A comparative study of the gastric ossicles of Trichodactylidae crabs (Brachyura: Decapoda) with comments on the role of diet and phylogeny in shaping morphological traits

Species of trichodactylid crabs

The specimens used in the present study are part of the collection of INALI (Instituto Nacional de Limnología, Santa Fe, Argentina) and INPA (Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil). We observed the stomachs of nine trichodactylid species of two subfamilies and three tribes (Table 1). The observations were made under stereoscopic microscope and scanning electron microscope (SEM). The locations and cephalothorax width and length of each specimen are listed in Table 1.

Preparation of feeding structures for light and scanning microscope observation

The cephalothorax of crabs were opened and stomachs were manually dissected under stereoscopic microscope. Gut content, coarse food remains and debris were washed with water with the aid of a squeeze bottle. To remove muscular remains and finely attached debris, feeding structures were cleaned in 10% KOH during 24 h, washed with distilled water and conserved in alcohol 70%.

Each dissected stomach was opened cutting the cardiac sac and exposing the gastric mill. The stomachs used for visualization under light microscope were stained with Alizarin Red 1% after the treatment with KOH 10%. Stomachs were submerged in a recipient with alcohol 70% and photographed with a Canon EOS Rebel T2i camera attached to a Leica S8APO dissecting microscope. Specific images were taken from the ossicles of the cardiac chamber: zigocardiac ossicles, uropyloric ossicle, pectineal ossicle and the cardiopyloric valve. Special emphasis was made on the lateral teeth, medial tooth, accessory teeth and the anterodorsal surface of the cardiopyloric valve (Fig. 1). The terminology used in the foregut descriptions followed those used by Meiss & Norman (1977). Dissected stomachs for analysis with scanning electron microscopy (SEM) were air dried for a minimum of 48 h in a desiccator containing silica gel with an indicator of dampness. Once mounted on a metal stub using double-sided tape and/or silver paint, samples were gold coated for 120 s using Combined Deposition System metal/carbon, SPI Supplies, AX-12157, operated under argon atmosphere (18 mA) for 120 s. Then, observations were made using a JSM-35C scanning electron microscope (JEOL, Shanghai, China), equipped with a system of digital image acquisition SemAfore, at an accelerating voltage of 20 kV.

Analysis of morphological traits

The morphological traits were encoded into categorical characters as listed in Table 2 according to those observed in the gastric ossicles (Table 3). The generated matrix was analyzed with a hierarchical clustering analysis with unweighted pair-group average using the Euclidean distance in Past 3.16 (Hammer, Harper & Ryan, 2001). The results obtained were compared with the systematic classification of Magalhães & Türkay (1996). The observations from morphological trait were contrasted with the natural diet of the trichodactylid species (T. borellianus, T. kensleyi, T. fluviatilis, Zilchiopsis oronensis (Pretzmann, 1978), D. pagei) with available information in the literature (Table 4). Zilchiopsis collastinensis was included in this case as a representative of the genus Zilchiopsis.

General considerations of the gastric mill of Trichodactylidae

The foregut of the studied trichodactylid crabs was equivalent with the general morphology of Brachyura. The gastric mill was a chewing apparatus formed by ossicles with chitin-covered teeth and other ossicle with supporting functions. These chitin-covered teeth protruded from the unpaired urocardiac ossicle and from the paired zigocardiac and pectineal ossicles. These structures, plus the cardiopyloric valve composed the chewing apparatus (Fig. 1). Despite the common features in the morphology of the gastric ossicles of the studied species, the morphology of the gastric teeth was different in each species.

Similarities and differences of the gastric mill of Trichodactylidae

Lateral teeth

The lateral tooth rises from the zigocardiac ossicle, a paired structure that extends ventrally and medially into the cardiac stomach. It is the major masticatory structure of the gastric mill. The lateral teeth of the studied trichodactylid species were strongly chitinized and calcified with a variable shape and number of cusps (Figs. 2A–2I) (Table 3). In Dilocarcininae, cusps were blunt or incisor-like (Figs. 2A–2F). D. pagei, D. septemdentatus, Poppiana argentiniana and Z. collastinensis were morphologically similar with blunt-like cusps that decreased in width posteriorly (Figs. 2A–2D). Juvenile or newly molted specimens exhibited more prominent and less worn cusp, such as a Z. collastinensis juvenile (Fig. 2D insert) and P. argentiniana (Fig. 2B). In Sylviocarcinus autralis and S. devillei, cusps had edges that were more incisive with intercalated dorsal and ventral cusps (Figs. 2E and 2F). In Trichodactylinae species, lateral teeth were quite different from Dilocarcininae because both ventral and dorsal cusps had sharp edges with a pronounced hollow between them (Figs. 2G–2I). Cusps also reduced in width posteriorly.

