Integr. Comp. Biol., 44:333–348 (2004) - [PDF Document] (2024)

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INTEGR. COMP. BIOL., 44:333348 (2004)

Evolution and Phylogeny of Gonad Morphology in Bony Fishes1

LYNNE R. PARENTI2,* AND HARRY J. GRIER*

*Division of Fishes, Department of Zoology, National Museum ofNatural History, MRC 159, Smithsonian Institution,P.O. Box 37012,Washington, D.C. 20013-7012

Fish and Wildlife Research Institute, 100 8th Avenue, SE, St.Petersburg, Florida 33701-5095

SYNOPSIS. Gonad morphology at the gross anatomical orhistological levels has long been studied byfisheries biologists toidentify annual reproductive cycles and length of breeding season,among other goals.Comparative surveys across vertebrate taxa havenot been detailed enough, however, to describe fully thedifferencesand similarities among gonads of bony fishes and other vertebrates,and to use gonad morphologyin phylogenetic systematic analyses. Anemerging constant among vertebrates is the presence of agerminalepithelium composed of somatic and germ cells in both malesand females. In females, the germinal epithe-lium lines the ovarianlamellae. In males, arrangement of the germinal epithelium intocompartments variesamong osteichthyans: basal taxa have ananastomosing tubular testis, whereas derived taxa have alobulartestis. The lobular testis is proposed as a synapomorphy ofthe Neoteleostei. The annual reproductive cycleis hypothesized tobe the source of morphological variation among testis types.Elongation of germinalcompartments during early maturation mayresult in a transition from anastomosing tubular to lobulartestes.In all male atherinomorphs surveyed, spermatogonia are restrictedto the distal termini of lobulesrather than being distributed alongthe lobule; there is an epithelioid arrangement of Sertoli and germcellsrather than a germinal epithelium. Arrest of thematuration-regression phases is hypothesized to lead to

formation of the atherinomorph testis. Atherinomorphs also havea distinctive egg with fluid, rather thangranular, yolk. Variationamong germinal epithelia is interpreted in a developingphylogenetic frameworkto understand evolution of gonad morphologyand to propose gonad characters for phylogenetic analyses.

INTRODUCTION

Gonad morphology at the gross anatomical or his-tological levelshas long been studied by fisheries bi-ologists to identify annualreproductive cycles, lengthof breeding season, onset ofreproductive maturity,spawning rhythms, fecundity and various otheraspectsof reproductive biology that can be applied tofisheriesquestions and concerns. Of necessity, these studieshavefocused on a restricted set of commercial or rec-reational fishingspecies, such as common snook, Cen-tropomus undecimalis (e.g.,Grier and Taylor, 1998;Taylor et al., 1998; Neidig et al., 2000),redfish or reddrum, Sciaenops ocellatus (e.g., Murphy andTaylor,1990), bonefish, Albula vulpes (e.g., Crabtree et al.,1997),and trout, Oncorhynchus species (Billard,1987), among others. Asecond field of investigationis the reproduction of marine fishes,many of whichhave long been known to switch sex and areher-maphroditic (e.g., Warner and Robertson, 1978; Has-tings, 1981;Cole, 1988, 1990). A third, well-studiedarea of fish reproductionis the modes of viviparity inthe atherinomorph ordersCyprinodontiformes (viz.,

Parenti, 1981), including the poeciliids (e.g., Rosenand Bailey,1963; Hoar, 1969), the four-eyed fishes,genus Anableps (e.g.,Turner, 1950), and the Mexicangoodeids (e.g., Miller andFitzsimons, 1971), and Be-loniformes, including the viviparoushalfbeaks (e.g.,Downing and Burns, 1995; Meisner and Burns,1997;

1 From the Symposium Patterns and Processes in the EvolutionofFishes presented at the Annual Meeting of the Society forInte-grative and Comparative Biology, 48 January 2003, atToronto,Canada.

2 E-mail: [emailprotected]

Meisner, 2001). Despite a few efforts aimed at usingreproductivecharacters in comprehensive classifica-tions of bony fishes (e.g.,Breder and Rosen, 1966),these areas of research remain relativelyindependent.Comparative surveys across vertebrate taxa have notbeenbroad or detailed enough to describe fully thedifferences andsimilarities among gonads of bonyfishes and other vertebrates, andto use gonad mor-phology routinely in phylogenetic systematicanalyses.

These are our aims.An emerging constant among vertebrates is thepres-

ence of a germinal epithelium (Grier, 2000, 2002;Grier and LoNostro, 2000). Almost all osteichthyanshave a germinal epitheliumcomposed of somatic andgerm cells in male and female gonads. Weclassify theatherinomorph testis as epithelioid based onrefinementof definitions of an epithelium as applied togonadmorphology. In teleosts, the germinal epithelium is ac-tivethroughout the life of the organism and is corre-lated withindeterminate reproduction of females.Among the Perciformes, fivereproductive classes havebeen described in males of common snook,Centro- pomus undecimalis (see Taylor et al., 1998), spottedseatrout, Cynoscion nebulosus (see Brown-Peterson,2003), cobia,Rachycentron canadum (see Brown-Pe-terson et al., 2002), and thefreshwater goby, Pado-gobius bonelli (as Padogobius martensi, seeCinquettiand Dramis, 2003):regressed, early maturation,midmaturation, late maturation, and regressionbased onthealternation of the germinal epithelium betweencontinuous anddiscontinuous types and the stages ofgerm cells present (Grier andTaylor, 1998; Taylor etal., 1998; Grier, 2002). These changes inthe germinalepithelium have also been used to describe annual

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TABLE 1. Survey of testis types of osteichthyans.*

Species Testis Type

Reference and/or

Material

CLASS SARCOPTERYGII

Order Coelacanthiformes

Latimeria chalumnae anastomosing tubular Millot et al., 1978

CLASS ACTINOPTERYGIISUBCLASS CHONDROSTEIOrderAcipenseriformesPolyodontidae

Polyodon spathula anastomosing tubular USNM/FMRI

SUBCLASS NEOPTERYGII

Order LepisosteiformesLepisosteidae

Lepisosteus platyrhinchus anastomosing tubular Grier, 1993

DIVISION TELEOSTEIOrder ElopiformesElopidae

Megalops atlanticus anastomosing tubular USNM/FMRI

CLUPEOCEPHALA

Order Clupeiformes

Clupeidae Dorosoma petenenseOpisthonema oglinum

anastomosing tubularanastomosing tubular

Grier, 1993Grier, 1993

OstariophyiOrder CypriniformesCyprinidae

Abbottina rivularis

Barbus kahajaniiDanio rerio Notemigonus crysoleucas

Notropis hypselopterus

anastomosing tubularanastomosing tubularanastomosingtubularanastomosing tubularanastomosing tubular

USNM 336887Grier et al., 1980USNM/FMRI; Maack and Segner,2003Grier et al., 1980Grier et al., 1980

