Joseph T. Eastman 1 * and Michael J. Lannoo 2

Transcription

1 JOURNAL OF MORPHOLOGY 269: (2008) Brain and Sense Organ Anatomy and Histology of the Falkland Islands Mullet, Eleginops maclovinus (Eleginopidae), the Sister Group of the Antarctic Notothenioid Fishes (Perciformes: Notothenioidei) Joseph T. Eastman 1 * and Michael J. Lannoo 2 1 Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, Ohio Indiana University School of Medicine Terre Haute, Indiana State University, Terre Haute, Indiana ABSTRACT The perciform notothenioid fish Eleginops maclovinus, representing the monotypic family Eleginopidae, has a non-antarctic distribution in the Falkland Islands and southern South America. It is the sister group of the five families and 103 species of Antarctic notothenioids that dominate the cold shelf waters of Antarctica. Eleginops is the ideal subject for documenting the ancestral morphology of nervous and sensory systems that have not had historical exposure to the unusual Antarctic thermal and light regimes, and for comparing these systems with those of the phyletically derived Antarctic species. We present a detailed description of the brain and cranial nerves of Eleginops and ask how does the neural and sensory morphology of this non-antarctic notothenioid differ from that seen in the phyletically derived Antarctic notothenioids? The brain of Eleginops is similar to those of visually oriented temperate and tropical perciforms. The tectum is smaller but it has well-developed olfactory and mechanoreceptive lateral line areas and a large, caudally projecting corpus cerebellum. Eye diameter is about twofold smaller in Eleginops than in many Antarctic species. Eleginops has a duplex (rod and cone) retina with single and occasional twin cones conspicuous centrally. Ocular vascular structures include a large choroid rete mirabile and a small lentiform body; a falciform process and hyaloid arteries are absent. The olfactory rosette is oval with lamellae, a large number for notothenioids. The inconspicuous bony canals of the cephalic lateral line system are simple with membranous secondary branches that lack neuromasts. In Antarctic species, the corpus cerebellum is the most variable brain region, ranging in size from large and caudally projecting to small and round. Stalked brains showing reduction in the size of the telencephalon, tectum, and corpus cerebellum are present in the deep-living artedidraconid Dolloidraco longedorsalis and in most of the deep-living members of the Bathydraconini. Eye diameter is generally larger in Antarctic species but there is a phylogenetic loss of cellularity in the retina, including cone photoreceptors. Some deep-living Antarctic species have lost most of their cones. Mechanosensation is expanded in some species, most notably the nototheniid Pleuragramma antarcticum, the artedidraconid genera Dolloidraco and Pogonophryne, and the deep living members of the bathydraconid tribe Bathydraconini. Reduction in retinal cellularity, expansion of mechanoreception, and stalking are the most noteworthy departures from the morphology seen in Eleginops. These features reflect a modest depth or deep-sea effect, and they are not uniquely Antarctic attributes. Thus, at the level of organ system morphology, perciform brain and sensory systems are suitable for conditions on the Antarctic shelf, with only minor alterations in structure in directions exhibited by other fish groups inhabiting deep water. Notothenioids retain a relative balance among their array of senses that reflects their heritage as inshore perciforms. J. Morphol. 269:84 103, Ó 2007 Wiley-Liss, Inc. KEY WORDS: brain histology; retinal histology; cephalic lateral line system; olfactory system With over 10,000 species, teleost fishes of the order Perciformes constitute 36% of known fish and 18% of known vertebrate species (Nelson, 2006). This phyletic profusion reflects a genotype and a body plan with an unparalleled capacity to generate diversity in both shallow marine habitats and in various freshwater habitats throughout the world. For example, perciforms constitute 9 of 10 families and about 75% of the species characteristic of tropical coral reef faunas (Bellwood and Wainwright, 2002). In addition, perciform cichlid stocks in East Africa have given rise to hundreds of species in some lakes and provide classic examples of adaptive radiations and species flocks (Kornfield and Smith, 2000; Kocher, 2004; Seehausen, 2006). In the temperate streams of eastern North America, darters of the percid subfamily Etheostomatinae constitute a radiation of nearly Contract grant sponsor: National Science Foundation; Contract grant number: ANT ; Contract grant sponsor: National Institutes of Health; Contract grant number: NS ; Contract grant sponsor: Indiana University School of Medicine. *Correspondence to: J.T. Eastman, Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, OH eastman@ohiou.edu Published online 27 September 2007 in Wiley InterScience ( DOI: /jmor Ó 2007 WILEY-LISS, INC.

