JOURNAL OF MORPHOLOGY 268:23–32 (2007) Reproductive Morphology of Brittanichthys axelrodi (Teleostei: Characidae), a Miniature Inseminating Fish From South America Robert Javonillo,1* John R. Burns,1 and Stanley H. Weitzman2 1 Department of Biological Sciences, The George Washington University, Washington, District of Columbia 20052 Division of Fishes, National Museum of Natural History, Smithsonian Institution, Washington, District of Columbia 20560 2 ABSTRACT Light and electron microscopy were used to investigate the morphology of reproductive characters in a characid fish, Brittanichthys axelrodi. Spermatozoa were found in ovaries of females, thereby confirming insemination in this species. Bony hooks can be found on the fourth unbranched ray and branched rays 1–4 of the anal fin and the unique sigmoidally-curved ray of the caudal fin in mature males. Testes have three distinct regions: an anterior spermatogenic region, an aspermatogenic middle region lined with a simple squamous epithelium and used for storage of mature spermatozoa, and a posterior region of coiled chambers lined with a high simple cuboidal epithelium. The most posterior region appears to be instrumental in the formation and storage of spermatozeugmata, unencapsulated sperm packets. Thus far, this tripartite testis morphology is unique among characids. The mature spermatozoon has an elongate nucleus (5 lm in length). A striated rootlet originates at the anterior end of the distal centriole and continues to the anterior tip of the cell. The striated rootlet wraps around the entire ventral area of the anterior part of the nucleus and appears to continue around the anterior tip of the nucleus and down the dorsal side as electron-dense material. Several large, spherical mitochondria (0.6 lm in diameter) with lamellar cristae overlap the posterior end of the nucleus and continue beyond together with the cytoplasmic collar that contains the flagellum which lacks axonemal fins. Each spermatozeugma is lanceolate in shape when sectioned mid-sagitally, with the core staining positively for mucopolysaccharides. In both sexes, the gonopore opens posterior to the anus, with the urinary pore having a separate opening posterior to the gonopore. Bands of skeletal muscle were found in the area of the male gonopore. These morphological features are likely linked to the reproductive mode of insemination, a trait that is so far as known, relatively rare among teleost fishes, but is proving increasingly frequent among certain groups of characid fishes. J. Morphol. 268: 23–32, 2007. Ó 2006 Wiley-Liss, Inc. family Alestidae, order Characiformes, occur in both Africa and South America (Zanata and Vari, 2005), leading biogeographers to hypothesize that the most recent common ancestor of all characiforms inhabited the supercontinent of Gondwanaland before its tectonic plates began to drift apart about 120 million years ago (Lundberg, 1993; Ortı́, 1997; Zanata and Vari, 2005). Insemination, whereby spermatozoa are introduced into the reproductive tract of a female, is, so far as known, rare among bony fishes in general, but is proving increasingly frequent among characids (Burns et al., 1995, 1997, 2000). We follow Meisner (2005) in recognizing a distinction between insemination and internal fertilization. This designation is due to the fact that the females of some sculpins (family Cottidae) are inseminated, yet fertilization of eggs does not occur until both the eggs and sperm are released by the female into seawater (Munehara et al., 1989). Thus, unless fertilized eggs are found in the ovary (thereby demonstrating fusion of egg and sperm, i.e., internal fertilization), the presence of spermatozoa in the reproductive tract of a female is termed insemination. When the presence of insemination is optimized onto a phylogeny of characid fishes (Burns et al., 1997; Malabarba and Weitzman, 2003; Weitzman et al., 2005), it appears to have multiple origins within an unnamed clade of species that synapomorphically lack supraorbital bones. Therefore, it is of great interest to recognize correlations among insemination and other morphological characters. Brittanichthys axelrodi (Fig. 1), a miniature characid, exhibits a remarkable number of reproductive specializations. Although rarely imported, KEY WORDS: Characidae; spermatozoa; spermatozeugmata; sperm ultrastructure; reproduction Characidae is a family of fishes that exhibits diverse morphologies and ecologies and that includes familiar forms such as piranhas and tetras. Although most characid fishes are endemic to South America, some of their close relatives in the Ó 2006 WILEY-LISS, INC. *Correspondence to: Robert Javonillo, Lisner Hall 340, Department of Biological Sciences, The George Washington University, 2023 G Street NW, Washington, DC 20052. E-mail: rjavonil@gwu.edu Published online 4 December 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jmor.10500 24 R. JAVONILLO ET AL. Fig. 1. Aquarium specimen of Brittanichthys axelrodi preserved in modified Karnovsky’s fixative. Mature male, SL 23.7 mm. Arrow indicates the curved caudal-fin ray. Note that some of the original red coloration has been retained. Scale bar ¼ 5 mm. this species has been known to aquarists as the blood-red tetra and is known from the Rio Negro system of Brazil. The only other species in the genus, B. myersi, is only known from the holotype, also collected in the Rio Negro basin. Brittanichthys is of ambiguous phylogenetic position within a group consisting of incertae sedis genera that also includes some genera formerly considered part of the characid subfamily Tetragonopterinae (Malabarba and Weitzman, 2003). This group is part of the aforementioned clade (diagnosed