Comparative gross encephalon morphology in Callichthyidae ( Teleostei : Ostariophysi : Siluriformes )

Species of Callichthyidae are easily recognized by the presence of a series of lateral bony plates along the body and a pair of barbels at the junction of the lips (Reis, 1998). They are grouped into eight genera (Ferraris, 2007) distributed throughout all major cis-Andean watersheds, as well as some trans-Andean drainages. The family is the sistergroup to a clade composed of Scoloplacidae, Astroblepidae and Loricariidae (de Pinna, 1998). Studies focused on phylogenetic relationships within Callichthyidae agree with its monophyly and the recognition of two main lineages among its representatives, i.e., Callichthyinae and Corydoradinae (Reis, 1998; Britto, 2003; Shimabukuro-Dias et al., 2004; Alexandrou et al., 2011). However, knowledge about their encephalon morphology remains unknown. Investigations on the neuroanatomy of Siluriformes date to the end of the 19th century and probably the first attempt to understand the encephalon is the work of Herrick, Herrick (1891) on some species of Ictaluridae. At this time, it was thought that Comparative gross encephalon morphology in Callichthyidae (Teleostei: Ostariophysi: Siluriformes)


Original article
Introduction Species of Callichthyidae are easily recognized by the presence of a series of lateral bony plates along the body and a pair of barbels at the junction of the lips (Reis, 1998).They are grouped into eight genera (Ferraris, 2007) distributed throughout all major cis-Andean watersheds, as well as some trans-Andean drainages.The family is the sistergroup to a clade composed of Scoloplacidae, Astroblepidae and Loricariidae (de Pinna, 1998).Studies focused on phy-logenetic relationships within Callichthyidae agree with its monophyly and the recognition of two main lineages among its representatives, i.e., Callichthyinae and Corydoradinae (Reis, 1998;Britto, 2003;Shimabukuro-Dias et al., 2004;Alexandrou et al., 2011).However, knowledge about their encephalon morphology remains unknown.
Investigations on the neuroanatomy of Siluriformes date to the end of the 19 th century and probably the first attempt to understand the encephalon is the work of Herrick, Herrick (1891) on some species of Ictaluridae.At this time, it was thought that 2 e170162 [2] siluriform brains are the most specialized among Actinopterygii.Besides the high accurate description, an insight of this contribution include that the encephalon stops to grow at a fish "moderate size" (Herrick, Herrick 1891:212) even if the body continues to gain biomass and independently of the size of the neurocranium.Several studies on the external morphology of the encephalon took place in the middle of the 20th century, followed by many papers published on the anatomy, physiology, cytoarchitecture, hodology and embryology of the encephalon and the peripheral nervous system (Finger, 2000 and references therein).Despite the establishment of phylogenetic systematics as the paradigm for investigating the evolutionary relationships of organisms (Hennig, 1966), few studies have used the central nervous system as sources of characters for vertebrate studies, particularly for fish.Although there is great amount of literature on the nervous system of fish (e.g., Davis, Northcutt, 1983;Northcutt, Davis, 1983), papers relating neuroanatomy to fish Systematics are scarce.The exceptions are the works of Eastman, Lannoo (1995, 1998, 2001, 2003a, 2003b, 2004, 2007, 2008, 2011) and Lannoo, Eastman (1995, 2000, 2006) on the encephalons and sensory systems of Notothenioidei groups; Albert et al. (1998) and Albert (2001) on Gymnotiformes.More recently, this subject drew attention of some researchers as the works of Pereira (2014, Characiformes); Rosa et al. (2014, miniaturization in Otothyris); Abrahão, Shibatta (2015, Pseudopimelodus bufonius); Pupo (2015, Loricarioidea) and Pereira, Castro (2016, Brycon orbignyanus); Angulo, Langeani (2017, Rineloricaria heteroptera); Abrahão et al. (2018, Pseudopimelodidae).Wiley, Johnson (2010) reviewed morphological synapomorphies for 118 major monophyletic groups of teleost fishes.This information was summarized by Datovo, Vari (2014: fig 1) in a single chart with only about 1% coming from neuroanatomical data.None of this information had been previously used as a synapomorphy of Siluriformes or even to characterize any of its families.In addition, Kotrschal et al. (1998) assigned that both history and ecology fashioned the vast number of encephalon shapes and, as expected, more closely related species share similar patterns of encephalon morphology.Although some homoplasies (manly on highly specialized taxa) can constrain the reliability of characters states, the authors claims that due to the current knowledge on fish brains, a phylogenetic perspective, in addition to purely descriptive, is fundamental to propose testable hypotheses and advance the understanding of how evolutionary forces act on the encephalon.
