Comparative morphology of the stomach ossicles of four freshwater crabs species of Dilocarcinini ( Crustacea , Decapoda , Trichodactylidae )

The morphology of the foregut ossicles of representatives of four genera of Dilocarcinini were described to assess the usefulness of the ossicles as possible morphological characters in studies on the intrafamilial relationships of Trichodactylidae. Stomachs of specimens of Dilocarcinus pagei Stimpson, 1861, D. septemdentatus (Herbst, 1783), Moreirocarcinus emarginatus (H. Milne Edwards, 1853) and M. laevifrons (Moreira, 1901) were heated for 60 minutes in 10% KOH solution and then were stained with Alizarin Red 1% added into the KOH solution. The foregut ossicles of Dilocarcinini consists of 11 ossicles of the gastric mill, 10 of the lateral supporting cardiac region, 4 of the cardio-pyloric valve, 6 supporting the dorsal pyloric stomach, 9 supporting the ventral pylorus and bulb, 3 supporting the supra-ampullary region, 5 supporting the lateral pylorus region, totaling 48 ossicles and the following 34 are similar in shape, size and degree of calcification. The morphological differences between the majority of the ossicles were not very clear among the species, showing inconsistency intrageneric differences, however, species of different genera showed small similarities between them. There is a clear need for including more taxa in a comparative analysis, Nauplius, 26: e2018024 CORRESPONDING AUTHOR Renata C. Lima-Gomes renatacslima@yahoo.com.br SUBMITTED 08 August 2017 ACCEPTED 04 May 2018 PUBLISHED 03 December 2018 Guest Editor Célio Magalhães DOI 10.1590/2358-2936e2018024 All content of the journal, except where identified, is licensed under a Creative Commons attribution-type BY. orcid.org/0000-0001-6412-7850 orcid.org/0000-0003-4858-2575 original arTiCle This article is part of the tribute offered by the Brazilian Crustacean Society in memoriam of Michael Türkay for his outstanding contribution to Carcinology Lima-Gomes and Magalhães 2 Morphology of stomach ossicles of Dilocarcinini Diagramação e XML SciELO Publishing Schema: www.editoraletra1.com Nauplius, 26: e2018024 combined with external morphology and perhaps even molecular data to properly evaluate the phylogenetic relationships within Trichodactylidae and their systematic position within Brachyura.


inTroduCTion
The Neotropical freshwater crab family Trichodactylidae comprises of 49 species distributed in 15 genera and two subfamilies (Ng et al., 2008).The suprageneric classification of the family was delineated by Rodríguez (1992) and reassessed by Magalhães and Türkay (1996), who used characters of the abdomen, the first male pleopod (gonopod) and the endofragmal skeleton as the basis of their classification system.These authors proposed a classification in which Trichodactylidae is divided into two subfamilies and two tribes [Trichodactylinae and Dilocarcininae (Dilocarcinini and Valdiviini)].The information currently available on the gastric system of trichodactylid crabs is restricted to descriptions of the foregut ossicles of Valdivia serrata White, 1847, Dilocarcinus septemdentatus (Herbst, 1783), and Sylviocarcinus pictus (H.Milne-Edwards, 1853) by Alves et al. (2010), and to the study of Lima-Gomes et al. (2017), which added some previously unnoticed ossicles to the description of the foregut ossicles of V. serrata and S. pictus.
The ossicles of crustacean stomachs, in addition to offering a possible indication of how the digestive processes work in each group, might provide valuable information on phylogenetic relationships between the groups (Patwardhan, 1935;Brösing et al., 2002;2007).In this study, we analyzed the stomachs of four Dilocarcinini species, Dilocarcinus pagei Stimpson, 1861, D. septemdentatus, Moreirocarcinus emarginatus (H. Milne Edwards, 1853), and M. laevifrons (Moreira, 1901) for comparing the morphology of their foregut ossicless and to assess the usefulness of the ossicles as possible morphological characters in studies on the intrafamilial relationships of Trichodactylidae.