by lack of supraorbitals). Mature male Brittanichthys have a unique type of caudal fin with the 12th principal caudal-fin ray thickened, elongate, sigmoidally-curved, and projecting slightly to one side (Figs. 1 and 2A; Malabarba and Weitzman, 1999: Figs. 9 and 10). This ray develops small bony hooks similar to those on fin rays of other characid species and which may serve as contact organs (Wiley and Collette, 1970; Weitzman and Fink, 1985: 29-30, Figs. 38–43, 47, 71–74). Another diagnostic character of Brittanichthys axelrodi is insemination (Burns et al., 2000; Burns and Weitzman, 2005). In this paper we confirm insemination and describe the possible morphological specializations associated with this reproductive mode in B. axelrodi. Our observations are based on light microscopy of ovaries, testes, and their associated ducts, and electron microscopy of sperm cells. These data will be added to the set of putative homologies that will be tested in forthcoming phylogenetic analyses. tissues were decalcified (when necessary), dehydrated, infiltrated and embedded in paraffin, sectioned at 5–7 lm and stained with a modified Masson’s trichrome (Schreibman, 1964). For glycol methacrylate sectioning, tissues were decalcified (when necessary), dehydrated in an ethanol series to 95%, infiltrated and embedded in glycol methacrylate, sectioned at 3.5 lm with a Sorvall Type JB-4 microtome, and stained with either toluidine blue (TB) or the periodic acidSchiff reagent/Harris hematoxylin (PAS/H) procedure. For scanning electron microscopy (SEM), the testis of a mature male (SL 27.8 mm) that had been fixed in a modified Karnovsky’s fixative, was dried in a Tousimis Autosamdri-815 critical point drier, teased apart with needles, coated with a gold-palladium mixture using a Denton Vacuum Desk II sputter-coater, and viewed with a LEO 1430VP scanning electron microscope. For transmission electron microscopy (TEM), testes had been initially fixed in a modified Karnovsky’s fixative and later stored under refrigeration. Testes were subsequently cut into small pieces (1 mm3), rinsed in phosphate buffer, and postfixed in 1% osmium tetroxide in phosphate buffer. These pieces were then rinsed in phosphate buffer, dehydrated in an ethanol series, infiltrated, and embedded in Araldite 502. Ultrathin sections were stained with aqueous uranyl acetate and lead citrate and examined with a JEOL JEM 1200 transmission electron microscope. Probably due to the unavoidable storage of tissues in fixative for extensive periods (initially at ambient temperature then under refrigeration), some sections had substantial artifacts on them, but still provided sufficient resolution to determine the ultrastructure of spermatozoa. MATERIALS AND METHODS We examined 11 specimens of Brittanichthys axelrodi Géry 1965 from lot USNM 221991 of the National Museum of Natural History (formerly United States National Museum), Smithsonian Institution, Washington, DC, USA . Eight were mature males (SL 21.4– 23.9 mm) and three mature females (SL 19.2–22.3 mm). In addition, six aquarium specimens were made available to us courtesy of Labbish Chao. These specimens, originally collected from the Rio Negro, Brazil, included five mature males (SL 23.2–27.8 mm) and one mature female (SL 21.6 mm). Each had been freshly fixed in a modified Karnovsky’s fixative (Ito and Karnovsky, 1968). For light microscopy, fish were initially fixed in 10% formalin and later transferred to 70% ethanol. For paraffin sectioning, Journal of Morphology DOI 10.1002/jmor Fig. 2. Fin rays bearing hooks in a mature male aquarium specimen of Brittanichthys axelrodi, SL 23.7 mm. A: Curved 12th principal caudal-fin ray bearing hooks (arrow) and unmodified rays in dorsal lobe of caudal fin (c). Scale bar ¼ 0.5 mm. B: Anal-fin rays bearing hooks (arrows). Scale bar ¼ 0.1 mm. REPRODUCTIVE MORPHOLOGY OF BRITTANICHTHYS 25 Fig. 3. Gonads of specimens of Brittanichthys axelrodi. LM. A: Mature female aquarium specimen, SL 21.6 mm. B,C,E: Mature male, SL 23.5 mm, USNM 221991. D,F: Mature male aquarium specimen, SL 23.2 mm. A: Ovary containing spermatozoa (sz) still partially aggregated into spermatozeugmata; o, previtellogenic oocyte; PAS/H. Scale bar ¼ 25 lm. B: Testis section showing anterior spermatogenic region (sg) and more posterior sperm storage region (st); TB. Scale bar ¼ 100 lm. C: Section through the most posterior coiled testis region showing spermatozoa (sz) in the lumen (l) of the tubules, as well as a tangential section through the simple cuboidal epithelium (e) lining the tubules; TB. Scale bar ¼ 25 lm. D: Section through the most posterior coiled testis region showing intact spermatozeugmata (arrows) in the lumen. Note the light PASþ staining of the cuboidal epithelium (e) lining the tubules; PAS/ H. Scale bar ¼ 25 lm. E: Section through the epithelium lining the most posterior coiled testis region showing engulfed pairs of spermatozoa (arrows) attached at their anterior tips; TB. Scale bar ¼ 5 lm. F: Longitudinal section through an individual spermatozeugma showing the PASþ core (arrow) to which the darkly staining sperm head pairs are attached. Lightly staining flagella (asterisks) comprise the outer covering of each spermatozeugma; PAS/H. Scale bar ¼ 5 lm. RESULTS Testis Structure Insemination was confirmed by the presence of spermatozoa in ovaries (Fig. 3A). Spermatozoa could be found in ovaries containing only previtellogenic oocytes, as well as ovaries with mature yolky oocytes, suggesting that insemination may occur prior to the time of oviposition in some cases. No specialized sperm storage areas were evident in the ovaries; spermatozoa generally occupied the lumina of branches of the ovarian cavity. Both the anal