Accordingly, the aims of the present study is to provide a phylogenetically-oriented description of the encephalon of Callichthyidae, considering representatives of each genus and key groups as found in Reis (1998) and Britto (2003).

Material and Methods
Examination of encephalon gross morphology was performed by the dissection of specimens preserved in 70° GL ethanol under a stereomicroscope.Representatives of all Callichthyidae genera and loricarioid families were sam- Nomenclature of encephalon subunits follows International Committee on Veterinary Gross Anatomical Nomenclature (2012) for general vertebrate structures and Meek, Nieuwenhuys (1998) for specific aspects of fish neuroanatomy.All specimens listed at Material examined session were dissected for removal of the encephalon.Data acquisition.Dissections for the removal of the encephalon were performed according to Abrahão, Pupo (2014), with the following steps: (1) removal of the skin flap on the nostrils; (2) removal of the skin of the head and predorsal area, exposing the skull roof; (3) release of the nasal organ from the olfactory chamber floor by cutting the ligaments between the anterior portion of the organ and the chamber, maintaining its attachment only by the nervus olfactorius (N.I); (4) release the bulbus olfactorius plus nervus olfactorius (N.I) from ligaments posterior to this organ, in the area anterior to the frontal bone; (5) removal of the nuchal plate and three adjacent pairs of dorsolateral plates (in Callichthyidae); (6) removal of the muscle tissue posterior to the head exposing the Weberian apparatus and associated structures; (7) incisions around the supraoccipital bone; (8) incisions between the frontal and sphenotic bones; (9)   lateralis anterior (N.lla), nervus linea lateralis posterior (N.llp)] of the cerebellum on both sides; (19) cross section of the nervus opticus (II) on both sides; and (20) removal of the encephalon with the aid of tweezers.This final step requires special attention because part of the auditory system and the hypophysis are ventral to the brainstem.
Figures of encephalon topography were made using a Leica DFC 450 digital camera attached to a Leica M205C auto-stacking multifocus stereomicroscope with the help of Leica Application Suite (version 4.8) software to obtain an "all-in-focus" image.All images were improved using the software Intensify (Macphun Software, San Diego, CA, 2016), Noiseless (Macphun Software, San Diego, CA, 2015) and Pixelmator version 3.6 (UAB Pixelmator team, Vilnius, Lithuania, 2016).

Results
A brief description of the gross morphology of the encephalon and its subunits are addressed below (Figs. 1, 2; Tab. 1).All character states in Tab. 1 refer exclusively to representatives listed in Material examined.
Lobus vagus.Among the representatives of Callichthyidae, the lobus vagus tumid with a roughly spherical shape (Figs. 1, 2, LV), and is larger than in other loricarioids (Fig. 3, LV), in which it is not as conspicuous and present a "V" shape.Exceptions of this were found in Loricaria and Loricariichthys.The anterior margin of the lobus vagus is not continuous with lobus facialis in Callichthyidae and has an anterior angular expansion positioned above the posterior portion of lobus facialis in Corydoradinae (Fig. 1, arrow), and is adjacent rather than dorsal to this lobe in Callichthyinae (Fig. 2).Another pattern was found among Loricariidae (e.g., Delturus carinotus, Hypancistrus sp., Hypostomus ancistroides, Liposarcus sp., Rhinelepis aspera), roughly pentagonal.Corpus cerebelli.Modifications of three features of the corpus cerebelli were observed among Callichthyidae: shape, volume, and position.In contrast with most of the Loricarioidea, the corpus cerebelli in callichthyids is tumid (Figs. 1, 2) and roughly spherical as several non-Siluriformes fishes (Kotrschal et al., 1998), suggesting that this modification could be exclusive to the family.In Corydoradinae, and mainly in representatives of Corydoras (Fig. 1, CC), there is a reduction in the volume of this portion of the cerebellum such that it is smaller than the tectum me- Tab. 1. Summary of the characters of gross morphology of the encephalon and its subunits, with its states and respectively taxa.included in clade 5 of Britto (2003: fig. 122: clade 5); in those cases, the corpus cerebelli is depressed, not swollen, and straight in lateral view (Pupo, 2015).