Order CharaciformesCharacidae

Gymnocorymbus ternetzi anastomosing tubular Grier et al.,1980

Order Siluriformes

PimelodidaeConorhynchus conirostris anastomosing tubular Lopeset al., 2004

Ictaluridae

Ictalurus natalis anastomosing tubular Grier, 1993

SUBDIVISION EUTELEOSTEI

ProtacanthopterygiiOrder SalmoniformesSalmonidae

Oncorhynchus mykissOncorhynchus kisutch

anastomosing tubularanastomosing tubular

USNM/FMRIGrier et al., 1980

NEOGNATHI

Esociformes

Esox lucius

Esox niger

anastomosing tubularanastomosing tubular

Grier et al., 1980; Grier, 1993Grier et al., 1980; Grier,1993

NEOTELEOSTEIParacanthopterygiiOrderPercopsiformesAmblyopsidae

Amblyopsis spelaea unrestricted lobular USNM 127055

Percopsidae

Percopsis omiscomaycus unrestricted lobular USNM 308217

Order OphidiiformesBythitidae

Dinematichthys sp. unrestricted lobular USNM 338466

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TABLE 1. (Continued)

Species Testis Type

Reference and/or

Material

Order LophiiformesLophiidae

Lophiodes mutilus unrestricted lobular USNM 322221

Order PolymixiiformesPolymixiidae

Polymixia lowei unrestricted lobular USNM 157839

SERIES ATHERINOMORPHAOrder AtheriniformesAtherinidae

Labidesthes sicculus Leuresthes sardina

Menidia beryllina

restricted lobularrestricted lobularrestricted lobular

Grier et al., 1980, 1990; USNM 108573USNM 177811Grier et al.,1980

Melanotaeniidae

Melanotaenia nigrans restricted lobular Grier et al., 1980

Phallostethidae

Gulaphallus bikolanus

Gulaphallus mirabilis Neostethus bicornis

Neostethus borneensis Neostethus lankesteri

restricted lobularrestricted lobularrestricted lobular

restricted lobularrestricted lobular

Grier and Parenti, 1994Grier et al., 1980; Grier and Parenti,1994Grier and Parenti, 1994

Grier and Parenti, 1994Grier and Parenti, 1994

Phenacostethus smithiPhenacostethus posthon

restricted lobularrestricted lobular

Munro and Mok, 1990; Grier and Parenti, 1994Grier and Parenti,1994

Order CyprinodontiformesAplocheilidae

Aphyosemion gardneri restricted lobular USNM 339706

Rivulidae

Pterolebias hoignei restricted lobular USNM 245947

Profundulidae

Profundulus guatemalensis restricted lobular USNM 134600

Fundulidae

Adinia xenicaFundulus chrysotus

Fundulus grandisFundulus seminolis Lucania goodei

restricted lobularrestricted lobular

restricted lobularrestricted lobularrestricted lobular

Grier et al., 1980Grier et al., 1980

Grier et al., 1980; USNM/FMRIGrier et al., 1980Grier et al.,1980

Goodeidae

Ameca splendens

Ataeniobius toweriCharacodon lateralis

Xenotoca eiseni

restricted lobularrestricted lobularrestricted lobularrestrictedlobular

Grier et al., 1980Grier et al., 1980Grier et al., 1980Grier etal., 1980; USNM 374494

Anablepidae

Anableps anablepsAnableps dowi

Jenynsia lineata Jenynsia multidentata

restricted lobularrestricted lobularrestricted lobularrestrictedlobular

Grier et al., 1980Grier et al., 1980Grier et al., 1980Martnezand Monasterio de Gonzo, 2002

Poeciliidae

Cnesterodon decemmaculatus

Gambusia affinis Heterandria formosaPoecilia latipinnaPoeciliareticulata

restricted lobular

restricted lobularrestricted lobularrestricted lobularrestrictedlobular

USNM 360480

Grier et al., 1980Grier et al., 1980Grier et al., 1980Grier etal., 1980

Poeciliopsis gracilisTomeurus gracilis

Xiphophorus helleri Xiphophorus maculatus

restricted lobularrestricted lobularrestricted lobularrestrictedlobular

Grier et al., 1980Grier et al., 1980; USNM 225463Grier et al.,1980Grier et al., 1980

Cyprinodontidae

Cyprinodon variegatus

Jordanella floridae

restricted lobularrestricted lobular

Grier et al., 1980Grier et al., 1980

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336 L. R. PARENTI AND H. J. GRIER

TABLE 1. (Continued)

Species Testis Type

Reference and/or

Material

Order BeloniformesAdrianichthyidae

Horaichthys setnai

Oryzias latipes

Oryzias matanensis

restricted lobularrestricted lobular

restricted lobular

Grier, 1984Grier, 1976

USNM 340428

Exocoetidae

Cypselurus heterurus Hirundichthys speculigerOxyporhamphusmicropterus

Prognichthys gibbifrons

restricted lobularrestricted lobularrestricted lobularrestrictedlobular

USNM 294785USNM 299274USNM 216327USNM 185882, 185883

Hemiramphidae

Dermogenys bispinna Dermogenys burmanica Dermogenysorientalis

Dermogenys pusilla Dermogenys siamensis

restricted lobularrestricted lobularrestricted lobularrestrictedlobularrestricted lobular

Downing and Burns, 1995Downing and Burns, 1995; Meisner,2001Downing and Burns, 1995Grieret al., 1980; Downing and Burns,1995Downing and Burns 1995; Meisner, 2001

Euleptorhamphus velox

Hemiramphus brasiliensis Hemirhamphodon chryopunctatusHemirhamphodon kapuasensis

Hemirhamphodon kuekenthali

restricted lobularrestricted lobularrestricted lobular

restricted lobularrestricted lobular

Grier et al., 1980Grier et al., 1980; USNM/FMRIDowning andBurns, 1995

Downing and Burns, 1995Downing and Burns, 1995 Hemirhamphodonphaisoma Hemirhamphodon pogonognathus

Hemirhamphodon tengah Hyporhamphus quoyi Hyporhamphusregularis

restricted lobularrestricted lobularrestricted lobularrestrictedlobularrestricted lobular

Downing and Burns, 1995Downing and Burns, 1995Downing and Burns,1995Grier et al., 1980Grier et al., 1980

Nomorhamphus brembachi

Nomorhamphus celebensis Nomorhamphus ebrardtii Nomorhamphusliemi

Nomorhamphus rossi

restricted lobularrestricted lobularrestricted lobularrestrictedlobularrestricted lobular

Downing and Burns, 1995; Meisner, 2001Downing and Burns,1995Downing and Burns, 1995; Meisner, 2001Downing and Burns,1995Downing and Burns, 1995; Meisner, 2001

Nomorhamphus towoetii Nomorhamphus vivipara

Nomorhamphus weberi Zenarchopterus buffonis Zenarchopteruscaudovittatus

restricted lobularrestricted lobularrestricted lobularrestrictedlobularrestricted lobular