and caudal fins of mature male Brittanichthys axelrodi possess rays that bear small bony hooks. On caudal fins, only the branched, posterior portions of the sigmoidally-curved rays bear such hooks (Fig. 2A). Hooks on the anal fins are limited to the fourth unbranched ray and branched rays 1–4, wherein each segment may bear multiple hooks (Fig. 2B). In small adult males, however, hooks are present on fewer rays. The gross morphology of the mature testis of Brittanichthys axelrodi is unique thus far among fishes of the family Characidae in having a ‘‘tripartite’’ testis (Fig. 4). The distinctiveness of the three regions comprising each of the two testes is obvious both at the gross (Fig. 4A) and light microscopical (Fig. 4B) levels. The most anterior part of the testis comprises the spermatogenic region containing germ cells in all stages of spermatogenesis (Figs. 3B and 4B). This region occupies 51% of total testis area of a midsagittal section. The middle region serves as a storage area for mature spermatozoa and the chambers that comprise this region are lined with a simple squamous epithelium (Figs. 3B and 4B). This region occupies 32% of total testis area of a midsagittal section. The most posterior portion of the testis consists of coiled chambers lined with a high simple cuboidal epithelium (Figs. 3C and 4B), and occupies Journal of Morphology DOI 10.1002/jmor 26 R. JAVONILLO ET AL. Fig. 4. A, dissected testis and B, histological section through same testis of a mature male Brittanichthys axelrodi, USNM 221991, SL 22.3 mm, showing the anterior spermatogenic region (sg), more posterior sperm storage region (st) and most posterior coiled testis region (arrows). Scale bar ¼ 1 mm. 17% of total testis area of a midsagittal section. As will be discussed below, this area appears to play a role in the formation of sperm packets referred to as spermatozeugmata. Together, the two nonspermatogenic regions occupy 49% of total testis area. Fig. 5. Testis of mature male Brittanichthys axelrodi aquarium specimen, SL 27.8 mm, showing lateral (1), dorsal (2) and oblique (3) views of spermatozoa. SEM. n, nucleus; m, mitochondria; arrows, cytoplasmic collar; arrowhead, exit of flagellum from cytoplasmic collar. Inset, ventral view of spermatozoon showing the attached cytoplasmic collar (arrows). Scale bar ¼ 2 lm. Sperm Ultrastructure The two centrioles are perpendicular to one another. A striated rootlet (Fig. 6, arrows) originates at the anterior end of the distal centriole and continues to the very tip of the cell. The striated rootlet wraps around the entire ventral area of the anterior part of the nucleus (Fig. 8) and appears to continue around the anterior tip of the nucleus and down the dorsal side as electron-dense material, with the striated characteristics absent in these areas (Figs. 6 and 7A). As the striated rootlet passes near the proximal centriole, the material that comprises it may be responsible for the poor resolution of the proximal centriole. The distal centriole gives rise to the single flagellum, whose initial segment is also located partially within a shallow nuclear fossa (Fig. 7D). More posteriorly, the flagellum is located within the canal of a cytoplasmic collar that is attached to the cell ventrally along its length (Figs. 6 and 7E). Several large, spherical mitochondria (0.6 lm in diameter) with lamellar cristae overlap the posterior tip of the nucleus (Fig. 6) and continue be- SEM demonstrates the overall shape of the mature spermatozoon (Fig. 5). The sperm head, which contains the elongate, flattened nucleus, is oblong and flattened, with a truncated tip. A centrally located, elongate cytoplasmic collar extends along most of the length of one side of the sperm head (Fig. 5, inset). For purposes of discussion we will consider this to be the ventral side of the cell. The anterior end of the sperm head sharply curves dorsally (Fig. 5). Several large, spherical mitochondria are located just posterior to the nucleus. Posterior to the mitochondria, the cytoplasmic collar continues for a distance of 0.75 lm before the flagellum exits from it (Fig. 5, arrowhead). With TEM, in midsagittal section the elongate nucleus (5 lm in length) is narrower along its anterior third and wider along most of the posterior two thirds (Fig. 6). A slight depression accommodates part of the distal centriole. The chromatin is highly condensed. The nucleus curves dorsally at its tip. In transverse section, the nucleus is flattened near the anterior end (Fig. 7A), somewhat triangular shaped more posterioly (Fig. 7C,D) and again more flattened along most of the rest of its length (Fig. 7E). The proximal centriole was never highly resolved, and was never found in the same section as the distal centriole. What we have identified as the proximal centriole (Fig. 7B) is always found in a region where the nucleus has a highly convoluted margin ventrally. The well-resolved distal centriole is partially contained within a shallow nuclear fossa (Fig. 7C). Journal of Morphology DOI 10.1002/jmor Fig. 6. A longitudinal section and oblique section through two mature spermatozoa of Brittanichthys axelrodi from male aquarium specimen, SL 27.8 mm. TEM. n1 and n2, nuclei; m, mitochondrion; dc, distal centriole; f, flagellum within cytoplasmic canal; arrows, striated rootlet. Note how the two cells are associated at their anterior tips. Scale bar ¼ 0.5 lm. REPRODUCTIVE MORPHOLOGY OF BRITTANICHTHYS 27 Fig. 7. TEMs through successively more posterior transverse sections through mature spermatozoa from testis of aquarium specimen of Brittanichthys axelrodi, SL 27.8 mm. A: Section near anterior tip of spermatozoon showing dense material (d) that appears to be continuous with