The brain of Brycon orbignyanus (Valenciennes, 1850) (Teleostei: Characiformes: Bryconidae): gross morphology and phylogenetic considerations

ABSTRACT

The brain of Brycon orbignyanus is described as a model for future studies of the gross morphology of the central nervous system in Characiformes. The study of brain gross morphology of 48 distinct taxa of Characiformes, one of Cypriniformes, two of Siluriformes and two of Gymnotiformes, allowed us to propose, for the first time, six putative brain synapomorphies for the Characiformes and also two possibly unique gross brain morphology characters for the Siluriformes. A detailed protocol for the extraction of the brain in Characiformes is also provided.

Keywords:
Comparative morphology; Encephalon; Ostariophysi; Otophysi; Putative synapomorphies

RESUMO

O encéfalo de Brycon orbignyanus é descrito como um modelo para futuros estudos da anatomia externa do Sistema Nervoso Central de Characiformes. O estudo da morfologia externa de 48 táxons distintos de Characiformes, um de Cypriniformes, dois de Siluriformes e dois de Gymnotiformes, permitiu-nos propor, pela primeira vez, seis prováveis sinapomorfias encefálicas e também duas possíveis características encefálicas para Siluriformes. Um protocolo detalhado para a dissecção e extração do encéfalo de Characiformes é também apresentado.

Introduction

During the last two centuries, diagnoses of fish taxa and hypotheses of their evolutionary relationships were almost exclusively based on osteological attributes (see Wiley & Johnson, 2010; Datovo & Vari, 2014). This extensive exploration of osteological features in bony fishes was really efficient in the delimitation of major Teleostei clades; notwithstanding, this almost exclusive focus on osteological features resulted in the "relatively minor attention" to other anatomical systems (Datovo & Vari, 2014), and very few studies have even tried to describe and analyze other major anatomical systems in fishes, such as the neuroanatomy, which according to Datovo & Vari (2014), based mostly on Wiley & Johnson (2010), represents approximately 1% of the synapomorphies currently recognized for teleosteans fishes.

Studies of comparative brain anatomy of teleosts focusing on phylogenetic relationships are scarce, and the first to combine brain features and cladistic methods was Northcutt (1984, 1985), who has shown that cladistics analytical tools could be used to find out the patterns resulting from the evolution of vertebrate brains (Abrahão & Pupo, 2014; Striedter, 2005). Previous to Northcutt's (1984, 1985) publications, studies of fish brain anatomy were focused on the relationship between ecological attributes and brain gross morphology, or simple descriptions - either total or partial - of chondrichthyan and teleostean brains (Ewart, 1888; Herrick, 1899, 1901; Evans, 1931, 1940; Miller & Evans, 1965; Nieuwenhuys, 1967).

Despite the scarcity of neuroanatomical features in phylogenetic analyses, a few neuroanatomical characters were found to be synapomorphic for some lineages of Teleostei. Some examples include the anterior brain position in relation to the cranial cavity in Gadiformes: Melanonoidei (Howes, 1993); the olfactory sensory epithelium arranged in sensory islets and absence of the saccus vasculosus in the Cyprinodontiformes and Atheriniformes (Yamamoto, 1982; Parenti, 1993, 2005); and the large dorsal telencephalon of the anterior portion of central nucleus and small medial portion, together with the absence of accessory optic tract and nucleus (except for the Sternopygidae), in addition to ampullary organs organized in rosettes in Gymnotiformes (Albert et al., 1998; Wiley & Johnson, 2010).

Characiformes contains approximately 2,100 valid living species, occurring mostly in the freshwaters of the Neotropical region (19 families) and less so in the African Sub-Saharan region of Africa (four families) (Reis et al., 2003; Oliveira et al., 2011). As with other teleostean groups, the clades within the Characiformes are diagnosed and have their evolutionary interrelationships hypothesized almost exclusively with osteological characters (Fink & Fink, 1981, 1996). The great diversity of lifestyles found in Characiformes is most probably reflected in the organization of their central nervous system (see Nieuwenhuys et al., 1998), thus, we hypothesize that the study of the brain gross morphology of characiforms can provide important insights into their biology, ecology, behavior, evolution and phylogenetic relationships, as pointed by Lisney & Collin (2006) for the Animal Kingdom, and their diversity of brain morphologies as a whole.