Downing and Burns, 1995Downing and Burns, 1995; Meisner,2001Downing and Burns, 1995; Meisner, 2001Grier and Collette,1987Grier and Collette, 1987

Zenarchopterus dispar Zenarchopterus dunckeri Zenarchopterusectuntio

Zenarchopterus gilli Zenarchopterus kampeni

restricted lobularrestricted lobularrestricted lobularrestrictedlobularrestricted lobular

Grier and Collette, 1987Grier and Collette, 1987Grier andCollette, 1987Grier and Collette, 1987Grier and Collette, 1987

Zenarchopterus novaeguineae

Zenarchopterus ornithocephala Zenarchopterus rasoriZenarchopterus robertsi

restricted lobularrestricted lobularrestricted lobularrestrictedlobular

Grier and Collette, 1987Grier and Collette, 1987Grier andCollette, 1987Grier and Collette, 1987

SERIES MUGILOMORPHAMugilidae

Agnostomus monticola

Mugil cephalusunrestricted lobularunrestricted lobular

USNM 318360USNM 101188; 111387; Grier et al., 1980

SERIES PERCOMORPHAOrder SynbranchiformesSynbranchidae

Synbranchus marmoratus unrestricted lobular LoNostro et al.,2003

Mastacembelidae

Mastacembelus armatus unrestricted lobular USNM 319481

Order GasterosteiformesGasterosteidae

Pungitius sinensis unrestricted lobular USNM 336886

Order PerciformesElassomatoideiElassomatidae

Elassoma evergladei unrestricted lobular USNM 357366

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TABLE 1. (Continued)

Species Testis Type

Reference and/or

Material

PercoideiCentrarchidae

Enneacanthus gloriosus

Lepomis macrochirus

Micropterus salmoidesPomoxis nigromaculatus

unrestricted lobularunrestricted lobular

unrestricted lobularunrestricted lobular

Grier et al., 1980Grier et al., 1980

Grier et al., 1980Grier et al., 1980

Centropomidae

Centropomus undecimalis unrestricted lobular Taylor et al.,1998

Percidae

Perca flavescens unrestricted lobular Grier et al., 1980

Rachycentridae

Rachycentron canadum unrestricted lobular Brown-Peterson et al.,2002

Gerreidae

Diapterus plumieri unrestricted lobular Grier et al., 1980

Serranidae

Centropristis striata unrestricted lobular USNM/FMRI

Sparidae

Archosargus probatocephalus unrestricted lobular Grier et al.,1980

Sciaenidae

Cynoscion nebulosus

Sciaenops ocellatus

unrestricted lobularunrestricted lobular

Brown-Peterson, 2003Grier, 1993

LabroideiLabridae

Thalassoma bifasciatum unrestricted lobular Koulish et al.,2002

Cichlidae

Cichlasoma nigrofasciatum Labeotropheus trewavasaeOreochromissp.Pterophyllum scalareSarotherodon aurea

unrestricted lobularunrestricted lobularunrestrictedlobularunrestricted lobularunrestricted lobular

Grier et al., 1980Grier et al., 1980Grier, 1993Grier et al.,1980Grier et al., 1980

Blennioidei

BlenniidaeOphioblennius steindachneri unrestricted lobular USNM292427

GobioideiOdontobutidae

Micropercops swinhonis unrestricted lobular USNM 336883

Microdesmidae

Microdesmus bahianus

Microdesmus dorsipunctatus

unrestricted lobularunrestricted lobular

Thacker and Grier, 2004Thacker and Grier, 2004

Gobiidae

Bathygobius lineatus

Neogobius fluviatilisPadogobius bonelliPandaka pygmaea

Tridentiger bifasciatus

unrestricted lobularunrestricted lobularunrestrictedlobularunrestricted lobularunrestricted lobular

Thacker and Grier, 2004Thacker and Grier, 2004Cinquetti andDramis, 2003Thacker and Grier, 2004Thacker and Grier, 2004

Schindleriidae

Schindleria praematura restricted lobular Thacker and Grier,2004

* Classification follows Nelson (1994), Johnson and Patterson(1996), and Parenti (2004), in part. See text for furtherexplanation.

male reproductive classes in the synbranchiformswamp eel,Synbranchus marmoratus (see Lo Nostroet al., 2003). Further,arrangement of the male ger-minal epithelium into compartments is afixed char-acteristic among taxa that may be used to definetestistypes (Grier, 1993).

One diagnostic character of the atherinomorph fish-es wasdescribed by Rosen and Parenti (1981, p. 11)as . . . spermatogonia. . . restricted to the distal endof the tubule immediately beneaththe tunica albugineawhereas other groups of teleosts have thespermato-gonia distributed along the length of the tubule. The

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338 L. R. PARENTI AND H. J. GRIER

previous year, Grier et al., (1980) identified this dis-tinctivetestis type and reported it in 31 atherinomorphspecies representingeach of the three currently rec-ognized orders (viz., Parenti,2004). The more taxo-nomically widespread condition was reported in19 te-leost species, including Esox and Oncorhynchus spe-cies, aswell as an array of ostariophysans and perco-

morphs (Table 1). Description and definition of testistypes inosteichthyans was reconsidered by Grier(1993) who concluded thatprimitive osteichthyanshave an anastomosing tubular testis, whereasderivedteleosts, including atherinomorphs, have a lobular tes-tis,and that the lobular testis could be divided intotwo types based ondistribution and arrangement ofspermatogonia. Germinal compartmentsthat extend tothe periphery of the testis and terminate blindlyaretermed lobules, not tubules (Figs. 1AD; 2AD; seeGrier, 1993, andDiscussion, below). Hence, the ath-erinomorph testis has arestricted distribution of sper-matogonia at the distal ends oflobules (Fig. 1A,C,D),in contrast to the perciform testis type,so-called

because of its initial description in fishes at onetimeclassified in the order Perciformes, such as the stripedmullet,Mugil cephalus (Fig. 1B), in which spermato-gonia are distributedalong the lengths of testis lobules.Surveys of gonad morphologyduring the past two de-cades have confirmed the presence of theunique, re-stricted testis type in atherinomorphs (viz., GrierandCollette, 1987; Grier and Parenti, 1994; Downing andBurns, 1995;Figs. 1, 2; Table 1). There have been noproposals, however,regarding the hierarchical level atwhich we can recognize thelobular testis as a syna-pomorphy.

Our collaboration began as an attempt to clarifyboth thedefinition of testis types and their distribu-

tion among bony fish taxa. It has expanded necessar-ily toinclude ovarian and egg characters as they alsovaryphylogenetically (Figs. 3, 4). Variation amonggerminal epithelia isinterpreted in a developing phy-logenetic framework to understandthe evolution ofgonad morphology and also to begin to proposego-nad characters that may be used in phylogenetic anal-yses.