the striated rootlet; n, nucleus. Scale bar ¼ 0.5 lm. B: Section through proximal centriole (pc); n, nucleus. Scale bar ¼ 0.5 lm. C: Section through distal centriole (dc); n, nucleus. Scale bar ¼ 0.5 lm. D: Section through initial portion of axoneme of flagellum (f); n, nucleus. Scale bar ¼ 0.2 lm. E: Section through more posterior section through cell showing more flattened nucleus (n) and flagellum (f) within cytoplasmic collar. Scale bar ¼ 0.5 lm. F: Section through three mitochondria (m) showing an oblique cut of the cytoplasmic collar containing the flagellum (f). Scale bar ¼ 0.5 lm. G: Section through flagellum (f) still located within cytoplasmic collar. Scale bar ¼ 0.2 lm. H: section through free flagellum (f) and posterior end of flagellum (arrow) showing unpaired microtubules. Scale bar ¼ 0.2 lm. yond together with the cytoplasmic collar that contains the flagellum (Fig. 7F). This would be equivalent to the midpiece region. Beyond the mitochondria, the cytoplasmic collar continues for about 0.75 lm (Fig. 7G) before the single flagellum leaves this organelle (Fig. 7H). The single flagellum lacks fins and has electron lucent central microtubules along its entire length. In the anterior portion, all microtubules of the peripheral doublets of the axoneme are electron lucent (Fig. 7D,E), whereas more posteriorly the A-tubules are electron dense (Fig. 7G,H). The total length of the flagellum is 37 lm. No accessory microtubules were observed in any part of the cell. Spermatozeugmata Within the testes, sperm are organized into discrete packets. Because these packets lack any distinct cellular or acellular covering, they are considered to be spermatozeugmata (Jamieson, 1978; Grier, 1981). Intact spermatozeugmata were found only in two of the sexually mature male specimens studied, suggesting that some type of cyclicity occurs in the testis. Each spermatozeugma is lanceolate in shape when sectioned mid-sagitally (Fig. 3D,F). PASþ material, which comprises the elongate core of each spermatozeugma, is also located among the spermatozoa of the packet (Fig. 3F). Each spermatoJournal of Morphology DOI 10.1002/jmor 28 R. JAVONILLO ET AL. mature female appeared to be closed to the exterior with a membrane across it (Fig. 9A). Male gonopores appeared to open to the exterior. Serial frontal sections of one mature male reveals that the extreme end of the gonopore appears to exit from a slightly raised area (compare Fig. 9C,D). Also, bands of skeletal muscle attach near this area. Perhaps this raised area serves as a genital papilla to aid in sperm transfer to the female. DISCUSSION Fig. 8. Tangential section through anterior region of mature spermatozoon from testis of aquarium specimen of Brittanichthys axelrodi, SL 27.8 mm, showing radiating striated rootlet (st) associated with ventral region of nucleus (n). TEM. arrow, cytoplasmic canal. Scale bar ¼ 0.25 lm. Although insemination has been demonstrated in Brittanichthys axelrodi and over 60 other characid species (Burns and Weitzman, 2005), the precise method of sperm transfer to the female is unknown. zeugma appears to be made up of pairs of spermatozoa that are joined at their tips, with the tips embedded in the PASþ core of the packet. The flagella of these spermatozoa cover the outside of each spermatozeugma (Fig. 3F). On many of the TEM’s analyzed, two sperm cells were often associated at their tips (Fig. 6). In addition, in one testis that appears to be in a postreproductive state, spermatozoa are being engulfed by the epithelium of the coiled posterior region, where many sperm cell pairs joined at their tips are evident (Fig. 3E). Thus, it appears that the joining of two sperm cells at their anterior tips may be the initial stage of spermatozeugma formation. Because fully formed spermatozeugmata were only found within the lumina of the coiled posterior testis region, it appears that this is the prime area for packet formation (Fig. 3D). The light PASþ staining of the abundant cytoplasm of the epithelial cells lining this region suggests that these cells may be the source of the PASþ material found within the spermatozeugmata. Positive staining with PAS indicates the presence of glycosaminoglycans (mucopolysaccharides) in the material that may serve as a binder for the cells comprising the sperm packet. No spermatozeugmata were available for either SEM or TEM. Relatively intact spermatozeugmata were observed within the ovarian cavity of one female (Fig. 3A). Gonopore Areas Because the mechanism of sperm transfer is currently unknown for any inseminating characid fish, we sectioned the gonopore regions of both mature males and females to see if any differences could be noted. In both sexes, the gonopore opens posterior to the anus (Fig. 9A,B), with the urinary pore having a separate opening posterior to the gonopore. No urogenital sinus is present. The gonopore of one Journal of Morphology DOI 10.1002/jmor Fig. 9. Sections through gonopore regions of mature specimens of Brittanichthys axelrodi. LM. Scale bar ¼ 200 lm. A: Mid-sagittal section through gonopore region of mature female, USNM 221991, SL 22.3 mm, showing anal opening (a) and gonopore (arrow) that appears to be covered by a thin layer of tissue. B: Mid-sagittal section through gonopore region of mature male aquarium specimen, SL 23.0 mm, showing intestine near anal (a) area and gonopore (arrow). C: Frontal section through gonopore region of mature male aquarium specimen, SL 22.2 mm, showing anal area (a), gonopore area (arrow), and urinary pore (arrowhead). D: Slightly more ventral section through same specimen as C showing anal area (a) and gonopore (arrow) on a raised ridge of tissue. REPRODUCTIVE MORPHOLOGY OF BRITTANICHTHYS