2 BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 85 Fig. 1. Cladogram of relationships within the notothenioid suborder (Near et al., 2004), pruned to the level of family, showing the Eleginopidae as the sister group of the Antarctic clade. Original cladogram is a strict consensus of four trees resulting from maximum parsimony analysis of the complete gene 16S rrna dataset and the analysis of this data demonstrates monophyly for the suborder (Near et al., 2004). Wedges are proportional to species diversity in each family with an updated list of species, currently numbering 131, posted at ohiou.edu/dbms-eastman. Geographic distributions also indicated, with Gon and Heemstra (1990) taken as authoritative for species with Antarctic distributions. Non-Antarctic species have distributions exclusively outside the Antarctic and Subantarctic Regions. Shading indicates clades with predominantly non-antarctic (blue), predominantly Antarctic (green), and mixed (purple) distributions. Antifreeze glycoproteins (AFGP) characteristic of the Antarctic clade are mapped. 200 species (Jenkins and Burkhead, 1994; Boschung and Mayden, 2004; Nelson, 2006). At the opposite extreme of the habitat spectrum, about 100 perciform species of the suborder Notothenioidei dominate the cold waters of the Southern Ocean surrounding Antarctica (Eastman, 1993). In the absence of competition from most other fish groups, this polar radiation has resulted in notothenioids monopolizing species diversity (76.6%), abundance (91.6%), and biomass (91.2%) on the Antarctic shelf (Eastman, 2005). This level of dominance by a single taxonomic group is unique among piscine shelf faunas of the world. For over a decade, we have been conducting a morphological survey of notothenioid brains and sense organs to examine the effects of phyletic diversification on the structure of these systems. Because of the presence of infrastructure (established field stations and available research vessels), the phyletically derived Antarctic species are more readily available. Therefore, we began our survey with Antarctic nototheniids (Eastman and Lannoo, 1995), then progressed to artedidraconids (Eastman and Lannoo, 2003a), bathydraconids (Eastman and Lannoo, 2003b), and channichthyids (Eastman and Lannoo, 2004; Fig. 1). A recent cruise in the periphery of the Southern Ocean allowed us to collect and study phyletically basal bovichtids (Eastman and Lannoo, 2007) and eleginopids, the sister group of the Antarctic clade. The monotypic family Eleginopidae is represented by Eleginops maclovinus, locally known as mullet or róbalo. Eleginops has a non-antarctic distribution and is common in southern South America and the Falkland Islands; this distribution may reflect an historic pattern on the South American component of the Gondwanan shelf. Unlike the Antarctic clade of notothenioids (see Fig. 1), eleginopinids did not become associated with the margins of the Antarctic plate and their subsequent evolution was little influenced by large-scale tectonic movements or by the cooling of the Southern Ocean (Eastman, 1993). As the sister group of the Antarctic clade (see Fig. 1), Eleginops is of interest in understanding notothenioid diversification because it represents the starting point for the notothenioid radiation. Eleginops maclovinus is a relatively large notothenioid reaching a maximum total length (TL) of 90 cm in the Falkland Islands (Falkland Islands Government, 2003; Brickle et al., 2005a). It has small, mobile subterminal jaws, small eyes, and small, cephalic lateral line pores (Figs. 2 and 7A). Although traditionally considered an opportunistic benthic omnivore in nearshore marine and estuarine habitats (Pequeño, 1989; Licandeo et al., 2006), recent research on a population from the Valdivia River in Chile indicates that both juveniles and adults are opportunistic carnivores capable of feeding in both marine and freshwater environments (Pavés et al., 2005). Amphipods and insects are the most abundant prey taxa in the Valdivia River, with plants and bryozoans being inadvertently consumed during feeding, as they serve as sites of refuge for crustaceans (Pavés et al., 2005). Unlike many other notothenioids, Eleginops maclovinus has a streamlined body with the free margin of the pectoral fin oblique rather than round, thus producing a higher aspect ratio (see Fig. 2). Eleginops has a relatively larger mass of Fig. 2. Live specimen of Eleginops maclovinus (SL 5 40 cm) showing general appearance and body shape

3 86 J.T. EASTMAN AND M.J. LANNOO red pectoral musculature and a greater capacity for sustained labriform swimming than other notothenioids (Fernández et al., 1999). The rapidity of its escape response is similar to eurythermal temperate non-notothenioids rather than to Antarctic notothenioids (Fernández et al., 2002) and its resting rate of oxygen consumption place it in an active category in comparison with sympatric non- Antarctic notothenioids (Vanella and Calvo, 2005). In the Falkland Islands, Eleginops is subject to an annual temperature range of 0 158C in the tidal creeks (Falkland Islands Government, 2003) and 4 118C in shelf waters (Arkhipkin et al., 2004). Other aspects of its life history distinguish Eleginops maclovinus from the stenohaline, sedentary, cold adapted Antarctic notothenioids. For example, it is one of only two euryhaline notothenioid species. Eleginops inhabits coastal waters, sounds, and tidal creeks in the Falkland Islands (Boulenger, 1900; Hart, 1946; Falkland Islands Government, 2003; Brickle et al., 2005a,b) and coastal waters, estuaries, and rivers in southern South America. Its distribution ranges from the Beagle Channel (548S) to approximately the Golfo San Matías, Argentina (408S) on the Atlantic coast (Gosztonyi, 1979) and Valparaiso, Chile (32 338S) on the Pacific coast (Gosztonyi, 1979; Pequeño, 1989; Ojeda et al., 2000). In Chile, Eleginops is dominant in both number and biomass in some estuaries, which also serve as breeding sites for adults and nursery grounds for young (Pequeño, 1981). Unlike the situation in Chile, the population of Eleginops in north central Patagonia has been observed spawning in shelf waters where bottom depths are m, with the young then migrating to inshore waters rather than estuaries to begin their adult lives (Cousseau et al., 2004; Dr. A.E. Gosztonyi, personal communication). In the Falkland Islands, juveniles reside in creeks or sounds and adults forage in larger creeks and sounds but migrate to spawn in shelf waters m deep, occasionally reaching the shelf break at 250 m (Falkland Islands Government, 2003; Brickle et al., 2005a,b). Some populations of Eleginops are therefore catadromous or marginally catadromous, as defined by McDowall (1988, p. 20, 33), since most of the life cycle is spent in fresh or brackish water and the spawning migration of adults takes them to the sea or to the mouths of estuaries or sounds to breed. Populations of Eleginops maclovinus in the Beagle Channel in Argentina (Calvo et al., 1992), in southern Chile (Licandeo et al., 2006) and in the Falkland Islands (Brickle et al., 2005b) exhibit a type of sex reversal known as protandrous hermaphroditism, with males in the Falklands predominating at a TL of cm and females at TL >53 cm. Eleginops has small pelagic eggs and the highest fecundity of any notothenioid (Brickle et al., 2005a). The larvae have never been caught and definitively identified in the Falkland Islands (Dr. Paul Brickle, personal communication). Unlike the Antarctic notothenioids, Eleginops is a rapidly growing species with a maximum age of 11 years (Brickle et al., 2005a). The status of Eleginops maclovinus as the sister group of the Antarctic notothenioids (see Fig. 1) is supported by phylogenetic analyses employing both morphological (Balushkin, 1992, 2000) and molecular data, including partial (Bargelloni et al., 2000) and complete (Near et al., 2004) mtdna gene sequences. Antifreeze glycoproteins (AFGPs) are a key innovation and a physiological necessity that allowed Antarctic notothenioids to survive and diversify in ice-laden seawater (DeVries and Cheng, 2005). The phyletically basal bovichtids and pseudaphritids (see Fig. 1), as well as Eleginops, do not possess AFGP gene sequences in their genomes (Cheng et al., 2003), indicating that they diverged before the tectonic isolation and associated cooling of Antarctica. The split between eleginopids and the Antarctic clade, the five families with AFGPs that inhabit the cold shelf waters of the continent, is variously estimated to have occurred at 5 14 million years ago (mya) (Chen et al., 1997), 27 mya (Bargelloni et al., 2000), or 40 mya (Near, 2004). The 40 mya estimate is based on a fossil calibration of what may or may not be an extinct eleginopid (Eastman, 2005). Near (2004) discusses reasons for the discrepancies among these dates. Irrespective of its divergence time, Eleginops maclovinus has not been subjected to polar environmental conditions. Because of this, and its sister group relationship to the Antarctic clade, it is an ideal subject for studying the morphology of notothenioid nervous and sensory systems that have not had historical exposure to the unusual Antarctic thermal and light regimes experienced by the phyletically derived species living on the high latitude shelf. Since a base of information on neural and sensory morphology is available for the Antarctic notothenioids (Eastman and Lannoo, 1995, 2003a,b, 2004; Lannoo and Eastman 1995, 2000), our focus here for Eleginops is to 1) present a detailed description of the brain and cranial nerves; 2) document the anatomy and histology of the brain, olfactory apparatus, retina, and branched membranous extensions of the cephalic lateral lines; 3) examine ocular vascular structures; and 4) compare eleginopid neural and sensory morphology with that of the phyletically derived Antarctic notothenioids. MATERIALS AND METHODS Specimens and Nomenclature We collected material during the ICEFISH cruise (No ) of the RV Nathaniel B. Palmer in the South Atlantic Ocean. The cruise began in Punta Arenas, Chile on May 17, 2004 and