MATERIALS AND METHODS

Gonad material was obtained from either freshlyfixed specimensor from specimens maintained in thefish collection of the NationalMuseum of Natural His-tory (USNM), Smithsonian Institution. Themuseummaterial is identified by the prefix USNM (United Stat-edNational Museum), followed by a catalogue num-ber. Material thatwas collected and processed at theFlorida Marine Research Institute(now the Fish andWildlife Research Institute) is identified by theabbre-viation USNM/FMRI. Voucher material will be de-posited in thepermanent collections of the USNM.Paddlefish, Polyodon spathula,gonads were obtainedfrom Kentucky State University. Rainbow trout,On-corhynchus mykiss, gonads were obtained from the ElZarco trouthatchery outside of Mexico City, Mexico.

Testis type was recorded in 136 osteichthyan speciesfrom our ownobservations, the literature or both (Ta-ble 1). Egg type wasobserved in a more limited setof taxa and is cited in the text.

The museum material is likely to have been fixedin formalin, acommon fixative starting in the late1800s. It is currently storedin 75% ethanol. Gonads

from fresh material were fixed in Bouins solutionor buffered 10%formalin. Formalin-fixed, alcohol-preserved whole fish specimensstored in museumcollections for decades proved as useful andreliablefor examination of histological structure of gonadsasrecently fixed material. Museum collections arethe only source ofgonads of certain taxa. When mu-seum specimens did not prove to beof high histo-logical quality, our opinion was that the initialfix-ation was at fault, not the prolonged storage in eth-anol.

Whole or sectioned gonads were embedded in plas-tic(glycolmethacrylate [Polysciences]) or paraffin.Tissue sectionswere cut at 68 m (paraffin) or 3.5

and 4 m (plastic). Paraffin sections were stainedwithhematoxylin and eosin; plastic sections were stainedw ith thion in o r m e ta n il y e llo w -p e rio d ic a c id / Schiffs(PAS) hematoxylin (Quintero-Hunter et al.,1991).

RESULTS

Testes

Anastomosing tubular testes are found throughoutprimitiveteleost taxa, ranging from the tarpon, Me-galops atlanticus (Fig.2E), the cypriniform, Abbottinarivularis (Fig. 2C), to the rainbowtrout, Oncorhynchusmykiss and the pikes and pickerels, genus Esox(see

Table 1 for references and material). In some histolog-icalpreparations of anastomosing tubular testes, thegerminalcompartments may appear somewhat lobular,as in Figure 2E, probablyowing to the plane of sectionthrough a three-dimensional tissue.Our descriptionsare based upon two-dimensional histologicalrepresen-tations of the three-dimensional germinal compart-ments. Atubular testis, which appears to be anasto-mosing, characterizesthe primitive sarcopterygian, thecoelacanth, Latimeria chalumnae,and the primitivenon-teleost actinopterygians, the paddlefish,Polyodonspathula, and the gar, Lepisosteus platyrhinchus(Table1).

Lobular testes characterize all fishes of the Neote-leostei thatwe have surveyed or for which we foundcitations (Figs. 1, 2A,B,D;Table 1). Lobular testesmay be further divided into restricted andunrestrictedtypes (Grier et al., 1980; Grier, 1993). In all maleath-erinomorph fishes surveyed to date, 79 species repre-sentingall three orders, including taxa with a range ofreproductive modes,spermatogonia are restricted tothe distal termini of lobules ratherthan being distrib-uted along the lobule (Fig. 1A,C,D; Table 1).Further-more, there is an epithelioid arrangement of Sertoliandgerm cells; that is, the germ cells and Sertoli cells

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FIG. 1. A. Cross section of the testis from the Gulf killifish,Fundulus grandis. The lobules terminate at the periphery of thetestis, wherespermatogonia (SG) are located. Proceeding proximally,meiotic germ cells are arranged almost in rows between juxtaposedlobules, andprimary spermatocytes (1SC), spermatids (ST), and sperm(SP) in spermatocysts or within the lumina of efferent ducts (ED)are observed.Bar 50 m. B. A lobule from the striped mullet, Mugilcephalus, collected in late October, approximately one month beforespawningcondition. Sperm (SP) accumulate within the lobule lumen.Spermatogonia (SG) are observed both at the distal terminus of thelobule andalso along the lateral walls as are spermatocystscontaining primary spermatocytes (1SC), secondary spermatocytes(2SC), and spermatids(ST). The germinal epithelium has becomediscontinuous as extensive regions of the lobule lackspermatocysts. Bar 50 m. C. Testis lobulesfrom the ballyhoo,Hemiramphus brasiliensis, have spermatogonia (SG) restricted totheir distal termini. Later stage developing sperm within

spermatocysts, primary spermatocytes (1SC) and spermatids (ST)are located progressively closer to the efferent ducts (ED) whichare filledwith sperm (SP). Bar 50 m. D. The distal termini oflobules from the testis of Fundulus grandis. Spermatogonia (SG),some dividing andin metaphase (M), are restricted to the lobuledistal termini. The borders of spermatocysts with primaryspermatocytes (1SC) are delineatedby lightly-staining Sertoli cells(SE). Bar 10 m. E. The efferent duct (ED) cells in the testis ofFundulus grandis contain eosinophilicsecretory material (brightpink). At the arrow, spermiogenesis, or release of sperm into theefferent ducts is occurring as Sertoli cell processesseparate, thelumen of the spermatocyst then becomes continuous with that of theefferent duct. Bar 10 m.

within the lobules do not border directly onto a lumen.Bydefinition, epithelia border a body surface, lumen,or tube (viz.,Grier, 2000; Grier and Lo Nostro, 2000).In atherinomorphs, theSertoli cells extend processes

across the widths of the lobules; spermatocysts, there-fore,extend across the lobules, and there is no lumenwithin the lobule(Fig. 1A,C,D). At spermiation, spermare voided from thespermatocyst the lumen of which

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340 L. R. PARENTI AND H. J. GRIER

FIG. 2. A. A lobule from the Everglades pygmy sunfish, Elassomaevergladei, illustrating primary spermatogonia (SG) withdifferent-sizednuclei. Some spermatogonia are in metaphase (M) oranaphase (A) along the wall of the lobule. Secondary spermatogonia(2SG) are withinspermatocysts. Bar 10 m. B. A testicular lobulefrom the trout-perch, Percopsis omiscomaycus, illustratingspermatogonia (SG) at thedistal terminus of and along the lobulewall. The lobule is occluded, and no lumen is observed, withspermatocysts containing synchronously-developing primaryspermatocytes (1SC). Bar 10 m. C. Testicular lobules from theChinese false gudgeon, Abbottina rivularis. Sperma-tocysts (CY)containing spermatocytes are numerous, and a few scatteredspermatogonia (SG) are located at the distal termini and alongthe

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341GONAD MORPHOLOGY IN BONY FISHES

FIG. 2. (Continued) lobule (L) walls. Bar 10 m. D. Testislobules of the beardfish, Polymixia lowei, have spermatogonia (SG)locatedalong their walls and at the distal termini. Spermatocytes(SC) and one testis ovum (TO) are observed. Bar 50 m. E. Theanastomosingtubular testis of the tarpon, Megalops atlanticus, isillustrated. Anastomosing tubules do not terminate at the testisperiphery, but rather formcontinuous, interconnected loops asobserved in the lobules shaded in turquoise. Lobules to the rightof those shaded appear to terminate atthe periphery of the testisowing to plane of section. The lobules in this reproductive fishare filled with stored sperm, and developing spermin spermatocystsare not observed, a characteristic of reproductive, synchronousspawning fish. Bar 100 m.

becomes continuous with that of the efferent duct (Fig.1E).