MaTerial and MeThods
Five uncatalogued specimens of each species of Dilocarcinus pagei, D. septemdentatus, Moreirocarcinus emarginatus, and two specimens of the M. laevifrons, adult and juvenile males and females, from the Crustacean Collection of the Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus (Tab.1), were used for the dissection of the stomachs.The specimens were separated according to sex and measured by carapace width (measured across the carapace at its widest point) and carapace length (measured along the midline from the frontal to the posterior margin).Their stomachs were obtained after removing the carapace and fixed in 10% formalin for 24 hours.For tissue maceration, stomachs were heated for 60 minutes in 10% Potassium Hydroxide (KOH) solution and further to 100°C (Mocquard, 1883;Brösing et al., 2002).Then the skeleton was stained with Alizarin Red 1% added into the KOH solution to facilitate visualization of the internal structures such as setae and ossicles (Brösing et al., 2002).Illustrations of the foregut ossicles were made in ventral, dorsal and lateral views, with drawings prepared with the aid of a camera lucida mounted on the stereoscopic microscope.
The nomenclature and abbreviations used in the morphological descriptions of the foregut ossicles follows Lima (2010), and a complete list was constructed with these names and abbreviations of all the described ossicles (Tab.2).

resulTs
In the species studied, the esophagus is connected with the antero-ventral portion of the cardiac chamber.This chamber is responsible for grinding and maceration of food particles (Mocquard, 1883;Patwardhan, 1935; Morphology of stomach ossicles of Dilocarcinini Nauplius, 26: e2018024  Meiss and Norman, 1977), its shape is wider than long, and consisting of the cardiac sac in the anterior portion and the ossicles of the gastric mill in the posterior portion.This cardiac sac is composed of a fairly thin chitin membrane.Ventrally there is the pyloric chamber, which comprises the posterior portion of the stomach, and is generally longer than wide and much smaller than the cardiac chamber.The pyloric chamber is responsible for the digestion, absorption and elimination of food particles (Mocquard, 1883;Patwardhan, 1935;Meiss and Norman, 1977;Brösing et al., 2002;Brösing, 2010).

Morphology of stomach ossicles of Dilocarcinini
Nauplius, 26: e2018024 The foregut ossicles consists of 11 ossicles of the gastric mill, 10 ossicles of the lateral supporting cardiac region, 4 ossicles of the cardio-pyloric valve, 6 ossicles supporting the dorsal pyloric chamber, 9 supporting ossicles of the ventral pylorus and ampullae, 3 supporting ossicles of the supra-ampullary, and 5 ossicles supporting the lateral pylorus region and cardiac-pyloric valve, totaling 48 ossicles for these species.
Of these 48 recognized ossicles in the four species studied (Figs.1-4), the following 34 are similar in shape, size and degree of calcification The description of all ossicles follows, and the differences among the studied species are highlighted.
Pyloric ossicle (III): unpaired, compressed, convex, moderately calcified, located in the dorsal part of the stomach, connecting the pro-pyloric (VI), exopiloric (IV) and urocardiac ossicles (VII); superior edge slightly bend; anterolateral angle with long process.The apex of the anterolateral angle process is conspicuous in D. pagei and M. laevifrons, but inconspicuous in D. septemdentatus and M. emarginatus.Subterminal process curved mesially.Deep median notch along coth shells.
Pre-pectineal ossicle (IX): paired, heavily calcified, divided into two distinct portions.Anterior portion narrower than posterior portion, thin and subcylindrical, located laterally in the cardiac stomach and connected to the post-pterocardiac ossicle (IIb), Anterior portion is as long as the posterior portion in D. pagei and M. laevifrons; only one-third the length of the posterior portion in M. emarginatus, and almost half the length of the posterior portion in D. septemdentatus.Posterior portion connected to the pectineal ossicle (VIII), devoid of lateral process.