4 BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 87 ended in Cape Town, South Africa on July 17, During a stop at Stanley ( S; W) in the Falkland Islands, we captured Eleginops maclovinus (Cuvier and Valenciennes, 1830) with a 30-m long beach seine in Fish Creek (water temperature 58C), Port Louis, East Falkland Island on May 29. These specimens were cm TL, cm standard length (SL) and most were males since they had not yet reached the length at which transformation to females typically occurs. In describing brain nuclei, we follow the nomenclature of authors included in Northcutt and Davis (1983) and Davis and Northcutt (1983) with the exception of the lateral line nerves. What have been traditionally termed the anterior and posterior lateral line nerves of fishes are actually complexes consisting of several distinct cranial nerves (Northcutt, 1989; Northcutt and Bemis, 1993). We follow Northcutt (1989) in distinguishing the anterodorsal and anteroventral lateral line nerves as innervating both canal and superficial neuromasts on the head. When describing ocular structures and vasculature, we use the nomenclature of Nicol (1989). Histology We fixed specimens onboard ship by transcardial perfusion of Bouin s fixative. After anesthetization in a solution of 3-aminobenzoic acid ethyl ester (MS-222, Sigma), the heart and bulbus arteriosus were exposed. Notothenioid saline solution (O Grady et al., 1982) was prepared, adjusted with NaCl to a concentration of 330 mosm/l, maintained at ambient seawater temperature, and perfused through the ventral aorta. Saline was followed by Bouin s fixative. During this perfusion, the gills were periodically irrigated with seawater of ambient temperature (58C). Three hours after perfusion, we removed brains and other tissues of interest and postfixed them in Bouin s. After several days of fixation, we transferred tissues to 70% ethanol for storage and transport. We subjected brains to the following protocol: dehydration in alcohol, clearing in butanol, and embedding in paraffin according to standard procedures (Kiernan, 1990). Embedded brains and spinal cords were cut in a transverse plane on a rotary microtome to produce sections lm thick. Sections were mounted on slides, dried, deparaffinized, stained with hematoxylin and eosin, dehydrated, and coverslipped using Cytoseal 60 as the mounting medium. We tried the traditional neurological stain for Nissl substance, 0.1% aqueous cresyl violet acetate, on these brains but slides would not adequately destain, probably because of the effects of fixation in Bouin s solution. We cut histological sections of the eyes of five specimens. In examining the eyes, we took dorsoventral strips from the central retina immediately temporal (lateral) to the optic disk. We also took transverse sections of the ventral retina in the area of the choroid fissure. We employed the histological protocol outlined earlier except that CitriSolv (Fisher Scientific) was substituted for butanol and sections were cut at 7 lm. In measuring retinal thickness, we excluded the optic nerve fiber layer. Sections were stained with hematoxylin and eosin, phloxine B, and methylene blue, Gomori s one step trichrome, or Bodian s Protargol for 24 h at 508C. We counted olfactory lamellae in six specimens of Eleginops maclovinus. We also examined transverse and longitudinal serial histological sections of the olfactory apparatus and of the head, with an emphasis on the membranous canals of the cephalic lateral line system. We used the same histological techniques outlined earlier. Other Morphological Procedures We examined the cephalic lateral line canal system to determine whether or not Eleginops maclovinus has membranous canals branching off of the bony canals. For this, we used specimens that had been cleared and stained with alizarin red S (Taylor, 1967) dissolved in 75% ethyl alcohol (Springer and Johnson, 2000). The head skin was left intact to preserve the membranous canals. We used yellow Microfil 1 (Flow Tech, Carver, MA), a liquid silicon rubber injection compound, to demonstrate ocular blood vessels. We injected fish in the caudal vein with 0.3 ml of heparinized (20 mg/ml) notothenioid saline and then returned them to the holding tank for 15 min. After anaesthetization, we placed the specimens ventral side-up on an iced surgical platform. We cut the bulbus-ventricle junction and cannulated the ventral aorta with a 34-cm length of either PE-50 tubing (0.96 mm OD) or PE-160 tubing (1.57 mm OD). We blunted the free end on a hot plate and secured the cannula with two sutures. The cannula was in turn connected to a 23-G or 18-G needle, an 84-cm extension tube and a 20-ml syringe. The entire apparatus was surrounded by ice to maintain a body temperature of 58C. We placed the syringe in either a Sage model 341B or a KD Scientific model 100 syringe pump and began the perfusion with notothenioid saline followed by Microfil. Flow rate was about ml/min. We allowed the Microfil to polymerize while maintaining the specimen on ice for about 1 h, and then preserved the specimen in 10% formalin with subsequent storage in 70% ethanol. Since Microfil did not fill hyaloid arteries at the vitreoretinal interface in Eleginops maclovinus, another means of assessing the presence or absence of these vessels was necessary. We removed a block of skull containing the eyes from four Microfil specimens and dissected away the cornea, lens, vitreous body, and some of the vitreous membrane. We introduced a reversible stain (a mixture of 1 part of 1% aqueous aniline blue and 10 parts of saturated aqueous picric acid) into the vitreous chamber for 12 min. We then rinsed with water and returned the specimen to 70% ethanol for additional dissection, examination, and storage. If present, the hyaloid arteries stain more darkly than the background and can be seen radiating from the falciform artery at the optic disk. RESULTS Brain and Cranial Nerve Anatomy In gross view, the brain of Eleginops maclovinus exhibits a mixture of reduced and hypertrophied structures (see Fig. 3). The olfactory bulbs are large and positioned rostral to the telencephalon. The telencephalic lobes are about twice the volume of the olfactory bulbs and roughly half the volume of the tectal lobes. Lobules of the telencephalon are pronounced, with dorsodorsal, dorsomedial, and dorsolateral nuclei prominent. The tectal lobes (vision) are small- to medium-sized, as are the inferior lobes (Fig. 3A). The corpus cerebellum (motor skill) is large, dorsally expanded, and caudally directed. The eminentia granularis and crista cerebellares, two structures associated with mechanoreceptive lateral line inputs, are well developed. A decussation of the crista cerebellares is present, bridging the fourth ventricle (Fig. 3B). Cranial nerves exhibit proportions atypical for perciforms but in accordance with Eleginops brain regions. The optic nerve is the largest-diameter cranial nerve and is pleated, but is relatively small for a perciform brain, only slightly larger than the olfactory nerve. The olfactory nerve is relatively large and exhibits a prominent bulge immediately rostral to the olfactory bulb (Fig. 3A). The anterior dorsal and anterior ventral lateral line nerve complexes are more than twice the diameter of the