Tectum mesencephali.
A gradual increase in the absolute size of this structure was noticed among the specimens of Corydoradinae analyzed.Nervus opticus (Fig. 1, N.II), could be thinner, equal to, approximately two times thicker, or have the diameter of its cross section three times or grea- sencephali, while representatives of Callichthyinae display a significant increase in the volume of this region, such that it is greater than the lobus vagus or tectum mesencephali (Fig. 2).In most of the representatives of Loricarioidea examined in this study the corpus cerebelli is positioned in the middle portion of the encephalon with its anterior area between the two mesencephalic tecta or even posterior to them.Only in Callichthyinae does the anterior margin of this structure extend anteriorly and is in contact with, or even with a small anterior portion dorsal to, the telencephalon.The anterior position of the corpus cerebelli of specimens of Callichthyinae is also observed among advanced siluriform families   Britto's (2003) hypothesis reveals an exclusive condition within the genus Corydoras with the thickness of this nerve being three times greater than the nervus olfactorius.This state seems to be positively associated with a gradual but significant increase in the volume of the tectum mesencephali, which suggests greater efficiency in visual perception.
Hypothalamus.The hypothalamus is the ventral most region of diencephalon.It is positioned posterior to the chiasma opticum, ventral to the truncus cerebri and tectum mesencephali, and posterior to the telencephalon, in ventral view (Fig. 1, Hyt).It can be divided into the hypothalamus itself, the lobus lateralis hypothalami and the lobus inferior hypothalami.Among the representatives of Loricarioidea, the lobus inferior hypothalami possesses an invagination in its posterolateral or posterior margin only in Corydoras, suggesting an exclusive condition for the genus.Its lateral margin is concave in Callichthyidae, Delturus and Hypancistrus while angular in Trichomycteridae and Scoloplax and convex in other Siluriformes.The hypophysis is rounded in all specimens examined of all families (Fig. 1, Hyp).This structure is anchored anteriorly to the hypothalamus.
Telencephalon.Despite the presence of this subunit in all Actinopterygii studied and the numerous studies that have contributed to understanding the organization of this structure, there is no consensus as to the limits of each of its inner nucleus and even homology with other vertebrates (Northcutt, Davis, 1983).Some attempts have been successful in determining the dorsomedial (Dm), dorsolateral (Dl), dorsocentral (Dc) and dorsoposterior (Dp) lobes using histology (e.g., Eastman, Lannoo, 2007;2008;2011).The telencephalon is the structure with the most variable shape.In general, representatives of Callichthyinae possess a short telencephalon with a rounded lateral edge (Fig. 2, Tel), while among specimens of Corydoradinae this structure is more elongate, has straight lateral margins and is roughly rectangular in shape (Fig. 1, Tel).Other families exhibit a more elongate telencephalon (Astroblepidae), and in a few representatives of Trichomycteridae it is even more elongate (e.g., Stauroglanis goldingi).

Bulbus olfactorius.
Callichthyidae has a sessile bulbus olfactorius (Figs. 1, 2, BO), while it is pedunculated in other loricarioids (Fig. 3, BO), in which is connected to the telencephalon via the nervus tractus olfactorius (Fig. 3, TOl).Some taxa in Loricariidae and Trichomycteridae may possess a greatly reduced tractus olfactorius, which is indicative of the presence of the sessile bulbus among a few species of these families, mainly the miniatures.The nervus olfactorius did not exhibit noticeable superficial variation (Fig. 1, N.I).