Among the Otophysi (composed by the Orders Cypriniformes, Characiformes, Siluriformes and Gymnotiformes), the study of the brain gross morphology has recently been addressed for gymnotiforms (Albert et al., 1998; Albert, 2001), in addition to the siluriforms Callichthyidae and Pseudopimelodidae (Abrahão & Shibatta, 2015), the Callichthyidae which was the subject of a Master's dissertation (Pupo, 2011) and the Characiformes studied in a Doctoral thesis (Pereira, 2014), both unpublished at this moment. These Otophysi brain studies have unequivocally shown that the central nervous system can be an important source of phylogenetically informative morphological characters, although relatively unexplored. Bearing that in mind, we have chosen Brycon orbignyanus , a member of the putative generalized and phylogenetically basal family Bryconidae (Roberts, 1969; Mirande, 2009, 2010; Oliveira et al., 2011), to be described as an example of a generalized Characiformes brain.

Material and Methods

Specimen preparation and brain dissection. The examined specimens belong to the following institution fish collections: LBP (Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista "Júlio de Mesquita Filho"); LIRP (Laboratório de Ictiologia de Ribeirão Preto, Universidade de São Paulo); MZUSP (Museu de Zoologia da Universidade de São Paulo); and UNT (Laboratório de Sistemática Ictiológica da Universidade Federal do Tocantins). The complete list of examined specimens is summarized in Table 1.

Table 1
Material examined in the present study. Asterisk represents type species of genus; SL = Standard length; HL = Head length.

Taxa	Catalog #	Morphometric data
		Total	Brain	Range (SL)	SL	HL
Acestrorhynchidae
Acestrorhynchinae
Acestrorhynchus falcatus*	LIRP 7639	12	2	(130.1-152.4)	130.24	39.86
Heterocharacinae
Heterocharax leptogrammus	MZUSP 55725	92	2	(22.7-32.1)	24.80	6.9
Anostomidae
Schizodon nasutus	LIRP 7151	2	1	(211.8-251.0)	211.80	48.17
Bryconidae
Bryconinae
Brycon orbignyanus	LIRP 6309	4	2	(170.8-183.4)	175.25	41.90
Salmininae
Salminus hilarii	LIRP 7084	2	1	(215.6-237.1)	237.10	65.07
Chalceidae
Chalceus erythrurus	LIRP 5955	2	1	(175-192)	192.00	50.40
Chalceus guaporensis	LIRP 8625	15	2	(140.1-152.7)	146.37	37.53
Characidae
Acestrorhamphinae
Oligosarcus pintoi	LIRP 7615	13	2	(75.6-83.3)	76.4	22.1
Aphyocharacinae
Aphyocharax dentatus	LIRP 2018	16	2	(27.8-57.0)	45.88	11.68
Aphyoditeinae
Microschemobrycon callops	LIRP 7544	200	5	(24.4-27.0)	25.86	6.82
Astyanax clade
Astyanax lacustris	LIRP 3243	7	2	(71.8-96.0)	85.4	20.9
Astyanax jordani	LBP 4586	5	1	(56.5-68.7)	61.0	15.9
Characinae
Charax leticiae	MZUSP 89106	119	2	(30.8-88.8)	88.11	27.63
Roeboexodon guyanensis*	MZUSP 94250	-	1	(57.2-51.8)	53.2	15.1
Exodon paradoxus*	LIRP 7535	174	2	(44.0-52.1)	51.30	14.90
Roeboides descalvadensis	LIRP 7624	11	2	(66.4-73.1)	68.54	17.89
Roeboides myersi	MZUSP 85208	96	1	(66.13-110.29)	76.00	23.10
Roeboides prognathus	MZUSP 6536	10	1	(72.35-89.09)	76.50	19.6
Cheirodontinae
Serrapinnus notomelas	LIRP 1819	64	2	(20.9-27.1)	26.53	6.37
Pristelinae
Hemigrammus marginatus	LIRP 4272	56	2	(19.6-27.9)	27.7	6.9
Moenkhausia sanctafilomenae	LIRP 2385	9	2	(37.2-57.3)	37.9	10.0
Rhoadsiinae
Rhoadsia altipinna*	LIRP 8157	16	1	(25.0-79.8)	72.83	21.79
Stervadiinae
Mimagoniates rheocharis	LIRP 6127	12	2	(29.6-51.1)	51.11	11.39
Stethaprioninae
Gymnocorymbus ternetzi	LIRP 6018	23	2	(27.1-41.7)	40.97	11.03
Tetragonopterinae
Tetragonopterus argenteus*	LIRP 5779	10	2	(48.9-57.0)	57.12	17.21
Probolodus heterostomus*	MZUSP 7904	30	1	(40.2-58.8)	46.6	12.2
Incertae sedis
Stygichthys typhlops*	LBP 8107	4	1	-	33.9	11.0
Chilodontidae
Caenotropus labyrinthicus*	LIRP 7537	76	2	(54.7-78.3)	70.36	18.71
Crenuchidae
Characidium fasciatum*	LIRP 10	24	2	(30.3-54.8)	39.68	9.27
Ctenoluciidae
Boulengerella cuvieri	LIRP 7536	5	1	(150.1-266.7)	156.86	50.36
Curimatidae
Steindachnerina brevipinna	LIRP 7505	56	2	(47.5-92.1)	78.22	19.92
Cynodontidae
Cynodon gibbus*	UNT 11753	04	1	(176.2-189.3)	188.10	41.55
Erythrinidae
Hoplerythrinus unitaeniatus*	LIRP 750	8	1	(97.1-134.5)	117.33	35.32
Gasteropelecidae
Carnegiella marthae	LBP 4199	211	2	(20.9-29.7)	28.62	7.61
Hemiodontidae
Hemiodus sterni	LIRP 7636	9	1	(96.2-135.0)	112.56	25.18
Iguanodectidae
Iguanodectinae
Iguanodectes spilurus	MZUSP 109455	100	2	(47.4-53.3)	51.30	10.00
Lebiasinidae
Pyrrhulininae
Pyrrhulina australis	LIRP 6049	10	2	(30.9-34.3)	32.91	7.99
Parodontidae
Apareiodon affinis	LIRP 7613	8	2	(98.8-101.1)	100.03	23.51
Prochilodontidae
Prochilodus lineatus	LIRP 7321	5	2	(135.6-185.6)	139.27	36.93
Serrasalmidae
Acnodon normani	UNT 2022	1	1	-	90.00	27.23
Catoprion mento*	MZUSP 8451	76	1	(29.74-107.45)	66.4	21.1
Serrasalmus maculatus	LIRP 8013	8	1	(80.7-84.2)	82.72	28.29
Utiaritichthys sennaebragai*	LIRP 8158	10	2	(30.4-114.5)	53.73	14.08
Triportheidae
Agoniatinae
Agoniates halecinus*	UNT 8759	2	1	(193.9-233.4)	193.90	39.62
Triportheinae
Triportheus nematurus	LIRP 7800	2	1	(79.8-106.8)	79.87	20.65
Citharinidae
Citharinus latus*	MZUSP 84480	17	1	(88.2-114.9)	104.70	33.45
Distichodontidae
Neolebias unifasciatus*	MZUSP 84476	223	2	(18.6-29.7)	26.6	7.5
Hepsetidae
Hepsetus odoe*	MZUSP 84469	6	1	(111.7-144.2)	112.4	38.2
Outgroups
Cypriniformes
Cyprinidae
Cyprininae
Cyprinus carpio*	LIRP 8923	2	2	(72.3-99.4)	72.3	23.8
Gymnotiformes
Gymnotidae
Gymnotus carapo	LIRP 7767	6	1	(129.0-135.8)	129.0	16.1
Sternopygidae
Sternopygus macrurus*	LIRP 4918	8	1	(127.3-115.3)	115.3	13.1
Siluriformes
Diplomystidae
Diplomystes mesembrinus	LBP 449	21	1	(71.9-126.8)	72.62	18.4
Pimelodidae
Pimelodus maculatus*	LIRP 6012	3	1	(67.4-86.2)	67.4	19.3

