

Region of the body
The presence of spicules in the species of Felimare and Felimida, which have previously been considered to be absent, make a deeper study on the body wall necessary in the remaining genera of this family. This necessity is supported by observations in two other Chromodorididae species: Noumea haliclona, in which Rudman (1984) points out their presence in juveniles, though lacking in adults, and Tyrinna delicata, in which spicules were observed in adults (Schrö dl & Millen 2001). The study should also include the juvenile stages due to the ontogenetic changes shown in the present work and should be extended to other families considered up to now as spicule-less. An explanation of the non-detection of spicules, though present, may be their low number, small size and, sometimes, deep position within the mantle tissue (Schrödl & Millen 2001). These authors also pointed out that spicules may also have been dissolved in material preserved for a long time.
These data on the presence, distribution, shape and ontogenetic changes of spicules could modify the phylogenetic interpretation of previous studies, such as those of Gosliner & Johnson (1999) and Alejandrino & Valdés (2006), or be useful for future research. Moreover, it would be necessary to carry out studies like those started by Penney (2006) to determine the function of these structures.