Ovaries and oocytes

The ovarian germinal epithelium is the origin of fol-licles inthe fish ovary (Grier, 2000), actively produc-ing folliclesthroughout the annual reproductive cycle

in the perciform Centropomus undecimalis. Morphol-ogy of thegerminal epithelium is identical in C. un-decimalis and thesynbranchiform Synbranchus mar-moratus (see Ravaglia and Maggese,2003). In the ath-erinomorph Gulf killifish, Fundulus grandis (Fig.3A),the ovarian germinal epithelium has essentially thesamefeatures as the above two species, and separatesthe ovarian lumenfrom the stroma. A consistent fea-ture of the ovarian stroma inteleosts is a conspicuousextravascular space of varying size, andsometimesseemingly nonexistent. If they do not dissolve inhis-tological preparation, the fixed fluids within theex-travascular space stain positively, if only lightly,forglycoproteins as does blood plasma. As capillaries are

leaky, that is, their endothelial cells are not joinedby tightjunctions, we infer that fluids within the ex-travascular space arederived from the circulatory sys-tem. Furthermore, theirdemonstration may be depen-dent upon type of fixative, rate ofpenetration of fix-ative, and stain (Grier, unreported).

The germinal epithelium is composed of epithelialcells thatbecome prefollicle cells when associatedwith oogonia, as inFundulus grandis (Fig. 3A). It issubtended by prethecal cells thatare identical to cellsin the ovarian stroma (Grier, unreported).Once an oo-gonium enters meiosis, it becomes an oocyte thatpro-gresses through the initial phases of the first meioticprophaseuntil the diplotene stage when division is ar-

rested, and the other events of oocyte maturation com-mence:primary oocyte growth (previtellogenesis [Pa-tino and Sullivan,2002], Fig. 3A,B), secondary oocytegrowth (vitellogenesis [Patinoand Sullivan, 2002],Fig. 3B,C), and final oocyte maturation (Figs.3D, 4A).During primary growth, the yolk nucleus appears asdocortical alveoli (Fig. 3B).

From the onset of formation of oocyte cytoplasmicyolk,osteichthyans have yolk that is distinctly granu-lar, i.e., duringvitellogenesis, the process of yolk for-mation, protein yolk isformed into spherical globules(Fig. 4B). Atherinomorphs have whatwe describe asfluid yolk; it is relatively uniform in contrast tothatof other osteichthyans (Figs. 3C,D, 4A). The differ-encebetween fluid and granular yolk is discerned read-ilyhistologically. Granular yolk becomes fluid duringthe final,pre-ovulatory events of oocyte maturation

(Wallace and Selman, 1981; Selman and Wallace,1989; Neidig etal., 2000), to which the term finaloocyte maturation (FOM) has beenapplied (Jalabertet al., 1977). In atherinomorphs, protein yolkappearsglobular at the oocyte surface, but these globulescon-tinuously fuse (Fig. 3C) to form a yolky mass thatstainspositively with the periodic acid Schiff reaction

for glycoproteins.The events of final oocyte maturation have notbeen

documented in atherinomorphs, but in Hemiramphusbrasiliensis(see McBride and Thurman, 2003; Fig.4A) there is both an increasein oocyte diameter anda reduction of the periodic acid Schiffstaining of yolk.In contrast to the fluid yolk of atherinomorphs,yolkis organized into globules in other taxa. In Mugil ce-phalus(Fig. 4B), yolk is distinctively globular and eo-sinophilic, notstaining with periodic acid Schiff. In Elassoma evergladei (Fig.4C), the yolk is periodicacid Schiff-positive, but globular.

DISCUSSION AND CONCLUSIONS

Our survey confirms the initial observation of Grier(1993) thatan anastomosing tubular testis character-izes basal osteichthyans,including basal teleosts,whereas a lobular testis characterizeshigher teleosts(Table 1). The lobular testis type is proposed as adi-agnostic or synapomorphic character of the Neoteleos-tei, asdiscussed below. Further, we confirm the resultsof Grier et al.(1980) that the restricted lobular typeof testis is diagnostic ofatherinomorph fishes.

Testis types in fishes have been defined poorly,based onexamination of too few species or indiscrim-inate application ofterms such that lobule and tu-bule were used interchangeably. Forexample, Grieret al. (1980) noted that the literature does notclearly

distinguish structural differences between lobularand tubulartesticular types, and that use of eitherterm varied by author. Thetestes of fishes, both thebasal osteichthyans and the neoteleosts,were termedtubular (Grier et al., 1980; Grier, 1981) deliberatelytoavoid confusion with the erroneous concept thatsome fishes havelobule boundary cells as Leydigcell hom*ologs. It was demonstratedsubsequently thatone teleost species, Esox lucius, which wasreportedto have lobule boundary cells as Leydig cell hom*o-logs, hada typical distribution of interstitial, hormone-secreting Leydigcells (Grier et al., 1989). The so-called lobule boundary cellswere Sertoli cells withinthe germinal compartment. Furthercomparative inves-tigations of testis morphology in teleosts led toa newinterpretation: the basal osteichthyans have anasto-mosingtubular testes in which the germinal compart-

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342 L. R. PARENTI AND H. J. GRIER

FIG. 3. A. The germinal epithelium ofFundulus grandis bordersthe ovarian lumen (OL). In it are observed an oogonium (OG)associatedwith a prefollicle cell (PFC), distal to which is anepithelial cell (E). Epithelial cells that are associated withoogonia are prefollicle cells.Beneath the germinal epithelium is aprethecal cell (PT). An extravascular space (EVS) is prominent, andprimary growth oocytes (PGOC),with dense-staining cytoplasm, arelocated beneath the germinal epithelium. Bar 10 m. B. In Fundulusgrandis the germinal epitheliumseparates the ovarian lumen (OL)from an extensive extravascular space (EVS) in which a primarygrowth oocyte, with cortical alveoli (ca),is observed. Its nucleoli(nu) are located around the periphery of the nucleus (n). A smallerprimary growth oocyte has a prominent yolknucleus (yn) within itscytoplasm, and the nucleoli are scattered within the nucleus (n).Cortical alveoli are also observed within a vitellogenic