5 88 J.T. EASTMAN AND M.J. LANNOO Fig. 3. Brain and cranial nerves of Eleginops maclovinus (SL cm) in left lateral (top) and dorsal (bottom) views To illustrate features of the rhombencephalon in dorsal view, the drawing was made from a slightly oblique dorsocaudal angle. Therefore structures such as the corpus of the cerebellum are not perfectly aligned in the two views of the brain. ADLL, anterodorsal lateral line nerve complex; AVLL, anteroventral lateral line nerve complex; CC, crista cerebellaris of the rhombencephalon; CCb, corpus division of the cerebellum; Dl, dorsolateral subdivision of the telencephalon; Dd, dorsodorsal subdivision of the telencephalon; Dm, dorsomedial subdivision of the telencephalon; EG, eminentia granularis division of the cerebellum; IL, inferior lobe of the diencephalon; OB, olfactory bulb; Pit, pituitary gland; PLL, posterior lateral line nerve complex; SN1, first spinal nerve; SN2, second spinal nerve; SV, saccus vasculosus; Tec, tectum of the mesencephalon; Tel, telencephalon; I, olfactory nerve; II, optic nerve; III, oculomotor nerve; IV, trochlear nerve; V, trigeminal nerve; VII, facial nerve; VIII, auditory/vestibular nerve; IX, glossopharyngeal nerve; X, vagus nerve. posterior lateral line nerve complex. The vagus nerve is large. The octaval nerve (hearing and balance), oculomotor, trochlear, and abducens nerves (eye movement), as well as the spinal nerves are proportional. Brain and Spinal Cord Histology Olfactory nerve and telencephalon. In gross view, the olfactory bulbs are sessile, positioned at the rostral base of the telencephalon. Before entering the olfactory bulbs, the olfactory nerves exhibit expansions. Histologically, these expansions consist of fiber bundles interspersed centrally with sparse groups of small cells (Fig. 4A). Cell types characteristic of the olfactory bulbs, including glomerular cells and the medial smaller cell layers (not labeled), appear caudal to the olfactory nerve expansions and rostral to the leading edge of the telencephalon (Fig. 4B). The rostral portion of the telencephalon is positioned over the caudal portion of the olfactory bulbs and consists primarily of the dorsal division, characterized by uniform, small cells. Cell groups present but not particularly prominent include the dorsodorsal, dorsomedial, and dorsolateral nuclei (Fig. 4C). At the level of the anterior commissure, the dorsodorsal, dorsomedial, dorsolateral, and dorsocentral nuclei are prominent, as are nuclei in the ventral division, including the ventrodorsal,