All specimens examined in the present study were adults to avoid the potentially confusing effects of developmental changes (Huber & Rylander, 1992). Standard length (SL) and head length (HL) were taken point to point with digital calipers on the left side of the specimens. Specimens were stained following the musculature dissection technique proposed by Datovo & Bockmann (2010), which allows a better visualization of cranial bones and its sutures in prepared specimens without any undesirable changes caused to their brains. The brains were then removed from the braincase using a protocol specifically developed and described below for bony fishes with laterally compressed skulls, like the characiforms.

Brain dissection protocol. To extract the brain of characiform fishes, the following dissection procedure was applied on both sides of the specimens heads:

To remove the lateral bones, branchial-basket and eyes, first scrape the epidermal layer (skin) on the opercle, orbital, facial, maxilla, premaxilla and dentary bones - scraping the epidermal layer (skin) and fat (e.g. Anostomoidea) of the neurocranium roof allows a better visualization of the cranial sutures. Remove the eyeball and associated musculature (Musculus rectus superior , Musculus rectus externus , Musculus rectus inferior , Musculus rectus internus , Musculus obliquus superior and Musculus obliquus inferior ), making a severing incision in the proximal region of eyeball muscles, as well as in the pedunculi bulbi (anterior portion of nervus opticus ). Cut free the maxillary, premaxillary and dentary bones. During the process, remove the anterior portion of the olfactory epithelium, located nearby the maxillary and premaxillary bones.

Remove the epaxial musculature located near the Weberian Apparatus and supraneural bones. Make an incision on the mid-posterior surface portion of the supraoccipital bone, and proceed to the first dorsal-fin ray removing the epaxial musculature until reaching the neural tube.