Medial tooth

The medial tooth rises at the posterior end of the urocardiac ossicle, an unpaired structure located in the dorsal midline of the cardiac stomach. The medial tooth of trichodactylid species was strongly chitinized and calcified with variable morphology (Figs. 3A–3I) (Table 3). In Dilocarcininae, D. pagei, D. septemdentaus, P. argentiniana and Z. collastinensis presented the medial tooth with transverse ridges that increased in size posteriorly with variable number of ridges according to the species (Figs. 3A–3D) (Table 3). In D. pagei and D. septemdentatus, these transverse ridges were more pronounced than in P. argentiniana and Z. collastinensis (Figs. 3A–3D). Sylviocarcinus australis and S. devillei were the most different species within Dilocarcininae, with a screw-like medial tooth with ridges that increased in size posteriorly (Figs. 3E and 3F). In Trichodactylinae, a cubical and blunt/smooth process with slight differences among species (Table 3) characterized the medial tooth. Moreover, laterally to the process, all species exhibited at least a pair of well-defined sub-terminal denticles (Figs. 3G–3I).

Accessory teeth

The accessory tooth rises from the pectineal ossicle, a paired structure located laterally in the cardiac stomach. In general, this was a tiny-sized structure moderately to slightly calcified in the studied Trichodactylidae specie. These features could affect the proper viewing because the accessory tooth might not conserve its original form after the preparation stages for SEM. The number of denticles varied according to the species (Table 3) although, in general, large groups of setae surrounded the accessory teeth also hindering the visualization. In Dilocarcininae, this structure seemed to be a bit more robust compared to Trichodatylinae (Figs. 3A–3F), with at least three denticles. Within this subfamily, the accessory tooth was very small in T. borellianus and T. kensleyi with only one prominent denticle (Figs. 4G and 4H). In T. fluviatilis, this structure exhibited at least three visible denticles (Fig. 4I).

Cardiopyloric valve

The cardiopyloric valve was located in the ventral midline of the stomach, in the transition of the cardiac and pyloric chambers. The anterodorsal edge of this structure comprised the encounter of the gastric mill and the entrance of the pyloric chamber with the valve. This region had a semicircle shape with numerous setae surrounding the anterodorsal and lateral borders and without tooth-like structures in all species (Figs. 5A–5I) (Table 3). However, valves differed in shape and relief among species. In Dilocarcininae, the anterodorsal surface of the valve of five studied species exhibited a W-design (Figs. 5A–5F) (Table 3). This notwithstanding, in Z. collastinensis it was not possible to confirm this trait in stereoscopic microscope due to the level of resolution (Fig. 5D). In D. pagei, this morphology was more pronounced, with the presence of two ridges of similar shape (Fig. 5A). In Trichodactylinae, there was no defined W-design (Figs. 5G–5I) but a concave region with less setae in T. kensleyi and T. fluviatilis (Figs. 5H–5I). In T. borellianus, the anterodorsal edge of the valve had less setae and smoother surface compared to Dilocarcininae (Fig. 5G).

Morphological traits

The clustering analysis calculated with categorical characters of the observed ossicles revealed that, in general, the species were grouped according the systematic classification of Magalhães & Türkay (1996) with a coefficient of correlation of 0.9775. Only Z. collastinensis was clustered more closely to the species of Dilocarcinini tribe (Fig. 6) in contrast the observations of the mentioned authors.