6 BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 89 Fig. 4. Brain histology (transverse sections) of Eleginops maclovinus (SL cm) from olfactory bulbs (A) through rostral spinal cord (V). Stain: Hematoxylin and eosin. Magnifications: A, B, G, 316.4; C, 316.8; D F, R, V, 310.8; H, T, 310.3; I, 39.2; J, L O, 37.7; K, P, Q 38.2; S, 313.7; U, AC, anterior commissure; CC, crista cerebellaris; CCb, corpus division of the cerebellum; CM, mammillary bodies; CP, central posterior nucleus of the thalamus; Dc, dorsocentral nucleus of the telencephalon; dcc, decussation of the crista cerebellaris; Dd, dorsodorsal nucleus of the telencephalon; Dl, dorsolateral nucleus of the telencephalon; Dm, dorsomedial nucleus of the telencephalon; Dp, dorsoposterior nucleus of the telencephalon; DP, dorsal posterior nucleus of the thalamus; E, endopeduncular nucleus; EG, eminentia granularis; G, nucleus glomerulosus; Ha, habenula; Hd, dorsal nucleus of the diencephalon; Hv, ventral nucleus of the diencephalon; IL, inferior lobe of diencephalon; LR, lateral recess of the inferior lobe; LT, lateral tuberal nucleus; MLF, medial longitudinal fasciculus; ND, nucleus diffusus of the inferior lobe; OB, olfactory bulb; PC, posterior commissure; PG, nucleus preglomerulosus; Pp, preoptic nucleus; RF, reticular formation; Sps, spinal sensory nucleus; SV, saccus vasculosus; Tec, tectum of the mesencephalon; TL, torus longitudinalis of the mesencephalon; TP, posterior tuberal nucleus; TS, torus semicircularis of the mesencephalon; VCb, valvula cerebelli; VM, dorsal medial nucleus of the thalamus; Xs, visceral sensory nucleus of the vagal nerve; CN I, olfactory nerve; II, optic nerve; III/IV, oculomotor/trochlear nerve complex; Vm, motor nucleus of the trigeminal nerve.

7 90 J.T. EASTMAN AND M.J. LANNOO Figure 4. (Continued.) and ventroventral nuclei (not shown; Fig. 4D,E). The anterior commissure is thick, and the white matter associated with this fiber bundle is extensive (Fig. 4D,E). Caudally, the dorsoposterior nuclei are medium sized and protrude ventrally, contributing to a top-heavy appearance (Fig. 4F). Cells in the dorsal region are notably differentiated by size and arrangement, and the dorsal portion of the dorsolateral nucleus forms a discrete lobe (Fig. 4F). In the caudal-most telencephalon, the ventral portion of the dorsolateral nucleus overlies the dorsoposterior nucleus (Fig. 4G). Diencephalon. The rostral preoptic area is small and consists of a parvocellular preoptic nucleus (Fig. 4E,F). Farther caudally, the endopeduncular nucleus forms a tight cluster of cells within the preoptic region (Fig. 4G,H). At the level of the rostral pituitary, cells of the magnocellular pre-

8 BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 91 Figure 4. (Continued.) optic nucleus are abundant in the dorsal preoptic area and the endopeduncular nucleus is large (Fig. 4G). In the central diencephalic region, the preoptic region contains both parvocellular and magnocellular cells (Fig. 4G,H). The habenula is medium-sized and thalamic nuclei, including the ventromedial nucleus, are present and exhibit a normal appearance. Cells comprising the lateral tuberal nucleus are large and form a prominent cluster (Fig. 4H). The pituitary is also large and appears well organized (not shown). At the level of the posterior commissure, narrow subependymal expansions are present (Fig. 4I,J), thalamic nuclei, including the dorsoposterior and centroposterior nuclei, and hypothalamic nuclei, including the dorsal and ventral nuclei, appear in normal positions