To completely disjoint and remove the cranial roof, first cut open the mesethmoid sutures with adjacent bones and remove it; then cut lengthwise the soft tissue of the frontal fontanel (the Erythrinidae and Lebiasinidae both have a completely ossified fontanel and thus bone will be cut instead of soft connective tissue); proceed backwards separating the paired parietal and supraoccipital bones, and then remove them totally. At this point the posterior portion of the brain corpus cerebelii and rhombencephalon should be visible. Cut free the pterotic and sphenotic bones, taking into account that both bones encase the lateral sides of the brain, making their inadequate removal prone to damage the brain. At the end of the aforementioned dissecting steps the brain should be almost completely exposed laterally and dorsally, and intact.

To completely extract the brain from the neurocranium, begin by making a severing incision on the posterior portion of medulla spinalis , posterior to the root the nervus vagus near and anterior to the vertical passing through the middle of the Weberian Apparatus and posterior to the imaginary line on the ventral surface of the insertion of the complex of the spino-occipitales nerves; the subsequent cuts of the cranial nerves must proceed in the following postero-anteriorly sequence, to avoid breaking inadvertently the anterior cranial nerves: sever the efferent (n. X) of lobus vagi ; the slim nervus abducens (n. VI) on the floor of the neurocranium; nerves from octavolateralis area (nervus trigeminus - n. V, nervus facialis - n. VII, nervus octavus - n. VIII, nervus linea lateralis anterior - nlla and nervus linea lateralis posterior - nllp); the nervus opticus (n. II) at the middle portion of the nervus passing through the floor of neurocranium where the chiasma opticum is located, and finally the nervus olfactorius (n. I). When the described procedure is complete, the brain should be completely free from the neurocranium.

Illustration and description. Brain illustrations were made using a pen tablet digital interface and image editing softwares applied to digital photographs made with a stereomicroscope and an attached digital camera. Colors in all illustrations are entirely arbitrary, not corresponding to the real colors of the anatomical structures illustrated. Brain descriptions were based on Meek & Nieuwenhuys (1998) and Striedter (2005), with a single modification: fish brains descriptions usually follow the posteroanterior direction (e.g. , Meek & Nieuwenhuys, 1998), but herein we chose to follow Striedter (2005) in adopting the anteroposterior direction, the most commonly used for vertebrates as a whole (see Bauchot et al., 1989; Striedter, 2005; Eastman & Lannoo, 2007, 2008 for fishes; ten Donkelaar, 1998a for amphibians; ten Donkelaar, 1998b for reptiles and Walsh & Milner, 2011 for avians). For illustration and description purposes, the brains were divided into telencephalon , mesencephalon , diencephalon , rhombencephalon and medulla spinalis (Fig. 1).

The anatomical brain nomenclature follows Meek & Nieuwenhuys (1998) and Striedter (2005); bone nomenclature follows Weitzman (1962), with the modifications proposed by Castro & Vari (2004) and musculature terminology follows Datovo & Vari (2013; 2014).

Fig. 1
Brain of Brycon orbignyanus (Characiformes: Bryconidae), LIRP 6309, 175.5 mm SL. Main encephalic divisons (Telencephalon , Diencephalon , Mesencephalon , Rhombencephalon + Medulla oblongata and Medulla spinalis ) in different colors. a. dorsal; b. lateral and c. ventral views.

Results

Brain gross morphology of Brycon orbignyanus (Figs. 1 and 2). The brain limits established for Brycon orbignyanus and also applied to other Characiformes examined in the present study are: the anterior portions of the bulbus olfactorius anteriorly, usually ending at the oval olfactory epithelium and the insertion of the complex of the spino -occipitales nerves on the ventral surface of the medulla spinalis , posteriorly. The encephalon is slightly elongate and narrow; slightly wider in its middle portion near the mesencephalon (tectum opticus ) and diencephalon . The brain occupies the cranial cavity almost entirely, from the region near the mesethmoid and lateral ethmoid to the region anterior to the third neural arch, not contacting the Weberian apparatus.

Fig. 2
Brain of Brycon orbignyanus (Characiformes: Bryconidae), LIRP 6309, 175.5 mm SL. a. dorsal; b. lateral and c. ventral views. Apt = Area postrema; Bol = bulbus olfactorius; Ch = chiasma opticum; Cocb = corpus cerebelli; Dien = diencephalon; Eg = eminentia granularis; Hyp = hypophysis; Hyt = hypothalamus; Lih = lobus inferior hypothalami; LobX = lobus vagi; Mo = medulla oblongata; Ms = medulla spinalis; Pob = nervus tractus olfactorius; Sv = saccus vasculosus; Tect = tectum opticum;; Telen = Telencephalon Tl = Torus lateralis; Tv = tela ventriculi; Cranial Nerves: nI = nervus olfactorius; nII = nervus opticus; nIII = nervus oculomotorius; nIV = nervus trochlearis; nV = nervus trigeminus; nVI = nervus abducens; nVII = nervus fascialis; nVIII = nervus octavus; nLLa = nervus lineae lateralis anterior; nLLp = nervus lineae lateralis posterior; nIX = nervus glossopharyngeus; nX = nervus vagus and; nSo = nervus spino-occipitales.