Mating, when insemination undoubtedly occurs, is generally a very rapid event in these quickly moving species, so detailed observations are difficult (Nelson, 1964). It was found that removal of the region of the anal fin bearing the hooks was the only intervention that prevented successful insemination in Corynopoma riisei (Kutaygil, 1959), suggesting that fin-ray hooks may be important in other inseminating species as well. Based on observations of mating Corynopoma riisei in aquaria, it was suggested that the male forms a pocket with its anal fin to enclose the female gonopore and facilitate sperm transfer (Richter, 1986). It is thought that the hooks on fin rays help to maintain close contact between partners during mating (Wiley and Collette, 1970). With respect to Characidae it is noteworthy that only within the lineage diagnosed by the presence of hooks is insemination also found. This lineage includes the clade diagnosed by lack of supraoritals, which in turn includes the group containing incertae sedis genera such as Brittanichthys (Malabarba and Weitzman, 2003; Weitzman et al., 2005). Because no observations of mating B. axelrodi are available, the function of the curved, hooked caudal-fin ray is also unknown. Males of most inseminating teleosts exhibit some form of intromittent organ. Modified anal fins, papillae, palps, and penes facilitate the transfer of sperm in these species (Meisner, 2005). Although no distinct intromittent organ was seen in Brittanichthys axelrodi, the slightly raised area around the exit of the male gonopore may serve that function to some degree. The extensive skeletal muscle in this area may also have some function during intromission, perhaps by allowing some manipulation of this region or even causing eversion of the genital area. The morphology of the gonopore area of B. axelrodi is comparable to that found in the goodeids Goodea atripinnis and Characodon lateralis. Males of these species lack the gonopodia distinctive of cyprinodontoids, yet still inseminate their mates by presumably contracting muscle masses around the urogential area and ejecting a jet of spermatophores (encapsulated sperm packets; Nelson, 1975). Successful insemination requires mates to be in close proximity such that spermatozoa either travel through the aquatic medium or are transferred directly from the male into the reproductive tract of the female. Characids and other ostariophysan fishes have long been known to secrete chemicals such as alarm substance (Pfeiffer, 1962). Brittanichthys and other characids may also secrete other types of chemical signals that mediate reproductive behaviors and therefore affect insemination. Although they are inseminating, male B. axelrodi develop neither gill glands (Burns and Weitzman, 1996; Bushmann et al., 2002) nor structures in the caudal fin that are thought to produce pheromones in other male characids (e.g., Glandulocaudinae and Stevardiinae sensu Weitzman et al., 2005). We also found no evidence of 29 club cells in association with either the anal-fin rays or caudal-fin rays of B. axelrodi. In other characids, including externally-fertilizing species and inseminating members of the Glandulocaudinae and Stevardiinae, club cells, which may produce pheromones, are found in association with hooks on the anal-fin rays (Weitzman et al., 2005). Division of each testis of Brittanichthys axelrodi into three distinct regions is thus far unique among characid fishes. Although never observed in externally fertilizing characids, some inseminating species have a large, aspermatogenic posterior testis that serves for sperm storage and, in some, sperm packaging (Burns et al., 1995; Weitzman et al., 2005). These storage areas may occupy 15.4–92.6% of total testis area (mid-sagittal sections; Burns et al., 1995) and they resemble histologically the middle region of the B. axelrodi testis, which occupies 32% of testis area. No characid species thus far analyzed was found to have a region similar to the coiled posterior portion of the B. axelrodi testis, which occupies a further 17% of testis area. Thus, B. axelrodi devotes a total of 49% of the area of each testis to nonsperm producing functions which appear to relate to the inseminating habit (see below). This suggests a strong selective pressure for insemination. In addition to increasing the probability of fertilization of the eggs, insemination may also allow the temporal and spatial separation of mating and oviposition, an advantage in environments that experience distinct rainy and dry seasons (Azevedo et al., 2000; Burns and Weitzman, 2005). The finding of spermatozoa in a B. axelrodi ovary containing only previtellogenic oocytes further supports this idea. Additionally, given that many inseminating species live in highly acidic waters of low conductivity, insemination may protect the sperm from osmotic shock and the detrimental effects of acidity (Burns and Weitzman, 2005). Within the midtestis region of Brittanichthys axelrodi some coalescence of spermatozoa does take place. However, it is the coiled posterior region that appears to be responsible for the final packaging of the sperm into distinct, unencapsulated packets (spermatozeugmata). In species of the subfamily Glandulocaudinae (sensu Weitzman et al., 2005), sperm packaging occurs within the posterior storage area (Pecio and Rafiński, 1994, 1999; Burns et al., 1995; Pecio et al., 2001), whereas in those members of the subfamily Stevardiinae (sensu Weitzman et al., 2005) that produce spermatozeugmata, packaging takes place within the spermatocysts in the anterior spermatogenic region (Burns et al., 1995; Pecio et al., 2005). Sperm packaging may increase the probability of insemination by