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FIG. 3. (Continued) oocyte (V). Bar 50 m. C. In vitellogenicoocytes of Fundulus grandis the yolk globules (y) fuse, being quiteseparateand small at the oocyte periphery and larger centrally.Because of staining characteristics, it is difficult to distinguishbetween fat globulesand cortical alveoli which become progressivelylighter-staining as oocyte development proceeds. The oocyte issurrounded by a zona pellucida(ZP), often called a chorion.Exterior to this is a follicle cell (FC) layer, and theca (T). Theoocyte nucleus (n) is dwarfed by the increasingsize of the oocyte.Germinal epithelium (GE). Extravascular space, (EVS). Bar 100 m. D.Portion of a mature oocyte from Fundulusgrandis wherein thecytoplasm is filled with periodic acid Schiff-positive fluid,protein yolk. Cortical alveoli (ca) are restricted to theperipheryof the oocyte, as is the nucleus (n), indicating that thisoocyte is preovulatory. The oocyte is surrounded by the zonapellucida (zp). Bar 100 m.

ments do not terminate at the testis periphery, but formhighlybranched, anastomosing loops or tubules. Theterm tubule wasretained as for mammals in whichtesticular tubules do not terminateat the testis periph-ery, but also form loops (viz., Grier, 1993).In neote-leosts, the germinal compartments may form anasto-mosingnetworks proximally, but distally they extend

to the periphery of the testis and terminate blindly.Thischaracter defines the lobular testis, which is eitherunrestricted(neoteleosts) or restricted (atherino-morphs) with regard to thedistribution of spermato-gonia within the lobules (Grier, 1993).Furthermore,the testes in Atherinomorpha do not form anastomos-ingnetworks, but the lobules may branch as they ex-tend from theefferent ducts to the periphery of thetestis. This may be anotheratherinomorph synapo-morphy, but support for more than this simpleproposalis lacking. The description of the germinal compart-mentsin atherinomorphs as lobular (Grier, 1993)replaces use of the termtubular (Grier et al., 1980;Grier, 1981; Nagahama, 1983; Rosen andParenti,

1981) and emphasizes the distinctive differences intestisstructure between basal and higher teleosts assubsequently definedby Grier (1993). The testis of theEverglades pygmy sunfish,Elassoma evergladei, aspecies examined here, is lobular, notanastomosingtubular as described for the banded pygmy sunfishElassoma zonatum by Walsh and Burr (1984). We in-terpret the testisof E. zonatum as unrestricted lobularand suggest that descriptionof the testis as anasto-mosing tubular was due to the inconsistentway inwhich these terms have been applied.

The unrestricted lobular testis is found throughouttheNeoteleostei (Table 1), including the paracanthop-terygianPercopsis (Fig. 2B), the beardfish Polymixia

(Fig. 2D), and all other neoteleosts, except for theath-erinomorphs and the diminutive gobioid Schindleria,discussedbelow. Basal, deep-sea neoteleosts in the or-ders Myctophiformes,Stomiiformes, and Aulopifor-mes (following the classification ofJohnson and Pat-terson, 1996) have not been surveyed; that theyhavean unrestricted lobular testis is a prediction open totest.

Atherinomorphs have been proposed by morpholo-gists to beclosely related to the percomorphs (Rosenand Parenti, 1981), tomullets (Stiassny, 1993), to agroup of taxa in a higher-levelteleost category, thesmegm*morpha (Johnson and Patterson, 1993),orwith unresolved relationships to the percomorphsandparacanthopterygians (Parenti, 1993). smegm*morphs

were considered by Johnson and Patterson (1993) toinclude, inaddition to the atherinomorphs, the mullets,Mugilidae, the swampand spiny eels, Synbranchifor-mes, the sticklebacks and relatives,Gasterosteiformes,and the pygmy sunfishes, Elassoma. We examinedrep-resentatives of each of the non-atherinomorph smeg-mamorphs andfound all to have an unrestricted lob-

ular testis (Table 1). Similarly, all paracanthopterygi-ansexamined have an unrestricted lobular testis (Table1).

That the restricted lobular testis of atherinomorphfishes is notfound in any of the other proposed smeg-mamorphs does not testsmegm*morph monophyly,but does refute a recent molecular hypothesisin whichatherinomorph monophyly was challenged (e.g., Chenet al.,2003). Atherinomorph monophyly has not beenquestioned bymorphologists since Gosline (1971), andhas been corroborated in arecent, broad-scale molec-ular analysis (Miya et al., 2003). Thelist of diagnosticmorphological characters for atherinomorphs isexten-sive and includes characters of the egg (viz., Parenti,

2004) to which we add fluid, rather than granular, yolk.Allother so-called smegm*morphs have globular pro-tein yolk withinvitellogenic and mature oocytes (Fig.4B,C). Only during finaloocyte maturation, precedingovulation, does the yolk become fluidasobservedthroughout vitellogenesis in atherinomorphs (Fig. 4A).Wealso note that atherinomorphs have periodic acidSchiff-positiveyolk, but the phylogenetic significanceof this observation remainsto be determined with fur-ther surveys.

Ultrastructural investigation has shown that oogoniaarescattered in the simple epithelium lining the ovar-ian cavity orlumen in common snook, Centropomusundecimalis (see Grier, 2000) andthe swamp eel, Syn-

branchus marmoratus (see Ravaglia and Maggese,2003). It is adiscontinuous germinal epithelium, fol-lowing definitions ofgerminal epithelia (continuousand discontinuous) developed forcommon snook(Grier and Taylor, 1998). Our examination of theovaryof Fundulus grandis, using plastic embedded tissue,revealedthe presence of oogonia that are scatteredwithin a germinalepithelium, similarly associated withepithelial and prefolliclecells as in the snook and theswamp eel. Contrary to Brummett et al.(1982), whoindicated that oocytes appear to be derived fromoo-gonia located immediately beneath the simple epithe-lium liningthe ovarian cavity, we conclude that theluminal epithelium in theovary of Fundulus is thegerminal epithelium. Furthermore, it isestablished that

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FIG. 4. A. Periphery of the ovary in Hemiramphus brasiliensiswith a preovulatory oocyte (POV) whose periodic acidSchiff-positive (PAS),protein yolk is lighter-staining than theprotein yolk in an oocyte that is not preovulatory. In bothoocytes, the protein yolk (y) is fluid. Thegerminal epitheliumseparates these two oocytes, and primary growth oocytes (PG), fromthe ovarian lumen (OL). Bar 50 m. B. In theovary of reproductiveMugil cephalus, the germinal epithelium (GE) separates the ovarianlumen (OL) from the stroma in which a prominentextravascular space(EVS) is observed. Within the germinal epithelium, two smallprimary growth oocytes (arrows) are observed. Largerprimary growthoocytes (PG) and mature oocytes (MOC) are surrounded by the EVS.Mature oocytes have globular protein yolk granules(yg) rather thanfluid yolk. These become fluid during final oocyte maturation, justbefore ovulation. Lipids (1) are circumnuclear in position,andcortical alveoli (ca) are peripheral. Bar 50 m. C. Portion of amature oocyte from the Everglades pygmy sunfish, Elassomaevergladei.The content of cortical alveoli is periodic acidSchiff-positive (purple). Globular yolk (y) is also periodic acidSchiff-positive, particularly theintensely-staining, small yolkglobules at the oocyte periphery. The germinal epithelium,separating the oocyte from the ovarian lumen (OL),is composed ofepithelial cells with flattened nuclei (E) and an oogonium (OG).Bar 10 m.