The olfactory epithelium is not considered as part of the telencephalon properly. In Brycon orbignyanus it is oval with a narrow support rod surrounded by 25 to 30 lamellae similar in size. The most rostral component of the telencephalon is the bulbus olfactorius . In B. orbignyanus , each nervus tractus olfactorius is composed of a slender and relatively elongated olfactory peduncle with a terminal expansion. The olfactory bulb is oval and elongate, narrower proximally and enlarged distally. The nervus tractus olfactorius is inserted directly on the ventral surface of the telencephalon .

The telencephalon is divided in two distinct parts: a conspicuous and well-developed area dorsale on its dorsal surface, and a small narrow area ventrale on its ventral surface, both parts widely and closely interconnected.

The diencephalon is well developed, located between the telencephalon and rhombencephalon , and composed of the following parts: the epithalamus and a pineal gland, both of which are inconspicuous and easily lost during dissection and located on the dorsal surface, but not in contact with the telencephalon and corpus cerebelli ; an oval and extremely reduced saccus vasculosus ; a vertically developed hypothalamus that is seen as a slight prominence on the ventral surface of the diencephalon , in ventral and lateral views; an oval lobus inferior hypothalami , kidney-shaped in ventral view and smaller than the hypothalamus ; and an oval small hypophysis stalked on the hypothalamus .

The thalamus dorsalis arises from grooves between the tecta optici and the telencephalon , being moderately developed along their extension and thicker than the olfactory tract. The chiasma opticum is located anteriorly to the posterior telencephalon margin.

The mesencephalon is composed of the tecta optici , tegmentum , torus longitudinalis , and the torus lateralis . The tecta optici are well developed, divided in two symmetrical rounded halves that equal to approximately one-third of the brain length; the tegmentum is small and totally covered by the tectum opticus , both in dorsal and lateral views and inconspicuous; the torus longitudinalis is totally inconspicuous (visible only in stained histological sections) located between the tecta optici , near the telencephalon complex; the torus lateralis is reduced and located in front of the lobus inferior hypothalami and the anterior portion of tectum opticum , with undefined limits, both in lateral and ventral views.

The rhombencephalon comprises the associated cerebellar complex and medullary areas; the crista cerebellaris region is almost inconspicuous, being just a slight prominence, when visible; the eminentia granularis is connected exclusively to the medial region of the corpus cerebelli ; the corpus cerebelli is spherical and smaller than the tectum opticum , being the dorsalmost structure of the brain; the lobus vagi is moderately developed and shaped like lateral wings attached to the basis of the corpus cerebelli ; the lobus facialis is inconspicuous and of very difficult visualization.

The medulla spinalis is cylindrical throughout its length, except for its anterior portion, near the corpus cerebelli , where it is flattened and cone-shaped, in dorsal view; the area postrema, usually is approximately trapeze-shaped in dorsal view, but its precise delimitation is possible only in stained histological preparations; the ventriculi quarti region is a semicircular area in dorsal view, with a slight concavity that abuts the corpus cerebelli posterior margin.

The cranial nerves are: rostrally, the nervus olfactorius (nI), ending in the bulbus olfactorius ; the moderately thick nervus opticus (nII), ending in the eyeball; the nervus oculomotorius (nIII), arising from the base of the midbrain crista cerebellaris region; followed by the moderately thick nervus trochlearis (nIV) innervating the eye extrinsic muscle (Meek & Nieuwenhuys, 1998); and the posteriormost nervus trigeminus (nV), extremely slender, with ramifications not amenable to observation by us. Also in the midbrain section, arising laterally from the base of the rhombencephalon , are the anteriormost nervus fascialis (nVII), followed by the nervus octavus (nVIII), nervus linea lateralis anterior (nlla), nervus glossopharyngeus (nIX), nervus linea lateralis posterior (nllp), and the posteriormost nervus vagus (nX). Also arising from the rhombencephalon , although from its ventral surface, is the nervus abducens (nVI) (Meek & Nieuwenhuys, 1998). The posteriormost nerve is the nervus spino -occipitalis , arising from the medullary base, wide at its origin and progressively narrow distally. It is probably not a real cranial nerve, but a ganglion located between the ear and the eye, being the anteriormost part of a prominent line of neuromasts that extends from head to tail (Ghysen & Dambly-Chaudière, 2004).