maintaining high sperm densities during transfer to the female (Ginzburg, 1968). A photograph of a male Corynopoma riisei, an inseminating species that does not appear to produce distinct sperm packets (Burns et al., 1995), shows what is reported Journal of Morphology DOI 10.1002/jmor 30 R. JAVONILLO ET AL. to be a ‘‘cloud’’ of sperm near the male immediately after spawning (Richter, 1986), suggesting that appreciable loss of sperm to the surrounding water may occur during sperm transfer in this species. Production of compact sperm packets may decrease such sperm loss to the environment. The overall morphology of the spermatozeugmata of B. axelrodi most closely resembles that of the packets produced by species in the stevardiine genus Xenurobrycon, where the sperm heads are also oriented inwardly and bound to a PASþ secretion, with flagella of those spermatozoa covering the outside of the packet (Burns et al., 1995; Burns and Weitzman, 2005). Spermatozeugmata were not present in all of the testes that we examined. Production of spermatozeugmata may therefore exhibit some form of cyclicity, perhaps based on the season. Unlike the spermatozoa with spherical nuclei that are typical of externally-fertilizing species (‘‘aquasperm’’ of Jamieson, 1991), the nuclei of spermatozoa from Brittanichthys axelrodi are elongate and flattened in some areas. The sperm of the inseminating stevardiine characids Corynopoma riisei, Diapoma speculiferum, Scopaeocharax rhinodus, and Tyttocharax tambopatensis and the inseminating glandulocaudine Mimagoniates barberi also have elongate nuclei with flat surfaces (Burns et al., 1998; Pecio et al., 2005). Though elongate the sperm nuclei of inseminating cheirodontine characids (e.g., Compsura gorgonae) are more rounded in transverse section (Burns et al., 1997). Of the 60þ species of Ostariophysi known to be inseminating, only six characid species (Attonitus irisae, Knodus sp., Kolpotocheirodon theloura, Planaltina britskii, Planaltina glandipedis, and Planaltina myersi) have sperm cells with spherical or slightly ovoid nuclei (Burns and Weitzman, 2005). Thus there may be a selective advantage for production of elongate sperm nuclei in inseminating species. Possible advantages were discussed by Burns and Weitzman (2005). First, more spermatozoa with elongate nuclei may be able to enter the female gonopore at a time compared to aquasperm. Second, a narrow cell is streamlined, which may facilitate travel through the narrow passages and secretions of the female reproductive tract. Third, elongation of the nuclei may increase the number of cells that can align and clump as they travel. Fourth, elongation of the nucleus may create a greater tendency toward forward movement (i.e., toward the female gonopore) and therefore result in decreased loss of sperm to the aquatic environment. The arrangement of centrioles in a spermatozoon is yet another ultrastructural character that displays interspecific variation. The long axes of centrioles may be parallel to one another as in Macropsobrycon uruguayanae (Burns and Weitzman, 2005), oblique as in Tyttocharax tambopatensis and Scopaeocharax rhinodus (Pecio et al., 2005), or perpendicular as in Diapoma (Burns et al., 1998), Pseudocorynopoma Journal of Morphology DOI 10.1002/jmor doriae (Burns and Weitzman, 2005), and Brittanichthys axelrodi. The functional significance of centriole arrangement is currently unclear. A striated rootlet, like that in the anterior portion of Brittanichthys axelrodi sperm, is not apparent in the sperm of all inseminating characids. However, striated rootlets are apparent in spermatozoa from another inseminating characid, Macropsobrycon uruguayanae (Burns et al., 1998), and in all families of elopomorph fishes except Muraenidae (Jamieson, 1991). Striated rootlets are also present in the spermatozoa of many invertebrates (e.g., Platyhelminthes; Justine, 1995), as well as in the spermatids of Postorchigenes (Platyhelminthes: Trematoda: Lecithodendriidae; Gracenea et al., 1997). Work by Yang et al. (2002, 2005) on striated rootlets of ciliated cells demonstrated that rootlets are critical structures for support of cilia and are composed of a large protein called rootletin. Although rootlets were not required for ciliary motility, their presence increased survival and enabled significantly more normal ciliated cells to withstand mechanical stress compared to mutant ciliated cells that lacked rootlets (Yang et al., 2005). In a spermatozoon, the rootlet may therefore act to stabilize the cell during periods of flagellar swimming. Successful propulsion of spermatozoa through the circuitous cavities of the ovary that may contain highly viscous fluids may benefit from robust anchorage at the base of the flagellum via a large striated rootlet. The diameter of sperm mitochondria of four externally fertilizing characid species available in our laboratory (Serrapinnus kriegi, S. calliurus, Aphyocharax anisitsi, and Knodus sp.) ranged from 0.2– 0.6 lm. The approximate diameters of the sperm mitochondria of Brittanichthys axelrodi were 0.6 lm, the upper end of this range. Having relatively large mitochondria may be advantageous in satisfying increased energy demands for sperm storage and/or movement of spermatozoa through the tortuous pathways of the ovary (Burns et al., 1995; Burns and Weitzman, 2005). Given that it is likely that eggs are fertilized either internally or close to the time of egg-laying, our finding of sperm in ovaries containing only previtellogenic oocytes suggests that some degree of sperm storage occurs in Brittanichthys axelrodi. There were no obvious structures for sperm storage in ovaries of female B. axelrodi, however. Other inseminating characids