The gastric ossicles of Dilocarcininae: morphological considerations

The gastric ossicles of Dilocarcinus spp., P. argentiniana and Z. collastinensis coincided in many features such as blunt-like cusps in the lateral teeth, transverse ridges in the medial tooth and the cardiopyloric valve with a W-shaped surface. Some slight differences among species were observed in the number and sharpness of cusps and transverse ridges. The morphology of the gastric ossicles of these species was different from Sylviocarcinus spp., which exhibited sharper cusps in the lateral teeth and a screw-like medial tooth. Alves, Abrunhosa & Lima (2010) also reported a screw-shaped urocardiac ossicle for S. pictus but did not present any illustration or image. Despite of specific dissimilarities, we found that these morphological traits resembled, overall, the gastric ossicles of other herbivorous crabs (Warner, 1977; Giddins et al., 1986; Allardyce & Linton, 2010). The trophic spectra found in the stomach content of D. pagei and Z. oronensis support the relation between morphological traits and a predominantly herbivorous diet.

These two trichodactylid species (D. pagei and Z. oronensis) incorporate an important percentage of plant material into their diet, besides some animal items and algae (Williner & Collins, 2002; V Williner, 2010, unpublished data) (Table 3). A diet enriched with vegetal remains requires a gastric mill with structures capable to disrupt the cellulose and hemicellulose. The cusps of the lateral teeth and the transverse ridges of the medial tooth of herbivorous crabs were significantly more pronounced than in species with omnivorous or carnivorous feeding habits (Warner, 1977; Giddins et al., 1986; Allardyce & Linton, 2010). The land crabs Gecarcoidea natalis (Pocock, 1888) and Discoplax hirtipes Lamarck, 1818 and the mangrove crab Neosarmatium smithi (Milne-Edwards, 1853) (cf. Giddins et al., 1986) had a predominant herbivorous diet exhibiting hooked spines in the dorsal cusps of the lateral teeth and transverse ridges in the medial tooth. In trichodactylid crabs, the hooked spines were absent and the other features were less pronounced. Despite the fact that decomposed vegetal remains are actually the main ingested food item in both groups (Greenaway & Linton, 1995; Giddins et al., 1986; Greenaway & Raghaven, 1998; Williner & Collins, 2002; V Williner, 2010, unpublished data), comparison should be made with criteria once both Trichodactylidae and Grapsoidea have different food availability and phylogenetic history (Martin, Crandall & Felder, 2009).

The other ossicles that compose the gastric mill, the cardiopyloric valve and the accessory teeth, seem to be simple structures compared to other decapod species (Caine, 1975; Kunze & Anderson, 1979; Williner, 2010; Brösing & Türkay, 2011). The cardiopyloric valve of Dilocarcininae exhibited a more or less pronounced W-like surface while the accessory teeth is a structure less developed than in the herbivorous crabs: G. natalis, D. hirtipes, and N. smithi. Both structures were previously described to retain the food in the cardiac chamber (Caine, 1975). However, the cardiopyloric valve may also act as a masticatory structure (Caine, 1975) while the accessory tooth may assist to push material into the gastric mill (Kunze & Anderson, 1979). Functional comparison with other brachyuran crabs requires further information of these structures in species with known natural diet.

The gastric ossicles of Trichodactylinae: morphological considerations

In Trichodactylinae, the overall morphology of the gastric mill was very similar among the observed species and quite different compared to that of Dilocarcininae. Species of the genus Trichodactylus analyzed in the present study exhibited lateral teeth with cuspidate cusps separated by a pronounced hollow and a medial tooth with a cubical and smooth process with well-defined sub-terminal denticles. These traits are quite specific of Trichodactylinae with some coincidences with other omnivorous and carnivorous crabs: Nectocarcinus tuberculosus Milne-Edwards, 1860 (cf. Salindeho & Johnston, 2003), Epixanthus dentatus (White, 1847) (cf. Cannicci et al., 1998), (Skilleter & Anderson, 1986; Heeren & Mitchell, 1997; Cannicci et al., 1998; Salindeho & Johnston, 2003; Allardyce & Linton, 2010). The observed morphological traits of the three Trichodactylus spp. analyzed in this study match well with an omnivorous trophic spectra previously described in the literature: T. borellianus (Williner & Collins, 2013; Carvalho, 2014), T. kensleyi (see Williner, Carvalho & Collins, 2014) and T. fluviatilis (Segadilha & Silva-Soares, 2015; Costa et al., 2016; Nogueira-Costa et al., 2016).