Morphology of stomach ossicles of Dilocarcinini
Nauplius, 26: e2018024 with two blades moderately calcified; anterior and posterior blades partially fused to lateral posterior cardiac plate and lateral-inferior cardiac ossicle, respectively; anterior blade with double row of bristles, posterior plate devoid of bristles.Lateral margin subcylindrical and calcified.Distal process connected to pectineal ossicle (VIII) and projected anterolaterally, surpassing distal end of mesial plate.
Quill of the postpectineal ossicle (Xa): paired, heavily calcified, J-shaped, located in lower part of the cardiac stomach; anterior end with compressed, subtriangular process projected dorsally.Anterior portion loosely connected to lower cardiac valve and to lateral posterior cardiac plate; posterior portion subcylindrical, completely fused to post-pectineal ossicle (VIII).Quill of the postpectineal ossicle (Xa) forming obtuse angle with lateral margin of the postpectineal ossicle (X).
Anterior lateral cardiac plate (XI): paired, moderately calcified, falciform, located in the anterior part of the cardiac sac.Anterior side devoid of longitudinal carina.
Inferior cardiac valve (XIIa): paired, moderately calcified, tapering, located at lateral part of bottom of cardiac stomach, loosely connected to posterior lateral cardiac plate (XII) and to quill of the postpectineal ossicle (Xa).Mesial part elongated, covered with tufts of long setae.
Inferior-lateral cardiac ossicle (XIII): paired, heavily calcified, located laterally in the cardiac stomach.Lower part with very long process, distinctly larger than width of the ossicle; posterior region strongly convex.
Subdentate ossicle (XIV): paired, heavily calcified, with conspicuous mesial bulge.Inferior part ending in two projections: anterior one acute, posterior one truncated, with clear semicircular mark in D. septemdentatus but inconspicuous in D. pagei, M. laevifrons and M. emarginatus.Inferior part of ossicle well tapered in D. septemdentatus, and a little tapered in D. pagei, M. emarginatus and M. laevifrons.Posterior margin without laminar expansion.
Anterior ossicle of cardio-pyloric valve (XVI): unpaired, lateral borders slightly calcified, located ventrally on the border between cardiac and pyloric stomachs, connected laterally to the inferior lateral cardiac ossicle (XIII) and posteriorly to the valve cardio-pyloric (v.c.p.).Posterior mesial part with heavily calcified, elongated process in D. pagei, and with short process in D. septemdentatus, M. laevifrons and M. emarginatus; mesial process straight, not bifurcated in any species.

Morphology of stomach ossicles of Dilocarcinini
Nauplius, 26: e2018024 Posterior mesopyloric ossicle (XX): paired, heavily calcified, semicircular; dorsolateral face with a crest.Total length of posterior mesopyloric ossicle measures half of the total length of anterior mesopyloric ossicle (XIX) in D. pagei and M. emarginatus, one third of the length in M. laevifrons, and approximately the same length of the anterior mesopyloric ossicle (XIX) in D. septemdentatus.
Posterior-inferior pyloric ossicle (XXVII): unpaired, mesially arched, strongly calcified in middle part, moderately so near borders, located in lower end part of pyloric stomach after the ampullae, connected anteriorly to inferior ampullary ossicle (XXIV) and posteriorly to posterior supra-ampullary ossicle (XXX).Lateral borders recurved lateroposteriorly.Maximum width of the posterior-inferior pyloric ossicle (XXVII) as wide as 0.5 time the maximum width of the ampullae.