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the ovarian germinal epithelium in the protogynousswamp eel,Synbranchus marmoratus, produces folli-cles during the female phaseof the reproductive cycle.During sex reversal, however, the samegerminal epi-thelium produces the initial male germ cellsbeforelobules are formed (Lo Nostro and Grier, 2002).Thisobservation introduces a new approach to study of the

mechanism of sex reversal in other protogynous spe-cies, forexample in the perciform families Serranidae,Labridae, andGobiidae.

Our investigation reveals that there is constant ori-gin ofovarian follicles from the germinal epitheliumamong taxa.Terminology should reflect this proposalof hom*ology. The histologytext book definition of afollicle (see Grier and Lo Nostro, 2000)precisely re-flects its origin from a germinal epithelium. Thefol-licle is composed of the germ cell, the oocyte, andsurroundingfollicle (granulosa) cells that originatefrom the epithelial(somatic) cells of the germinal ep-ithelium. The follicle issurrounded by a theca, derivedfrom the stromal compartment of theovary (Grier,

2000) and is always separated from this compartment,throughoutdevelopment and final oocyte maturation,by a basem*nt membrane. Asa basem*nt membraneseparates an epithelium from the underlyinglaminapropria or supportive tissue, so it also separatesthefollicle from the theca. The follicle basem*nt mem-brane isderived from that underlying the germinal ep-ithelium (Grier,2000). The term follicle complex(Grier and Lo Nostro, 2000) hasbeen proposed to in-clude the follicle, basem*nt membrane, and thetheca,including its blood vessels. The various definitions ofafollicle within the fish literature are based primarilyon functionrather than form, however. These defini-tions of a follicle includethe surrounding theca, with

(Redding and Patino, 1993; Sullivan et al., 1997) orwithout(Tyler and Sumpter, 1996) the basem*nt mem-brane.

Terminology may be confusing due to our ignoranceof comparativemorphology, the use of synonyms, thestill-emerging physiologicaland molecular eventscausing oocyte growth, the now documented roleof agerminal epithelium in follicle formation and in sexreversal(vide supra), and an array of egg types in fish-es that is onlydealt with superficially herein. The termfinal oocyte maturationhas not become irrelevantand misleading, as argued by Patino andSullivan(2002). Final oocyte maturation is used commonly todescribechanges in oocytes leading to ovulation ob-served in common snook(Neidig et al., 2000) andother marine fishes (viz., Brown-Petersonet al., 1988,2002) and is embedded in the fisheries literature.Oo-cyte changes leading to ovulation include cytoplasmicand nuclearevents (as in Patino and Sullivan, 2002),and, in our opinion, theseevents mark final oocytematuration prior to ovulation. All of thechanges infollicles as they mature can be viewed as maturationorgrowth.

Definitions based on hom*ology of form can aid inunderstandingreproduction across a broad array of

taxa. For example, mammals have an advanced follic-ularmorphology not found in teleosts. As above, theteleost follicleconsists merely of the oocyte and itsencompassing, monolayer offollicle (granulosa) cells,defined as a primordial follicle (viz.,Van Blerkomand Motta, 1979). In mammals, however, the folliclecellsdivide, become many cells deep, surround the

oocyte to produce a primary follicle. Then, a fluid-filled spacedevelops, to form the antrum (Gray et al.,1995) of a tertiaryfollicle. From the standpoint ofcomparative anatomy, the fishovarian follicle corre-sponds to the mammalian primordial follicle,andall oocyte maturation occurs within the hom*ologue ofthemammalian primordial follicle. Furthermore, asin fish (Grier,2000), mammalian follicles originatefrom a germinal epithelium(Zamboni, 1972), i.e., theyare hom*ologues. Recent studies ofmammalian repro-duction confirm that follicle cells in sheepdevelopfrom cells of the germinal epithelium (Heywood et al.,2002),and that a possibly active germinal epitheliumis present in mice(Johnson et al., 2004: fig. 2). We

anticipate that additional hom*ologous characteristicswithinreproductive systems across a broad array oftaxa are yet to berevealed.

The annual reproductive cycle is hypothesized to bethe source ofmorphological variation among testistypes. Five reproductiveclasses have been describedin males of the common snook,Centropomus unde-cimalis (see Taylor et al., 1998), seatrout,Cynoscionnebulosus (see Brown-Peterson, 2003), the cobia,Ra-chycentron canadum (see Brown-Peterson et al.,2002), the swampeel, Synbranchus marmoratus (seeLo Nostro et al., 2003), and thefreshwater goby, Pa-dogobius bonelli (see Cinquetti and Dramis,2003): re-gressed, early maturation, mid maturation, late matu-

ration, and regression. These are identified by the al-ternationof the germinal epithelium between contin-uous and discontinuoustypes and the stages of germcells present (Grier, 2000).

The transition between basal and neoteleosts ismarked bynumerous physiological and morphologicalmodifications, such as type4 tooth attachment (Fink,1981) and acellular bone (Parenti, 1986),charactersthat Esox shares with neoteleosts (Parenti, 1986;John-son and Patterson, 1996). Teleost evolution was de-scribed aspaedomorphic by Fink (1981) because somecharacters observed inadults of more advanced tele-osts, such as tooth attachment mode,approximate theearly developmental stages of primitive teleostfishes.The evolutionary transition from an anastomosing tu-bular toa lobular testis could have resulted from theelongation of thetestis germinal compartments duringthe early maturation class whenthe testis enlarges pri-or to the breeding season. The processcould haveevolved as a simple change in formation of thesup-porting basem*nt membrane of the germinal epitheli-um.Similarly, restriction of spermatogonia to the dis-tal ends oflobules in the atherinomorphs is mirroredin perciforms by theestablishment of distal epithelioidcords of Sertoli cells andspermatogonia in cobia testes

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346 L. R. PARENTI AND H. J. GRIER

during the regression and regressed classes of the an-nualreproductive cycle (Brown-Peterson et al., 2002).Clusters ofspermatogonia become established duringthe same reproductiveclasses in common snook (Grierand Taylor, 1998). During regressionin these perci-forms, but not in atherinomorphs, spermatogoniaalsobecome established along the walls of lobules. There

is a marked change in the arrangement of Sertoli cellsinatherinomorphs compared to other fishes. Sertolicells extendprocesses across the lobules, and a lobulelumen is absent. Wehypothesize that the difference inthe way in which Sertoli cellprocesses bridge thewidths of the lobules, as in Fundulus grandis(Fig. 1A)and Hemiramphus brasiliensis (Fig. 1C), preventsthecolonization of the lateral lobule walls by spermato-goniatheyare only observed at the distal termini ofthe lobules. Evolution ofthe atherinomorph testis type,the strongest evidence supportingatherinomorphmonophyly (viz., Parenti, 2004), is hypothesized toen-tail mechanisms that prevent the repopulation of sper-matogoniaalong lobule walls during regression and

when regressed. Atherinomorphs may be said to havea regressedtestis that undergoes a functional mat-uration.