Trichodactylus spp. exhibit a natural diet with great importance of both vegetal and animal components (Williner & Collins, 2013; Carvalho, 2014; Williner, Carvalho & Collins, 2014; Segadilha & Silva-Soares, 2015; Costa et al., 2016; Nogueira-Costa et al., 2016). An efficient exploitation of these trophic resources requires a gastric mill capable of grinding soft material and also disrupting fibrous structures. The blunt medial tooth observed in Trichodactylus spp. revealed great similarity with the omnivorous rock crab N. tuberculosus (Salindeho & Johnston, 2003) and the carnivorous xanthoid crab E. dentatus (Cannicci et al., 1998). This notwithstanding, the lateral teeth of Trichodactylus spp. exhibited ventral and dorsal cuspidate cusps of similar morphology, whereas in others carnivorous and omnivorous crabs (E. dentatus, Geograpsus grayi Milne-Edwards, 1853, G. crinipes, Ozius truncatus Milne Edwards, 1834, Leptograpsus variegatus (Fabricius, 1793), N. tuberculosus, Pseudocarcinus gigas (Lamarck, 1818)), the dorsal surface is a ridge-like structure (Skilleter & Anderson, 1986; Heeren & Mitchell, 1997; Cannicci et al., 1998; Salindeho & Johnston, 2003; Allardyce & Linton, 2010).

In coincidence with what was observed in Dilocarcininae, the cardiopyloric valve and the accessory teeth of Trichodactylinae were simple structures compared to anomurans (Dardanus setifer (Milne-Edwards) (Kunze & Anderson, 1979); Clibanarius vittatus (Bosc), Petrochirus diogenes (L.) (Caine, 1975); Aegla uruguayana (Williner, 2010)), hermits crabs (Clibanarius taeniatus (Milne Edwards) (Kunze & Anderson, 1979)), and brachyuran (Discoplax gracilipes Ng & Guinot, 2001, Cardisoma armatum Herklots, 1851, Epigrapsus notatus (Heller, 1865) (Lima, 2010)). Comparing both subfamilies, the cardiopyloric valve of Trichodactylinae had a smoother anterodorsal surface. If this structure is involved in the food maceration, the absence of a W-shaped surface coincides with a diet with less importance of vegetal items than Dilocarcininae. The accessory teeth were a tiny-size structure in both subfamilies and seem to be less developed in Trichodactylinae. However, this structure was different from other crabs with varied trophic habit, possibly due to this morphological trait being a trichodactylid-owned feature, as per our findings and the mentioned literature.

Comments on phylogenetic and functional aspects of gastric ossicles

Our results, together with the information available in the literature about the morphology of the gastric armature, strongly support the hypothesis that this structure conserves much of the phylogenetic history of a taxon (Felgenhauer & Abele, 1989; Brösing, Richter & Scholtz, 2006; Brösing, 2010; Brösing & Türkay, 2011). However, and as mentioned by Brösing & Türkay (2011): “The morphology of the foregut ossicles and the attached gastric teeth have to be evaluated separately”. Despite being evident that the gastric ossicles have a conservative pattern (e.g., number of ossicles) even being used in the construction of phylogenies (Felgenhauer & Abele, 1989; Brösing, 2010), it is striking that species phylogenetically distant and with similar trophic habit exhibit similar morphological traits of the gastric teeth (Schaefer, 1970; Caine, 1975; Dall & Moriarty, 1983; Felgenhauer & Abele, 1985; Felgenhauer & Abele, 1989; Allardyce & Linton, 2010; Brösing & Türkay, 2011).

In the Trichodactylidae species, we were able to identify morphological traits of the gastric teeth that were, overall, similar to that of other phylogenetically distant crabs of similar trophic habit. The transverse ridges, typical of herbivorous crabs such as G. natalis, D. hirtipes (cf. Allardyce & Linton, 2010) and N. smithi (cf. Giddins et al., 1986), were present in all analyzed Dilocarcininae species with variable number and ridges in the medial tooth and cardiopyloric valve. The stomach content analysis of D. pagei and Z. oronensis revealed that these species ingested predominantly vegetal material (Williner & Collins, 2002; V Williner, 2010, unpublished data), as could be expected based on the gastric mill. In Trichodactylinae, the species exhibited blunt and smooth structures (the medial tooth and the cardiopyloric valve) such as those found in N. tuberculosus (cf. Salindeho & Johnston, 2003) and E. dentatus (cf. Cannicci et al., 1998). These traits are observed in species with less fibrous contents in their diet (Salindeho & Johnston, 2003; Allardyce & Linton, 2010). The three Trichodactylus spp. examined in this study presented an omnivorous feeding habit with high frequency of animal items (Williner & Collins, 2013; Williner, Carvalho & Collins, 2014; Carvalho, 2014; Segadilha & Silva-Soares, 2015; Costa et al., 2016; Nogueira-Costa et al., 2016). However, the vegetal remains were also an important trophic resource for Trichodactylus spp. Functionally, the cuspidate lateral teeth could supplement the absence of cuspidate structures in the medial tooth and the cardiopyloric valve, aiding in the disruption of fibrous content. These observations provide more examples of crabs that exhibited similar trophic habits and morphological features despite not being closely related species.