that appear to be capable of sperm storage also lack obvious ovarian structures for sperm storage. Female Corynopoma can deposit fertile eggs after being separated from males for seven months (Nelson, 1964). Burns and Weitzman (2005) also reported that female Corynopoma riisei, Mimagoniates barberi, M. lateralis, M. microlepis, Tyttocharax tambopatensis, and Pseudocorynopoma doriae are capable of sperm storage. The nature of the relationship between sperm morphology and long-term viability within a female remains to be demonstrated. REPRODUCTIVE MORPHOLOGY OF BRITTANICHTHYS The flagella of Brittanichthys axelrodi sperm do not possess axonemal fins, which is consistent with the observations of sperm ultrastructure from other otophysan fishes (Jamieson, 1991). The only exception known to date in Characiformes is Chilodus punctatus of the family Anostomidae (Pecio, 2003). Sperm storage areas are found in the testes of Brittanichthys axelrodi, as well as all Glandulocaudinae and Stevardiinae. Elongate binding collars containing flagella are found in mature spermatozoa of B. axelrodi, as well as the stevardiine genera Corynopoma, Diapoma, and Pseudocorynopoma (Burns et al., 1998) and Gephyrocharax and Chrysobrycon (unpublished data), and the cheirodontines Odontostilbe dialeptura and O. mitoptera (unpublished data). Elongate binding collars are present in the spermatids of the glandulocaudine genus Mimagoniates (Pecio and Rafiński, 1994, 1999; unpublished data) and the stevardiine genera Tyttocharax and Scopaeocharax (Pecio et al., 2005); however, all but the anterior portions of the collars degenerate before the mature spermatozoa are released into the sperm ducts. The overall morphology and location of the mitochondria in B. axelrodi, mainly posterior to the nucleus, more closely resembles the condition observed in spermatozoa of inseminating cheirodontines (Burns et al., 1998; unpublished data) than in inseminating glandulocaudines and stevardiines, where mitochondria are found along the elongate nuclei (Pecio and Rafiński, 1994, 1999; Burns et al., 1998; Burns and Weitzman, 2005). In addition to B. axelrodi, the inseminating cheirodontine Macropsobrycon uruguayanae also has a striated rootlet attached to the distal centriole (Burns et al., 1998) of the spermatozoon. Brittanichthys axelrodi exhibit a unique combination of characters that has led to numerous discussions of relationship (e.g., Géry, 1965; Weitzman and Fink, 1985; Weitzman and Menezes, 1998; Malabarba and Weitzman, 1999), but at this time we hesitate to suggest what if any known genus is sister to Brittanichthys. It is clear from the above that B. axelrodi possesses characters that are similar to those of inseminating species from several lineages. Insemination may therefore have multiple evolutionary origins within Characidae. Like correlations among traits of the reproductive system, evolutionary miniaturization may entail a suite of convergent changes in other aspects of morphology. Forthcoming phylogenetic analyses will incorporate other morphological characters to help us ascertain the close relatives of Brittanichthys, the number of times insemination evolved in the family, and whether similarities among B. axelrodi and other inseminating characids are homologous. ACKNOWLEDGMENTS The authors thank Labbish Chao for providing aquarium specimens. We also are grateful to Michael Goulding (Rainforest Alliance, USA) for sup- 31 plying the museum specimens. Fernando Alvarez Padilla was most helpful in providing assistance with the scanning electron microscope. LITERATURE CITED Azevedo MA, Malabarba LR, Fialho CB. 2000. Reproductive biology of the inseminating glandulocaudine Diapoma speculiferum Cope (Teleostei: Characidae). Copeia 2000:983–989. Burns JR, Weitzman SH. 1996. Novel gill-derived gland in the male swordtail characin. Corynopoma riisei (Teleostei: Characidae: Glandulocaudinae). Copeia 1996:627–633. Burns JR, Weitzman SH. 2005. Insemination in ostariophysan fishes. In: Uribe MC, Grier HJ, editors. Viviparous Fishes. Homestead, FL: New Life Publications. pp 107–134. Burns JR, Weitzman SH, Grier HJ, Menezes NA. 1995. Internal fertilization, testis and sperm morphology in glandulocaudine fishes (Teleostei: Characidae: Glandulocaudinae). J Morphol 224: 131–145. Burns JR, Weitzman SH, Malabarba LR. 1997. Insemination in eight species of cheirodontine fishes (Teleostei: Characidae: Chierodontinae). Copeia 1997:433–438. Burns JR, Weitzman SH, Lange KR, Malabarba LR. 1998. Sperm ultrastructure in characid fishes (Teleostei: Ostariophysi). In: Malabarba LR, Reis RE, Vari RP, Lucena ZMS, Lucena CAS, editors. Phylogeny and Classification of Neotropical Fishes. Porto Alegre, Brazil: Museu de Ciências e Tecnologia, PUCRS. pp 235–244. Burns JR, Weitzman SH, Malabarba LR, Meisner AD. 2000. Sperm modifications in inseminating ostariophysan fishes, with new documentation of inseminating species. In: Norberg B, Kjesbu OS, Taranger GL, Andersson E, Stefansson SO, editors. Proceedings of the Sixth International Symposium on the Reproductive Physiology of Fish. Bergen, Norway: Institute of Marine Research and University of Bergen. p 255. Bushmann PJ, Burns JR, Weitzman SH. 2002. Gill-derived glands in glandulocaudine fishes (Teleostei: Characidae: Glandulocaudinae). J Morphol 253:187–195. Géry JR. 1965. A new genus from Brazil-Brittanichthys, a new, sexually-dimorphic characid genus with peculiar caudal ornament, from the Rio Negro, Brazil, with a discussion of certain cheirodontin genera and description of two new species, B. axelrodi and B. myersi. Trop Fish Hobbyist 13:13–24, 61–69. Ginzburg AS. 1968. Fertilization in Fishes and the Problem of Polyspermy. Moscow: Academy of Science USSR. 366 pp. Gracenea M, Ferrer JR, González-Moreno O, Trullols M. 1997. Ultrastructural study of spermatogenesis and the spermatozoon in Postorchigenes gymnesicus (Trematoda, Lecithodendriidae). J Morphol 234:223–232. Grier HJ. 1981. Cellular organization of the testis and spermatogenesis in fishes. Am Zool 21:345–357. Ito S, Karnovsky MJ. 1968. Formaldehyde glutaraldehyde fixatives containing trinitro compounds. J Cell Biol 36:168. Jamieson BGM. 1978. Rhyacodrilus arthingtonae, a new species of freshwater oligochaete (Tubificidae) from North Stradbroke Island, Queensland. Proc R Soc Qld 89:39–43, pls 5–6. Jamieson BGM. 1991. Fish evolution and systematics: Evidence from spermatozoa. Cambridge, UK: Cambridge University Press. 