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Olfactory organ.Within Loricarioidea, only specimens of Callichthyidae possess a circular olfactory organ (Fig. 5), while it is oval in Astroblepidae and elliptical in other Loricarioidea.All examined representatives of Callichthyidae have less than 15 lamellae, while other loricarioids exhibit more than 30.The volume of each lamella of the nasal organ increases in a medial to distal direction (from the center to the edge of rosettes) in Callichthyidae.Also in Callichthyinae, each lamella is flat and attached to the floor of the organ throughout its extension, which differs from Corydoradinae where the distal margin of the lamellae is detached from the chamber floor.In specimens of Corydoras hastatus, the olfactory epithelium does not have lamellae, but instead possesses small crests (Fig. 5c).

Discussion
The obtained data of the gross morphology of the encephalon allows some patterns of the organ in Callichthyidae and related families to be discussed.It corroborates Herrick (1891) conclusions about the cerebellum that, although its variation, it is suited to characterize families and genera, which means it is phylogenetic informative.
Although the characters presented herein have some limitations in terms of scope, they can help resolve the relationships of groups with uncertain topologies, mainly at the levels of family, subfamily, and genera, as well as raise hypotheses regarding processes of evolutionary convergence.The species Ochmacanthus alternus (27.3 mm SL), Stauroglanis gouldingi (22.9 mm SL), Trichomycterus hasemani (15.1-15.2mm SL), and Tridentopsis pearsoni (19.7 mm SL) possess a reduction in the number of olfactory lamellae (3 to 8) relative to other representatives of Trichomycteridae.Scoloplax distolothrix (14.8 mm SL), and Scoloplax empousa (12.8 mm SL) bear three lamellae, while Corydoras hastatus (15.4 mm SL) lack lamellae altogether but possesses small elevations of the olfactory epithelium (Fig. 5c).These species are considered to be miniature, suggesting that this drastic reduction in lamellae number could be related to the process of miniaturization (sensu Weitzman, Vari, 1988).
The following neuroanatomical conditions are unique to Callichthyidae: (1) nasal organ circular in shape in dorsal view; (2) olfactory organ with fewer than 15 lamellae; (3) volume of nasal organ lamellae increasing in a medialdistal direction; (4) sessile positioning of bulbus olfactorius; (5) spherical central portion of the corpus cerebelli;  size of the tectum mesencephali; (4) corpus cerebelli posterior to the tectum mesencephali; (5) decreased size of the corpus cerebelli; (6) lobus facialis ventral to corpus cerebelli; (7) lateral portion of lobus facialis angular, dorsal to the lobus vestibulolateralis; and (8) anterior margin of the lobus vagus above the posterior portion of the lobus facialis.The distributions of these character states support the groups originally proposed by Hoedeman (1952), which were supported by the morphology-based phylogenies proposed by Reis (1998) and Britto (2003) and the molecularbased phylogeny proposed by Shimabukuro-Dias et al. (2004) and Alexandrou et al. (2011).
According to Britto (2003), most of the species of the subfamily Corydoradinae studied herein are incertae sedis within Corydoras.The existence of a nervus opticus three or more times thicker than the nervus olfactorius suggests a group within the genus comprised of Corydoras polystictus, C. julii, C. trilineatus, C. multimaculatus, C. melanistius, C. hastatus and C. tukano.Furthermore, the first four belong to lineage 9 of Alexandrou et al. (2011), of which lineage 8, including C. melanistius, is its sister-group.In fact, a gradual increase in the volume of the tectum mesencephali can be observed within Corydoras, mainly in these species.This condition also occurs in representatives of Dianema.The state in which the nervus opticus is thinner than the nervus olfactorius occurs in Callichthys callichthys and Lepthoplosternum pectorale.According to observations of the habit of these animals, species of the genus Callichthys are associated with muddy bottoms, while species of Dianema occur in the middle of the water column, a behavior also observed for Corydoras hastatus.Considering a scenario where a thin nervus opticus constitutes an ancestral condition, and taking into account the topology of Reis (1998), there could be a gradient towards increased thickness of this nerve among the genera of the subfamily Callichthyinae, reflecting adaptation from benthic to mid-water swimming.