9 92 J.T. EASTMAN AND M.J. LANNOO and proportions. Nucleus preglomerulosus is normally proportioned. The inferior lobes are small to medium sized. Farther caudally, the nucleus glomerulosus is large and circular shaped, the posterior tuberal nucleus crosses the midline (Fig. 4K,L). Immediately ventral to the posterior tuberal nuclei the mammillary bodies meet along the third ventricle. At this level the inferior lobes are medium sized, cells of the nucleus diffusus are small and sparse, and the lateral recess is positioned within the lobe. The saccus vasculosus is small. Farther caudally, at the level of the oculomotor nerve (Fig. 4L,M), cells within the nucleus diffusus are well organized. Near the caudal-most inferior lobes, nucleus diffusus cells are less well organized and the lateral recesses become subpial (Fig. 4N). Mesencephalon. The tectum is small and unusually proportioned. Rostrally (Fig. 4I) the superficial white matter, consisting of the superficial white and gray zone, the central zone, and the deep white zone, is proportional along the dorsal and lateral tectal surfaces, but thickens to form a deep neuropil ventrally. At about the mid-tectal level, the tectum remains small and thin. The thick appearance of the white matter is no longer apparent, but the ventrolateral portion of the tectum makes an acute angle (Fig. 4J). The rostral portions of the torus longitudinales are present. The rostral torus semicircularis is flattened along its dorsal surface it protrudes very little into the tectal ventricle (Fig. 4K). Caudally, the torus semicirculares form narrow lobes that project deeply into the tectal ventricle (Fig. 4L N). Motor neurons of the oculomotor complex are large but sparse at about the midtectal level (Fig. 4L). At the level of oculomotor nerve exit (Fig. 4M) oculomotor nuclei are numerous. Farther caudally (Fig. 4N) they are again less numerous but clustered, and positioned immediately dorsal to the medial longitudinal fasciculus. Cerebellum. At its rostral-most level the valvula cerebellum is single lobed, consisting of a central molecular layer and lateral granule cell regions (Fig. 4L). Just caudally, at the level of oculomotor nerve exit (Fig. 4M), the valvula consists of two large, stacked lobes, which caudally become broader, with lobules separated dorsoventrally a morphology that does not appear to be artifact and that we have not seen before (Fig. 4N). The rostral corpus cerebellum is tall, comprising more than 50% of the dorsal extent of the brain at the level of the rostral rhombencephalon (Fig. 4O,P). Farther caudally, the corpus becomes broad (Fig. 4P,Q); the eminentia granulares appear as large lateral granule cell masses (Fig. 4Q). At the point where it becomes a lobe, the corpus and the vestibulolateral lobe are large and well differentiated; the crista cerebellaris is large (Fig. 4R). As it forms a lobe, the corpus is large, with about the same cross-sectional area as the brainstem proper (Fig. 4S). Rhombencephalon. The brainstem of Eleginops is typically proportioned. Rostrally, neurons of the trigeminal motor nuclei are prominent (Fig. 4O Q). The rostral portions of the crista cerebellares are large (Fig. 4R) and at the level of the mid-fourth ventricle form a decussation (Fig. 4S). At the level of the caudal rhombencephalon, the fourth ventricle is again open dorsally, the crista cerebellares become small, and the sensory nuclei of the vagal nerve are present (Fig. 4T). At this level, subependymal expansions (arrows) are prominent and contain cell bodies and axons. At the level of the brainstem junction with the spinal cord, the spinal sensory nuclei are moderately sized (Fig. 4U). Subependymal expansions are prominent and form a septum below the central canal. The rostral spinal cord is narrower dorsally than it is ventrally; motor neurons are large (Fig. 4V). Sensory Systems Visual system Eye size. At 11% 13% of head length (HL) or 3% of SL, the eye diameter of Eleginops maclovinus (Figs. 2 and 7A) is about twofold smaller than in many other notothenioids. For comparison, eye diameter of the artedidraconid Dolloidraco longedorsalis is 31 37% of HL, bathydraconids are 16 31%, and channichthyids are 19 22% (Eastman and Lannoo, 2003a,b, 2004). Ocular vasculature. The generalized teleostean eye (Walls, 1942) as well as the phyletically basal notothenioid eye, exemplified by Bovichtus diacanthus (Eastman, 2006; Eastman and Lannoo, 2007), possess three ocular vascular structures supplying the retina and ocular adnexa: the choroid rete mirabile, the lentiform body (also a rete), and the falciform process. In Eleginops maclovinus the embryonic choroid fissure is closed and therefore the falciform process is not present (Fig. 5A). There is a well-developed choroid rete (Fig. 5B) but this was not filled by our ventral aortic perfusions of Microfil because the perfusate is unable to pass distal to the capillary bed of the pseudobranch. The ophthalmic artery, a vessel originating in the pseudobranch, supplies the choroid rete. The eye of Eleginops also receives blood from a second vessel, the optic artery (60 lm diameter). This vessel, a branch of the carotid complex, is the afferent supply to the lentiform body and was filled by Microfil (Fig. 5C). The lentiform body, located ventral to the optic nerve (Fig. 5B,C), is a small complex (1.2 mm by 0.5 mm) of two to three orders of short, small diameter (15 lm) arterial capillaries. Since the venous capillaries are extrinsic and part of the general choriocapillaris network, they were not filled by Microfil. The falciform artery (also

10 BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 93 Fig. 5. Ocular vascular structures and retinal histology of Eleginops maclovinus. A: After removal of cornea and lens, vitreous chamber of left eye shows falciform artery (containing yellow Microfil) at the optic disk continuing as the main vessel to the retractor lentis muscle. The main vessel is superficial to the site of the fused choroid fissure. Hyaloid arteries are absent at the vitreoretinal interface. B: Removal of sclera and argentea from back of left eye displays choroid rete mirabile (unfilled) and lentiform body (filled with Microfil). C: Enlargement of B showing detail of arterial vasculature of lentiform body. Optic artery is the afferent vessel and falciform artery the efferent vessel. D, E: Transverse histological sections showing predominance of cone photoreceptors and layering of central retina. Stains: D, Gomori; E, Bodian. Magnifications: A, 36.7; B, 34.5; C, 315.6; D, E, cc, choriocapillaris; cr, choroid rete; fa, falciform artery; lb, lentiform body; mv, main vessel; oa, optic artery; ocn, oculomotor nerve; od, optic disk; on, optic nerve; r, retina; rl, retractor lentis muscle; 1, retinal pigment epithelium; 2, outer segments of photoreceptors; 3, inner segments of photoreceptors; 4, external limiting membrane; 5, external nuclear layer; 6, internal nuclear layer; 7, ganglion cell layer; 8, optic nerve fibers. 60 lm diameter) is the efferent vessel of the lentiform body (Fig. 5C); it enters the optic nerve and continues into the vitreous chamber of the eye as the main vessel (31 38 lm diameter) supplying blood to the retractor lentis muscle (Fig. 5A). This vessel is occasionally double. Microfil preparations