Gross brain morphology in other characiforms. Some features related to the forebrain (telencephalon and diencephalon ) rhombencephalon (corpus cerebelli ) and the tecta optici are conserved in the examined taxa, thus allowing us to compare and perceive their variation within 48 taxa of Characiformes and five other non-characiform otophysan taxa herein studied (Table 1). The following similar areas of the gross brain morphologies across all the examined taxa have been considered as clearly perceived and homologous areas: 1) presence or absence of the lobus facialis ; 2) degree of development of the lobus vagi , corpus cerebelli and tecta optici ; 3) width of rhombencephalon ; and 4) external morphology of area postrema (see Discussion).

The telencephalon (pallium ) is the most variable brain area in terms of size and shape, as observed in several other actinopterygian taxa (see Northcutt, 1981), due to the formation of a highly differentiated superficial layer of gray matter after the eversion of pallium (Northcutt, 1981), as we have also observed both in Characiformes and outgroup taxa, making almost impossible to identify a generalized morphological pattern for the area. The corpus cerebelli together with the telencephalon are the most variable parts of the actinopterygian brains regarding their size and shape (Nieuwenhuys, 1982) and present a unique additional structure known as eminentia granularis in teleosteans (Nieuwenhuys, 1967), as observed in characiforms.

On the other hand, in Characiformes the rhombencephalon is moderately developed, (i.e. length and height) with a modest corpus cerebelli , different from the rhombencephalon observed by us in the Cypriniformes, Siluriformes and Gymnotiformes taxa, which present comparatively larger corpus cerebelli .

The area postrema region on the dorsal surface of rhombencephalon presents a unique shape in all the characiform taxa examined, usually with a slight depression, quite different from its shape in the comparative taxa of Cypriniformes, Siluriformes and Gymnotiformes examined (Figs. 2-5).

In the present study, we have observed a considerable diversity in the corpus cerebelli of the Characiformes, always much reduced when compared to the corpus cerebelli of the examined Gymnotiformes and Siluriformes.

The tecta optici form the roof of the midbrain in teleostean fishes and are considered the main visual center in fishes (Northmore, 2011). In the examined taxa, it showed an enormous variation of its relative size, probably related to the relative importance of vision in the various taxa. Nevertheless, several authors affirmed that, in vertebrates, the relative size of parts of vision apparatus is totally correlated to size of the image on retina and visual information reaching the brain (Garamszegi et al., 2002; Howland et al., 2004). According to our results, Characiformes and Cypriniformes usually present normally developed and similar optic structures, differing mostly in size, while the examined Siluriformes and Gymnotiformes taxa possess much reduced optic structures.

The lobus vagi is inconspicuous or almost absent in almost all Characiformes examined. When visible, the lobus vagi is shaped as small lateral wings emerging from the rhombencephalon base, as in Brycon orbygnianus (Figs. 1 and 2), except in the Chilodontidae which have a large lobus vagi , very similar in shape and size to the one found in our representative taxon of the Cypriniformes.

Remarkably the lobus facialis in the Characiformes is barely observable, clearly different from the conspicuous structure found in all the Cypriniformes, Siluriformes and Gymnotiformes examined.

In addition, we also propose herein two apparently exclusive brain features of the Siluriformes among all the examined taxa, but we consider that more studies must be done to establish their real phylogenetic signals, increasing the number of taxa representing this order (Figs. 1-3and 5): (1) olfactory rosette elongate and well developed (Fig. 6c) vs . the olfactory rosette approximately circular and moderately developed in Characiformes, Cypriniformes and Gymnotiformes (Fig. 6a, b and d); and (2) 60 olfactory lamellae present vs . a comparatively reduced number of lamellae, not surpassing 30, in the representatives of the Cypriniformes (16 lamellae), Characiformes (25-30 lamellae), and Gymnotiformes (15 lamellae) (Fig. 6a-d). Taking into account the well-known fact that the Cypriniformes and Characiformes are usually primarily diurnal and visually oriented, whereas the Siluriformes and Gymnotiformes are primarily nocturnal and/or inhabitants of very turbid waters, being oriented mainly by the senses of smell and tact in the case of siluriforms (see Caprio, 1978), and almost exclusively by electroreception, in the case of gymnotiforms (Albert et al., 1998; Albert, 2001), it is to be expected the presence of more complex and developed olfactory rosettes in the Siluriformes.