There is scant information on the mechanism of lob-uleelongation during gonad maturation betweenspawning seasons, andpractically nothing is knownabout the process of regression. It hasrecently beenproposed that fish testes shift betweenmeiosis-domi-nated to mitosis-dominated cell divisions duringtheirannual reproductive cycles (Grier, 2002). The supplyofspermatogonia, from which meiotic germ cells arederived, becomesprogressively exhausted betweenearly maturation, mid maturation,and late maturation.It has not been appreciated that in the latterpart of the

annual reproductive cycle, especially regression, thetestes areactively preparing for the next reproductivecycle, i.e., thelobules become repopulated by sper-matogonia. The same is true inthe teleost ovary, atleast in Centropomus undecimalis, in which thepro-cess of folliculogenesis was interpreted using ovariesfromregressed fish (Grier, 2000). There is significantcell divisionduring the regressed class in Synbranchusmarmoratus that involvesboth Sertoli cells and sper-matogonia (Lo Nostro and Grier, 2003).Sertoli cellshave been demonstrated to divide in theatherinomorphPoecilia latipinna (see Grier, 1993: fig. 25), andthewrasse, Thalassoma bifasciatum (see Koulish et al.,2002).Mitotic cell division in fish testes during theregression andregressed classes, as defined by Tayloret al. (1998), has hardlybeen investigated. In light ofgrowth processes during theseclasses, however,Brown-Peterson et al. (2002) suggested that thetermresting stage no longer be used. Active cell divisionlikelytakes place in fish gonads throughout the annualreproductive cycle,even when not spawning.

There appear to be two locations within the testesof perciformsfrom which spermatogonia are derived:in common snook (Grier andTaylor, 1998) and cobia(Brown-Peterson et al., 2002), clusters orelongation

of the lobules composed of spermatogonia and Sertolicells becomeestablished at the distal ends of lobules.Divisions of these cellsresult in lobule elongation(Grier, 1993). Lobule growth wasinferred to occur viaa branching process; in common snook,anastomosingtestis morphology results from fusion of laterallobulewalls (Grier and Taylor, 1998). We have not observed

anastomosing of the lobules in any atherinomorph.Spermatogoniaalso repopulate the lateral walls of

testicular lobules. By the end of the breeding season,thepopulation of spermatogonia is exhausted, thesecells having formedsperm. There are always scatteredspermatogonia along the lobulewalls that apparentlydo not divide, however. They compose adiscontin-uous germinal epithelium, one that spans reproduc-tiveseasons and are the prima facie evidence for apermanent germinalepithelium in fishes (Grier, 1993;Lo Nostro et al., 2003). Duringthe regression and re-gressed classesthe mitosis dominatedclassessper-matogonia within the discontinuous germinal epithe-liumbecome mitotically active, repopulate the lateral

lobule walls and form a continuous germinal epithe-lium, againcomposed of spermatogonia and Sertolicells. The term epithelioidwas first applied to adescription of the testes in the cobia inreference tocords of spermatogonia and Sertoli cells that growfromthe distal termini of the lobules during the re-gressed class inthe annual reproductive cycle (Brown-Peterson et al., 2002).Because they lack a lumen,these cells do not fulfill the criteriathat define an ep-ithelium (Grier, 2000; Grier and Lo Nostro,2000). Theepithelioid arrangement of cells at the distal terminioflobules is transitory. When a lumen develops, thesecells composea germinal epithelium.

Changes in the male germinal epithelium, used to

define annual reproductive classes first in commonsnook (Tayloret al., 1998), cannot be used to defineannual reproductive classesin females. The basic dif-ference between the sexes that preventsthis simplecharacterization is the early initiation of meiosis infe-males. In common snook, the germinal epithelium isactivethroughout the year, producing follicles wherethe oocyte is inarrested meiosis, diplotene of the firstmeiotic prophase, and thegerminal epithelium is al-ways discontinuous. In males, onlydiploid spermato-gonia persist in the regressed class and thegerminalepithelium is continuous, becoming discontinuous dur-ingmid maturation and late maturation. The annualalternation betweencontinuous and discontinuous ger-minal epithelia in the maleteleost germinal epitheliumpermits the use of changes to be usedfor definingannual reproductive classes.

These proposals emphasize that morphologicalchange within thetestis during the annual reproductivecycle can lead to theformation of distinct testis types.Interestingly, Schindleria, agobioid fish that has beencharacterized as . . . the most radicallyontogeneti-cally truncated fish (Johnson and Brothers, 1993,p.469), reportedly has a restricted lobular testis (ThackerandGrier, 2004). This corroborates our hypothesis that

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arrest of the late maturation-regression phases leadstoformation of the atherinomorph testis type; that is, itis likethe paedomorphic testis of mature adult Schin-dleria.

Testis type provides another way to distinguishamong model fishorganisms: the zebrafish, Danio re-rio, has an anastomosing tubulartestis, whereas the

atherinomorph medaka, Oryzias latipes, has a restrict-ed lobulartestis (Table 1). We predict that the fugu,Takifugu rubripes, aderived percomorph, has an un-restricted lobular testis. Ourproposal is that simplechanges in the annual reproductive cyclehave resultedin these different testis types. The genetic basisofthese differences is unknown. Identifying morpholog-icalvariation, and proposing the source of that mor-phologicalvariation, is a first step in understandingunderlying geneticmechanisms.

ACKNOWLEDGMENTS

We are grateful to Francesco Santini and GustavoYbazeta,University of Toronto, for organizing the

symposium on teleost fishes and for inviting us to par-ticipate.Helen F. Wimer, USNM, skillfully preparedthe bulk of thehistological sections. She was grantedaccess to plastic embeddingand sectioning facilitiesby William D. Swaim, Director, CellularImaging CoreFacility, National Institute of Dental andCraniofacialResearch, NIH. Steve Mims, Kentucky State Univer-sity,Rich McBride, FMRI, and Maria del Carmen Uri-be, UNAM, Mexico City,helped obtain gonad mate-rial. Llyn French, FMRI, graciouslyprepared the fig-ures and helped edit the manuscript. Jeffrey M.Clay-ton, USNM, and Yvonne Waters, FMRI, providedinvaluabletechnical assistance.

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