11 94 J.T. EASTMAN AND M.J. LANNOO TABLE 1. Cell counts a in the central area of the retina comparing Eleginops maclovinus with members of other notothenioid families both non-antarctic and Antarctic Species Habitat depth b (m) Retinal thickness (lm) Cones Rods Cones 1 rods Ratio cones:rods c Cells in internal nuclear layer Ganglion cells Convergence ratio (cones 1 rods:ganglion cells) d Bovichtidae (non-antarctic) Bovichtus diacanthus : :1 B. variegatus : :1 Cottoperca gobio : :1 Eleginopidae (non-antarctic) Eleginops maclovinus : :1 Nototheniidae (non-antarctic) Notothenia angustata : :1 N. microlepidota < : :1 Nototheniidae (Antarctic) Pagothenia borchgrevinki : :1 Trematomus newnesi : :1 T. bernacchii : :1 T. pennellii : :1 T. eulepidotus : :1 Bathydraconidae (Antarctic) Parachaenichthys charcoti : :1 Gymnodraco acuticeps : :1 Channichthyidae (Antarctic) Chionodraco hamatus : :1 Pagetopsis macropterus : :1 Champsocephalus gunnari : :1 a Counts are mean number of nuclei for three replicates in an area of 100 lm along the various layers of one Bodian-stained histological section, viewed at 31,000. Counts for B. variegatus and for non-bovichtids from Eastman (1988), Eastman and Lannoo (2003b, 2004, 2007), and Lannoo and Eastman (2000). b Gon and Heemstra (1990), with some maximum depth records from Eastman and Hubold (1999). c Values for cone:rod ratios in notothenioids (modified from Eastman, 1988): high (1:2 4), moderate (1:5 11), and low (1:14 57). d Values for convergence ratios in notothenioids (Eastman, 1988): high (58:1), moderate (30 12:1), and low (10 5:1). Arrangement is phylogenetic and interfamilial comparisons are among species with the shallowest depth ranges. Data were from adult specimens. of Eleginops did not display any hyaloid branches of the falciform artery at the vitreoretinal interface nor were these vessels encountered in gross staining of the vitreous chamber or in histological sections of the retina. Given the scarcity or absence of hyaloid arteries, the dense capillary network of the choriocapillaris on the sclerad surface is the primary blood supply to the retina of Eleginops (Fig. 5D,E). Retinal cellularity. The retina of Eleginops maclovinus is thicker and more cellular than those of the Antarctic notothenioids but less cellular than the phyletically basal bovichtids (Table 1). The magnitude of the phylogenetic loss in cellularity in notothenioids is expressed by totaling the cells in the external (cones 1 rods) and internal nuclear layers and then taking a mean for the species in the families. Antarctic species have 3.1-fold fewer cells than bovichtids and 2.3-fold fewer cells than Eleginops. This reduction in cellularity is attributable primarily to the loss of photoreceptors. Retinal histology. The layering in Eleginops maclovinus is typical for fishes and the retina is duplex, containing both cone and rod photoreceptors (Fig. 5D,E). Single cones, with occasional twin cones interspersed, dominate the central retina and are recognizable by their acidophilic ellipsoids (Figs. 5D and 6A). These are 4 5 lm and 9 10 lm in diameter, respectively. Cones are more numerous in the central than in the peripheral retina. The thickness of the external nuclear layer indicates rods, recognizable by thin basophilic myoids within their inner segments, are also numerous, although in the central retina they are confined to narrow gaps between cones (Fig. 6A). Silver staining of nuclei provides an appreciation of cell density and resolves the horizontal, bipolar, and amacrine cell populations of the internal nuclear layer (Fig. 5E). Olfactory apparatus Nasal sacs and olfactory rosette. The nasal sac has a single relatively large opening at the end of an extremely short tube (Fig. 7A) and, as is common in perciforms (Hansen and Zielinski, 2005), dorsal and ventral accessory nasal sacs communicate with the nasal sac (Fig. 6B). As in all other notothenioids except the bovichtid Cottoperca gobio (Eastman and Lannoo, 2007), Eleginops maclovinus has an oval rosette with olfactory lamellae arranged perpendicular to a central raphe (Fig. 6B), the Type G arrangement in the classification of Yamamoto (1982). Eleginops possesses a modal number of lamellae, a large number for a notothenioid. The lamellae are lm thick

12 BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 95 Fig. 6. Retinal histology and olfactory anatomy and histology of Eleginops maclovinus. A: Enlargement of Figure 5D showing detail of photoreceptors. Ellipsoids of cone inner segments stain red and outer segments light orange with Gomori s trichrome. Thin myoids of inner segments of rods are basophilic and located between cones. B: Dorsolateral view of left nasal sac, rosette, and olfactory lamellae of a 39 cm SL specimen. Arrows indicate communications between nasal sac and dorsal and ventral accessory nasal sacs. Anterior is to the left. C, D: Transverse section of olfactory lamellus showing nature of epithelium and large fasciculi of the olfactory nerve in the connective tissue core of the lamellus. E, F: Olfactory epithelium on one side of lamellus showing cell types, especially darkly stained nuclei of primary olfactory neurons in apical epithelium. Dendrites of primary olfactory neurons converge on small fasciculi of the olfactory nerve in the basal epidermis. Stains: A,C,E, Gomori; D,F, Bodian. Magnifications: A, 31,100; B, 36.6; C, D, 3140; E, F, b, bone of cephalic lateral line canal; bc, basal cells; cr, central raphe of olfactory rosette; ct, connective tissue; d, dermis of head skin; dans, dorsal accessory nasal sac; e, epidermis of head skin; la, lamellae of olfactory rosette; muc, mucous cells; n, nerves; ns, nasal sac; p, melanin pigment; pon, primary olfactory neurons; ro, rod (myoid); sc, supporting cells; sco, single cone (ellipsoid); sk, head skin; tco, twin cone (ellipsoid); v, blood vessels; vans, ventral accessory nasal sac; 2, outer segments of rod photoreceptors; 3, inner segments of rod photoreceptors; 4, external limiting membrane; 5, external nuclear layer. and increase in number ontogenetically at the anterior margin of the rosette (left portion of Fig. 6B). For example, a 15 cm SL specimen has lamellae whereas a 43 cm SL specimen has Olfactory epithelium. Both sides of the lamellae are faced with a pseudostratified columnar epithelium (Fig. 6C,D) that is lm thick but thins to lm in the non-olfactory area of the tips. The core of the lamellae contains abundant fascicles of olfactory nerve fibers and pigment cells (Fig. 6C,D). This dark pigment is also grossly evident in a surface view of the lamellae (Fig. 6B). The olfactory epithelium lacks secondary folds and consists of basal cells, supporting cells and, although the entire cell is not evident, primary olfactory neurons (Fig. 6E,F). The nuclei of these