Fig. 3
Brain of Cyprinus carpio (Cypriniformes: Cyprinidae), LIRP 8923, 72.3 mm SL. a. dorsal; b. lateral and c. ventral views. Apt = Area postrema; Bol = bulbus olfactorius; Ch = chiasma opticum; Cocb = corpus cerebelli; Dien = diencephalon; Eg = eminentia granularis; Hyp = hypophysis; Hyt = hypothalamus; Lih = lobus inferior hypothalami; LobVII = lobus facialis; LobX = lobus vagi; Mo = medulla oblongata; Ms = medulla spinalis; Pob = nervus tractus olfactorius; Tect = tectum opticum; Telen = Telencephalon; Tl = Torus lateralis; Tv = tela ventriculi. Cranial Nerves: nI = nervus olfactorius; nII = nervus opticus; nIII = nervus oculomotorius; nIV = nervus trochlearis; nV = nervus trigeminus; nVI = nervus abducens; nVII = nervus fascialis; nVIII = nervus octavus; nlla = nervus lineae lateralis anterior; nllp = nervus lineae lateralis posterior; nIX = nervus glossopharyngeus; nX = nervus vagus and; nSo = nervus spino-occipitales.

Fig. 4
Brain of Diplomystes mesembrinus (Siluriformes: Diplomystidae), LBP 449, 72.62 mm SL. a. dorsal; b. lateral and c. ventral views. Apt = Area postrema; Bol = bulbus olfactorius; Ch = chiasma opticum; Cocb = corpus cerebelli; Dien = diencephalon; Eg = eminentia granularis; Hyp = hypophysis; Hyt = hypothalamus; Lih = lobus inferior hypothalami; LobVII = lobus facialis; LobX = lobus vagi; Mo = medulla oblongata; Ms = medulla spinalis; Pob = nervus tractus olfactorius; Tect = tectum opticum; Telen = Telencephalon; Tl = Torus lateralis; Tv = tela ventriculi. Cranial Nerves: nI = nervus olfactorius; nII = nervus opticus; nIII = nervus oculomotorius; nIV = nervus trochlearis; nV = nervus trigeminus; nVI = nervus abducens; nVII = nervus fascialis; nVIII = nervus octavus; nlla = nervus lineae lateralis anterior; nllp = nervus lineae lateralis posterior; nIX = nervus glossopharyngeus; nX = nervus vagus and; nSo = nervus spino-occipitales.

Fig. 5
Brain of Gymnotus carapo (Gymnotiformes: Gymnotidae), LIRP 7767, 129.0 mm SL. a. dorsal; b. lateral and c. ventral views. Apt = Area postrema; Bol = bulbus olfactorius; Ch = chiasma opticum; Cocb = corpus cerebelli; Dien = diencephalon; Eg = eminentia granularis; ELL = electrosensory lateral line lobus; Hl = lateral nucleus of hypotalhamus; Hyp = hypophysis; LobX = lobus vagi; Mo = medulla oblongata; Ms = medulla spinalis; ndl = lateral portion of nucleus diffusus; nE = nucleus electrosensorius; Sv = saccus vasculosus; Tect = tectum opticum; Tv = tela ventriculi; Vcocb = valvula cerebellum. Cranial Nerves: nI = nervus olfactorius; nII = nervus opticus; nV = nervus trigeminus; nVI = nervus abducens; nVII = nervus fascialis; nVIII = nervus octavus; nlla = nervus lineae lateralis anterior; nllp = nervus lineae lateralis posterior; nIX = nervus glossopharyngeus; nX = nervus vagus.

Fig. 6
Olfactory epithelium of representatives of Otophysi. a. Cyprinus carpio (Cypriniformes); b. Brycon orbignyanus (Characiformes); c. Pimelodus maculatus (Siluriformes) and; Sternopygus macrurus (Gymnotiformes). Scale bars = 1 mm.

Discussion

As pointed by Striedter (2005), even homologous brain regions differ in size, shape, position, cytoarchitecture, histochemistry, connections, and/or function across the major vertebrates groups. Striedter (2005), nevertheless, also pointed that the forebrain, corpus cerebelli and tectum opticum are conserved homologous regions of vertebrate brains. Thus, it is not unexpected that we have found the same unequivocally identifiable and conserved homologous regions in the brains of representatives of the 22 families of Characiformes analyzed in this study.

Due to the taxonomic diversity and ecological plasticity of characiform fishes, combined with our aim to make available a first description of the external brain morphology of a characiform, we chose a representative of this order possessing putatively generalized and ancestral-like external brain morphology, since the external brain form of Brycon probably resembles that of the ancestral of all characiforms. Taking these considerations together with the availability of specimens for the inevitable and destructive brain removals, we have opted to use specimens of Brycon . Among the 48 taxa of Characiformes examined by us (Table 1), B. orbignyanus possess the external brain morphology more similar to the representative of the Cypriniformes, the sister order of all remaining Otophysi, especially regarding their respective olfactory bulbs, telencephalon and corpus cerebelli (Figs. 1-3). Also, in many significant past papers the genus Brycon was considered "primitive", "generalized" or phylogenetically basal in the Neotropical Characiformes, or at least in relation to the Characidae, sharing several osteological features with basal African groups (see Weitzman, 1962; Buckup, 1998; Mirande, 2009, 2010; Malabarba & Weitzman, 2003 and Oliveira et al., 2011).