319 pp. Justine J-L. 1995. Spermatozoal ultrastructure and phylogeny in the parasitic platyhelminthes. In: Jamieson BGM, Ausio J, Justine J-L, editors. Advances in Spermatozoal Phylogeny and Taxonomy, Vol 166. Paris: Mém Mus Natn Hist Nat. pp 55–86. Kutaygil N. 1959. Insemination, sexual differentiation and secondary sex characters in Stevardia albipinnis Gill. Hidriobiol Univ Istanbul Fen Fak Mecumuasi Ser B 24:93–128. Lundberg JG. 1993. African-South American freshwater fish clades and continental drift: Problems with a paradigm. In: Goldblatt P, editor. Biological Relationships Between Africa and South America. New Haven Connecticut: Yale University Press. pp 156–199. Journal of Morphology DOI 10.1002/jmor 32 R. JAVONILLO ET AL. Malabarba LR, Weitzman SH. 1999. A new genus and species of South American fishes (Teleostei: Characidae: Cheirodontinae) with a derived caudal fin, including comments about inseminating cheirodontines. Proc Biol Soc Wash 112:410–432. Malabarba LR, Weitzman SH. 2003. Description of a new genus with six new species from southern Brazil, Uruguay and Argentina, with a discussion of a putative characid clade (Teleostei: Characiformes: Characidae). Comun Mus Ciênc Tecnol PUCRS Sér Zool 16:67–151. Meisner AD. 2005. Male modifications associated with insemination in teleosts. In: Uribe MC, Grier HJ, editors. Viviparous Fishes. Homestead, FL: New Life Publications. pp 167–192. Munehara H, Takano K, Koya Y. 1989. Internal gametic association and external fertilization in the elkhorn sculpin, Alcichthys alcicornis. Copeia 1989:673–678. Nelson GG. 1975. Anatomy of the male urogenital organs of Goodea atripinnis and Characodon lateralis (Atheriniformes: Cyprinodontoidei), and G. atripinnis courtship. Copeia 1975: 475–482. Nelson K. 1964. Behavior and morphology in the glandulocaudine fishes (Ostariophysi, Characidae). Univ Calif Pub Zool 75: 59–152. Ortı́ G. 1997. Radiation of characiform fishes: Evidence from mitochondrial and nuclear DNA sequences. In: Kocher TD, Stepien CA, editors. Molecular Systematics of Fishes. San Diego: Academic Press. pp 219–243. Pecio A. 2003. Spermiogenesis and fine structure of the spermatozoon in a headstander, Chilodus punctatus (Teleostei, Characiformes, Anostomidae). Folia Biol (Kraków) 51:55–62. Pecio A, Rafiński J. 1994. Structure of the testes, spermatozoa and spermatozeugmata of Mimagoniates barberi Regan, 1907 (Teleostei: Characidae), an internally fertilizing, oviparous fish. Acta Zool 75:179–185. Pecio A, Rafiński J. 1999. Spermiogenesis in Mimagoniates barberi (Teleostei: Ostariophysi: Characidae), an oviparous, internally fertilizing fish. Acta Zool 80:35–45. Pecio A, Lahnsteiner F, Rafiński J. 2001. Ultrastructure of the epithelial cells in the aspermatogenic part of the testis in Mimagoniates barberi (Teleostei: Characidae: Glandulocaudinae) and the role of their secretions in spermatozeugmata formation. Ann Anat 183:427–435. Journal of Morphology DOI 10.1002/jmor Pecio A, Burns JR, Weitzman SH. 2005. Sperm and spermatozeugma ultrastructure in the inseminating species Tyttocharax cochui, T. tambopatensis, and Scopaeocharax rhinodus (Pisces: Teleostei: Characidae: Glandulocaudinae: Xenurobryconini). J Morphol 263:216–226. Pfeiffer W. 1962. The fright reaction of fish. Biol Rev 37:495–511. Richter H-J. 1986. The swordtail characin, Corynopoma riisei. Trop Fish Hobbyist 35:44–49. Schreibman MP. 1964. Studies on the pituitary gland of Xiphophorus maculatus (the platyfish). Zoologica 49:217– 243. Weitzman SH, Fink SV. 1985. Xenurobryconin phylogeny and putative pheromone pumps in glandulocaudine fishes (Teleostei: Characidae). Smithson Contrib Zool 421:1–121. Weitzman SH, Menezes NA. 1998. Relationships of the tribes and genera of the Glandulocaudinae (Ostariophysi: Characiformes: Characidae) with a description of a new genus Chrysobrycon. In: Malabarba LR, Reis RE, Vari RP, Lucena ZMS, Lucena CAS, editors. Phylogeny and Classification of Neotropical Fishes. Porto Alegre, Brazil: Museu de Ciências e Tecnologia, PUCRS. pp 159–180. Weitzman SH, Menezes NA, Evers H-G, Burns JR. 2005. Putative relationships among inseminating and externally fertilizing characids, with a description of a new genus and species of Brazilian inseminating fish bearing an anal-fin gland in males (Characiformes: Characidae). Neotrop Ichthyol 3:329– 360. Wiley ML, Collette BB. 1970. Breeding tubercles and contact organs in fishes: Their occurrence, structure, and significance. Bull Am Mus Nat Hist 143:143–216. Yang J, Liu X, Yue G, Adamian M, Bulgakov O, Li T. 2002. Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet. J Cell Biol 159:431–440. Yang J, Gao J, Adamian M, Wen X-H, Pawlyk B, Zhang L, Sanderson MJ, Zuo J, Makino CL, Li T. 2005. The ciliary rootlet maintains long-term stability of sensory cilia. Mol Cell Biol 25:4129–4137. Zanata AM, Vari RP. 2005. The family Alestidae (Ostariophysi, Characiformes): A phylogenetic analysis of a trans-Atlantic clade. Zool J Linn Soc 145:1–144.