One of the most impressive conditions found in the present study involves the shape and position of the lobus facialis in Callichthyidae.This structure is considered to be involved in chemoreception because of its connection to taste buds on the surface of the body of catfish, especially on the head, lips and barbels (Butler, Hodos, 2005).Additionally, a significant increase in the lobus vagus can be observed in Callichthyidae.This center is responsible for gustatory and tactile senses in the oropharyngeal cavity.These two features suggest, in general, a high capacity for chemical perception of the environment.Kohda et al. (1995) recorded an interesting behavior in Corydoras aeneus involving females drinking sperm for the fertilization of oocytes.Later, Kohda et al. (2002) reported that female Corydoras aeneus exhibited no preference for males regarding size or aggressiveness.It is possible that the significant increase of these two encephalon lobes is related to the reproductive mode of this species, such as a chemical role (perhaps through pheromones) instead of a physical or behavioral preference.
Studies of the central nervous system almost invariably lead to issues relevant to animal behavior.More specifically, for those who study patterns, the association between structure and behavior is almost inevitable.Despite the myriad of possible interpretations of the subject, some consensus has been established on the positive relation between the size and efficiency of the encephalon subdivisions (Kotrschal et al., 1998).First, it is hard to imagine that the volume increase of a given portion of the encephalon, especially in fish, would not mean a more complex function of this area for realizing senses or processing information.Examples among teleost fish include: (1) the lobus vagus and the sense of taste in the oropharyngeal cavity; (2) lobus facialis and the chemoreception on barbels, lips and head surface; (3) cerebellum and the receipt and processing of stimuli (mechanical, electrical, motor, proprioceptive, lateral line, etc); (4) tectum mesencephali and vision; (5) telencephalon and part of olfaction, memory and processing of information from other centers; and (6) bulbus olfactorius and the olfactory organ and smell (Davis, Northcutt, 1983;Northcutt, Davis, 1983;Meek, Nieuwenhuys, 1998).In this sense, some questions and possibilities for future work are: (1) to test the relationship between parts of the encephalon and animal behavior, considering a family-level survey whose members have a broad spectrum of body sizes, habitat uses and diets, and with reasonable knowledge about their phylogeny; and (2) test taxonomic and geographic variation in parts of the central nervous system and assess the influence of microhabitat variables.
Material examined.Callichthyidae: Aspidoras albater Nijssen, Isbrücker MZUSP 50157, 2 ex., 21.8-26.8 mm SL;A. microgaleus Britto MZUSP 86842, 2 ex., 23.8-28.8 mm SL;A. poecilus Nijssen, Isbrücker MNRJ 11716, 2 ex., 26.1-27.6 Lobus facialis.In Callichthyidae, the lobus facialis (Figs.2, 4, LF) rises from the floor of the fourth ventricle, with its lateral face detached from the medial face of the ventricle.In other Loricarioidea and several Siluriformes, these lobes are fused with the edge of the fourth ventricle and with the lobus vagus.In representatives of Callichthyinae, the corpus cerebelli (Fig.2, CC) is more anterior and the lobus facialis is completely exposed in dorsal view, as in other catfish lineages.In specimens of the Corydoradinae (Fig.1, CC), the corpus cerebelli is displaced posteriorly with the lobus facialis ventral and almost completely covered by it, in dorsal view.The three divisions of the lobus facialis (lateral, intermediate and medial portions) can be seen superficially in Callichthyidae specimens(Figs.2, 4, LF).The lateral portion varies in shape and can be a dorsal swelling present only in the Callichthyinae (Fig.2, LF), or a lateral angular expansion which end advances above the lobus vestibulolateralis, present in the Corydoradinae (Fig.4, LF).
(6) lobus facialis detached from the medial margin of the lateral walls of the fourth ventricle; and (7) swelling of the lobus vagus.The monophyly of the two subfamilies of Callichthyidae is also supported by this study.The following conditions are exclusive to Callichthyinae: (1) nasal organ lamellae flattened dorsoventrally; (2) telencephalon short with a curved lateral edge; (3) corpus cerebelli adjacent or above the posterodorsal margin of the telencephalon; (4) increased volume of the corpus cerebelli; (5) lateral por-tion of the lobus facialis in the shape of a dorsolateral callosity; and (6) anterior margin of the lobus vagus adjacent to the lobus facialis.The exclusive features observed in Corydoradinae are: (1) distal area of nasal organ lamellae detached from the nasal chamber floor; (2) telencephalon with straight edges and roughly rectangular; (3) increased