The use of B. orbignyanus external brain morphology as a surrogate of the hypothetical ancestral external brain morphology of the Characiformes should be done warily, since ours is a preliminary analysis encompassing just part of the many known taxa of Characiformes. Notwithstanding the presence of conserved homologous regions in the brains of all taxa of Characiformes, variation found in the characiform brain gross morphology allows us to consider their brain as a rich source of phylogenetically useful characters.

Bearing that in mind, we herein propose six putative synapomorphic brain features for the Characiformes, not found in the examined Cypriniformes, Siluriformes and Gymnotiformes (Figs. 3-5, respectively): (1) area postrema shaped as an inverted triangle, wider anteriorly and narrowing posteriorly (Figs. 1-2) vs . area postrema equally narrow throughout its full length, with inconspicuous limits in the Cypriniformes, Siluriformes and Gymnotiformes (Figs. 3-5, respectively); (2) width of the rhombencephalon not exceeding the width of the midbrain, both in dorsal and ventral views (Figs. 1-2) vs . rhombencephalon wider than midbrain due to the larger size of the corpus cerebelli in the Cypriniformes, Siluriformes and Gymnotiformes (Figs. 3-5, respectively); (3) lobus vagi less developed (except in the Chilodontidae) (Figs. 1-2) vs . lobus vagi well-developed in the Cypriniformes, Siluriformes and Gymnotiformes (Figs. 3-5, respectively); (4) lobus facialis inconspicuous; when visible, a small oval structure attached to the basis of the corpus cerebellaris (Figs. 1-2) vs . lobus facialis well-developed and visible in Cypriniformes and Siluriformes (Figs. 3-4, respectively), and developed but completely hidden by the uniquely large corpus cerebellaris in the Gymnotiformes (Fig. 5); (5) corpus cerebelli rounded, and elongate vertically (Figs. 1-2) vs . corpus cerebelli horizontally elongate in the Siluriformes and Gymnotiformes (Figs. 4 and 5, respectively) and moderately developed and dorsally pointed in the Cypriniformes (Fig. 3); (6) tectum opticum horizontally elongate, in contact with the anterior margin of the corpus cerebelli except in Stygichthys typhlops (a blind troglobitic species) (Figs. 1-2) vs . tectum opticum in the Cypriniformes vertically elongate and not in contact with the anterior margin of the corpus cerebelli , and relatively reduced in Siluriformes and Gymnotiformes (Figs. 3-5).

Conclusions

The initial premise in our comparative study of the brain gross morphology of the Characiformes reflected the commonly held belief that the Central Nervous System in fishes was highly conserved throughout their evolution (e.g. , Northcutt, 1984, 2002; Butler & Hodos, 2005). That was not what we found in our study, where the form of the brains has shown surprisingly variation among the examined taxa of Characiformes, having provided six phylogenetically useful characters. Thus, the CNS morphology in the Characiformes, as most probably also in other otophysan orders, is unquestionably worth exploring, as was the case for other non-osteological sources for morphological information (e.g. , Datovo & Castro, 2012; Datovo & Vari, 2014).

Acknowledgments

We are grateful to Mario de Pinna (MZUSP), Claudio de Oliveira (LBP), Paulo Lucinda (UNT-UFT) for loan of specimens for dissection. To Aléssio Datovo (MZUSP) for helping with illustrations in digital interface. To Flávio Bockmann (LIRP-USP), George Mattox (UFSCar-Sorocaba), Marcelo Britto (MNRJ-UFRJ) and Wilfred Klein (FFCLRP-USP), for the very useful criticisms and suggestions offered as members of TNAP's Doctoral Thesis (RMCC Advisor) Examination Committee. To the project "Fundos para o Fortalecimento da Conservação e Pesquisa em Coleções do Departamento de Biologia da Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto" (CNPq process nº 09/54931-0, Flávio Bockmann Coordinator) for the use of a FAXITRON digital x-ray set. TNAP was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and RMCC was financed by Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq Proc. 307554/2010-4). This study was partially supported by the FAPESP Thematic Projects "Phylogenetic relationships in the Characidae (Ostariophysi: Characiformes) (FAPESP 04/09219-6, RMCC Coordinator and Principal Investigator), and "South America Characiformes Inventory - SACI" (FAPESP 11/50282-7, Naércio Menezes Coordinator), of which RMCC is a Principal Investigator and TNAP an Associated Investigator.

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Publication Dates

• Publication in this collection
  2016

History

• Received
  28 Apr 2015
• Accepted
  20 June 2016