13 96 J.T. EASTMAN AND M.J. LANNOO Fig. 7. Cephalic lateral line anatomy and histology of Eleginops maclovinus. A: Head showing small eyes and inconspicuousness of bony lateral line canals, membranous tubules and pores. B: Lateral view of four of left infraorbital bones of alizarin-stained and cleared specimen (SL mm) showing the arrangement, size and degree of ossification of the bony canals. Since some of the head skin is intact, origin of two membranous canals is evident (arrows). Membranous canals also originate from the three bony canals on io1. Pores are not visible and situated peripherally at the ends of membranous canals. C: Transverse section of a bony infraorbital canal includes longitudinal section of a membranous canal. The section through the bony canal is at the periphery of a neuromast. Bone and scales stain red. D: Enlargement of longitudinal section of membranous canal from C showing location in the dermis superficial to scales and the absence of bone and neuromasts. Stains: C,D, Gomori. Magnifications: A, 30.9; B, 33.2; C, 323; D, 370. b, bone of cephalic lateral line canal; bc, lumen of bony canal; ct, subcutaneous connective tissue; d, dermis of head skin; e, epidermis of head skin; io, infraorbital bones; mc, membranous canal (canaliculus); n, nerve (to neuromast); nm, neuromast; s, scale; sk, head skin. neurons; however, are recognizable because they are more apically located than are the nuclei of the supporting cells (Hansen and Zielinski, 2005). Furthermore, nuclei are larger and darker with a denser pattern of chromatin (Fig. 6F), and their axons are bundled together in the basal epithelium (Fig. 6E). Mucous cells are located in the apical epithelium (Fig. 6C,E). Cephalic lateral line system. Since our specimens of Eleginops maclovinus were subject to ab-

14 BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 97 rasion during seining and the long transport back to aquaria on the ship, we cannot comment definitively on the number and distribution of superficial neuromasts. A few are present on the dorsal surface of the head in the interorbital and narial areas, but they are probably not numerous. The bony canals of the cephalic lateral line have the typical actinopterygian pattern (Northcutt, 1989) and specific descriptions are available for Eleginops (Gosztonyi, 1979; Andersen, 1984). Based on development and size, the bony canals of Eleginops are most similar to the simple 5 narrow type in the classification of Webb (1989). They are inconspicuous on the surface of the head (Fig. 7A) and, as exemplified by the infraorbital series, canal segments are ossified with the exception of superficial areas near their ends (Fig. 7B). In a 260 mm SL specimen, they measure mm diameter at the center and mm at the flared ends. Some notothenioids, including Eleginops, possess additional or secondary branches of the bony canals in the form of membranous canals in the dermis of the skin. These have been termed canaliculi in notothenioid systematic work (Jakubowski, 1971; Andersen, 1984; DeWitt et al., 1990). They are straight and unbranched in Eleginops of the size range that we examined, although it is possible they become more complexly branched during ontogeny as has been documented in a number of groups including clupeids and bovichtids (Stephens, 1985; Eastman and Lannoo, 2007). Along the infraorbital canal, some of membranous canals arise between and perpendicular to the bony segments while others are continuous with the bony canals (Fig. 7B). Membranous canals have a lumen diameter of 0.4 mm at their origins (Fig. 7B). The pores are situated at the ends of membranous canals rather than between bony segments of the main canal. Pores are small and inconspicuous and some of those on the snout are slit-like. Mandibular pores are lm in diameter. Histological transverse sections of the region ventral to the eye show the deeply situated bony infraorbital canal with a portion of a neuromast as well as a longitudinal section through a membranous canal (Fig. 7C). The latter is contained in a meshwork of loose dermal collagen fibers superficial to the scales and the denser basal dermis (Fig. 7C,D). Membranous canals are lined by a cuboidal epithelium containing numerous mucous cells (Fig. 7D) and neuromasts are not present. DISCUSSION Few Studies Have Addressed Brain Variability in Perciforms Perciforms are visual specialists with a welldeveloped tectum (Demski, 2003) and, as exemplified by coral reef fishes, their behavior and sensory world are dominated by vision (McFarland, 1991). Most habitats occupied by perciforms are relatively shallow and well illuminated. Despite their speciosity and prominence in many aquatic ecosystems, surprisingly few surveys have focused on variability in brain morphology among perciforms. In one study, drawings of only three representative species sufficiently encapsulated the variation in brain morphology encountered in a survey of 17 species of pomacanthids (angelfishes) and 35 species of chaetodontids (butterflyfishes), typical coral reef fishes (Bauchot et al., 1989). As with many perciforms, chaetodontids exhibit a telencephalon with prominent lobation, and with tectal (visual), corpus cerebellum (motor) and octavolateralis (auditory and mechanoreceptive) areas well developed; in contrast the olfactory bulbs are less robust. In two other studies, among 189 cichlid species from the three major African Great Lakes, interspecific variation in brain regions is similar among species from each of the lakes, with the greatest variation in association areas such as the telencephalon, followed by the regions responsible for mechanoreception, olfaction, and vision (van Staaden et al., 1994/95; Huber et al., 1997). Brain and Sense Organ Morphology in Non-Antarctic Eleginops maclovinus Compared with Antarctic Notothenioids Except for the shape of the corpus cerebellum, a highly variable feature among teleosts (Meek and Nieuwenhuys, 1998), and enhanced development of olfactory areas, the brain of Eleginops maclovinus is not substantially different from the brains of the temperate and tropical perciforms mentioned earlier. Nor is the brain of Eleginops fundamentally different than that of the moronid Dicentrarchus labrax (Mediterranean sea bass), another marine perciform (Cerdá-Reverter et al., 2001a,b). Because Eleginops is the sister group of the Antarctic notothenioids, the neural starting point for the notothenioid radiation appears to have been a normal (typical) perciform brain. Our work to date has focused on the brains and sense organs of the phyletically basal non-antarctic bovichtids (Eastman and Lannoo, 2007) and of four of the five phyletically derived Antarctic families Artedidraconidae, Harpagiferidae, Bathydraconidae, and Channichthyidae (Eastman and Lannoo, 2003a,b, 2004). Eleginops maclovinus provides the final piece of the puzzle in interpreting the neural and sensory morphology of notothenioids. Do the brains and sense organs of Antarctic notothenioids differ from those of their non-antarctic sister group? With Eleginops as the reference for the ancestral state, we will note departures in the anatomy and histology of the major brain divisions and associated cranial nerves and sensory systems in members of the Antarctic clade.