Morphology of stomach ossicles of Dilocarcinini
Nauplius, 26: e2018024 heavily calcified bar, posterior portion laminated with a terminal process.
Posterior supra-ampullay ossicle (XXX): paired, heavily calcified, leaf shaped, located vertically in posterolateral portion of pyloric stomach, after ampullae and above inferior-posterior pyloric ossicle; upper portion fully fused to posterior pleuropyloric ossicle, lower part firmly attached to inferior-posterior pyloric ossicle.Upper part with laminated process projected laterally.

disCussion
The stomach is composed of seven sets of ossicles that together form 48 ossicles, but the early authors who studied this system did not recognize all of these ossicles (Mocquard, 1883;Cochran, 1935;Maynard and Dando, 1974;Meiss and Norman, 1977;Brösing et al., 2002).Since the studies of Mocquard (1883) only the mesocardiac, exopyloric, pro-pyloric, zygocardiac, and urocardic ossicles have been frequently mentioned.The definition of the remaining ossicles that constitute the foregut ossicles varied depending of each author and, therefore, the nomenclature used in early studies had to be modified.The incorrect identification of the ossicles and the proposals of new ossicles without justification can cause instability in the nomenclature and thus generate difficulties in comparing the results obtained by different authors (Lima, 2010).
Most of the ossicles are very similar in the four species studied.The shape of the ossicles did not vary either in relation to the stage of development or the sex of the individuals, nor did we observe a variation in the number of ossicles in younger individuals, in the same way as in the studies of Lima-Gomes (2013) with Trichodactylidae species.Nevertheless, in some cases the ossicles of young specimens, regardless of sex, were more decalcified than in the adults, but still conspicuous, such as the pyloric ossicles, the anterior inferior-pyloric ossicle and some plaques.This is also the case for D. septemdentatus, thus differing from the results of Alves et al. (2010), who only recognized 37 ossicles.
According to Mocquard (1883), the pyloric ossicle is an unpaired ossicle, presenting a membranous longitudinal fissure, but Cochran (1935) described it as a paired ossicle.Maynard and Dando (1974), studying lobsters (Panulirus White, 1847 and Homarus Weber, 1795) and crabs (Callinectes Stimpson, 1860), referred to the pyloric ossicle as being an unpaired and paired ossicle, respectively, depending upon the animal under study.In the present study, we considered that the pyloric ossicle is paired because it had a membranous fissure, which would lead to a complete separation of the ossicles (Figs.1A, 2A, 3A, 4A).The ossicle differed only in relation to the apex of the anterolateral angle among the four species studied, being conspicuous in D. pagei and M. laevifrons, and inconspicuous in D. septemdentatus and M. emarginatus.

Morphology of stomach ossicles of Dilocarcinini
Nauplius, 26: e2018024 emarginatus and M. laevifrons bearing dorso-lateral face devoid of carina).However, this pattern does not hold for another feature of this very same ossicle: the mesial end teeth are separate in D. pagei, D. septemdentatus, and M. laevifrons, but fused in M. emarginatus.Other examples of such inconsistent patterns can be found in the morphological description of the ossicles above.
Therefore, the overall resemblance of the ossicles of the stomachs among the species of Dilocarcinini studied herein and those of Valdiviini (see Lima-Gomes et al., 2017) does not seem to provide sufficient support to consider the ossicles as good characters for phylogenetic studies of the Trichodactylidae, since these similarities would generate many homoplasies.Although these characters were useful for corroborating the monophyly of Gecarcinidae (see Lima, 2010) and Lithodidae (see Olguin, 2016), they may not be as useful for separating groups within the trichodactylid crabs.However, a more comprehensive study that includes representatives of the other genera of both subfamilies remains necessary for a conclusive evaluation.

aCKnowledgeMenTs
This paper is dedicated to the memory of the late Prof.Dr. Michael Türkay for his outstanding contributions to the taxonomy of freshwater crabs and for calling our attention (in special, CM) to the importance of an integrative approach in taxonomic studies.We are gratefull to Dr. Sammy De Grave (Oxford University Museum of Natural History) for reviewing the English wording.The authors also thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq): RCLG for a doctoral scholarship (140191/2014-3); and CM for an ongoing research grant (304736/2015-5).

Table 2 .
Nomenclature and abbreviations used in the morphological descriptions ossicles of the stomachs of four species of Dilocarcinini.