Considering the consistent relationship found between the gastric teeth traits of trichodactylid species with known feeding habits, it is plausible to predict that the analyzed Dilocarcininae species with unknown trophic spectra exhibit a predominantly herbivorous diet. Despite the tendency towards more herbivore or carnivorous trophic habits, trichodactylids are generally omnivores (Collins, Williner & Giri, 2007), which suggests that they are capable of finding, capturing, ingesting and digesting prey of different trophic levels and behaviors, morphologies and chemical compositions (Diehl, 2003). This implies that they must have behavioral, sensory, morphological and physiological adaptations that allow them to identify and incorporate into the diet a varied range of foods of both animal and vegetable origins (Eubanks, Styrsky & Denno, 2003). From a morphological perspective, we observed that the stomach of the studied crabs, while showing features more adjusted to an herbivorous or carnivorous diet, presented a morphology that allows the mechanical digestion of a variety of food of animal and vegetable origin. Indeed, the design that we appreciate today must be a trade-off between the ability to gather food items available in the environment and the ability to digest, in order to favor the continuity of that design (Turner, 2007). As mentioned by Stevens & Hume (1995): “In any one taxon, each of these digestive organs will have morphological characteristics that functionally reflect the evolutionary history and contemporary ecology of the taxon”.

Since the continental invasion of trichodactylid ancestors in the post-Gondwanan period (Yeo et al., 2008; Cumberlidge & Ng, 2009), the ability to conquer new freshwater environments was shaped by habitat heterogeneity and complicated topography and hydrology (Yeo et al., 2008). It follows, in this context, that those morphological traits that facilitated an opportunistic diet may be favored (Waloszek et al., 2007). Nowadays, the trophic spectra of trichodactylid crabs exhibit omnivorous and opportunistic trophic habit with variable importance of vegetal and animal items according to the species (Collins, Williner & Giri, 2007; Williner & Collins, 2013; Williner, Carvalho & Collins, 2014; Carvalho, 2014; Segadilha & Silva-Soares, 2015; Carvalho et al., 2016; Costa et al., 2016; Nogueira-Costa et al., 2016). This trophic habitat is in concordance with the availability of trophic resources (Carvalho et al., 2016) typical of large rivers of South America subjected to constant variation due to the high renewal of water of these pulsatile systems (Junk, Bayley & Sparks, 1989; Neiff, 1996).

The digestive capacity of trichodactylid crabs or any other taxon must be counteracted by factors other than morphology to compensate differences in the quality of food. Similarity in diet but pronounced differences in gastric mill morphologies could indicate a partial analysis of what the completely digestive process would be, or indicate different approaches to process the same food. The adjustments to digest a determined food will be constrained by the phenotypic plasticity of a taxon, which in turn depends on the evolutionary time scales changes in the genotype (Linton & Greenaway, 2007; Piersma & Van Gils, 2011). The enzymatic response to specific nutrients of each species (Fernández-Gimenez, 2013), the ingestion of bacteria with the food (Harris, 1993), the feeding preferences (Constantini & Rossi, 2001), are a few examples of complementary factors that must be studied before any solid conclusions can be made about the role of dietary and historical components in shaping morphological traits. Future studies should concentrate on the efforts in quantify the role of phylogeny and function in shaping morphological traits of the gastric ossicle through analysis of phylogenetic signal, defined as the “tendency for related species to resemble each other more than they resemble species drawn at random from the tree” (Blomberg & Garland, 2002).