Hiroyuki Munehara

Sympatric speciation in a genus of marine reef fishes

Sympatric speciation has been contentious since its inception, yet is increasingly recognized as important based on accumulating theoretical and empirical support. Here, we present a compelling case of sympatric speciation in a taxon of marine reef fishes using a comparative and mechanistic approach. Hexagrammos otakii and H. agrammus occur in sympatry throughout their ranges. Molecular sequence data from six loci, with complete sampling of the genus, support monophyly of these sister species. Although hybridization occurrs frequently with an allopatric congener in an area of slight distributional overlap, we found no F1 hybrids between the focal sympatric taxa throughout their coextensive ranges. We present genetic evidence for complete reproductive isolation based on SNP analysis of 382 individuals indicating fixed polymorphisms, with no shared haplotypes or genotypes, between sympatric species. To address questions of speciation, we take a mechanistic approach and directly compare aspects of reproductive isolation between allopatric and sympatric taxa both in nature and in the laboratory. We conclude that the buildup of reproductive isolation is strikingly different in sympatric vs. allopatric taxa, consistent with theoretical predictions. Lab reared hybrids from allopatric species crosses exhibit severe fitness effects in the F1 or backcross generation. No intrinsic fitness effects are observed in F1 hybrids from sympatric species pairs, however these treatments exhibited reduced fertilization success and complete pre‐mating isolation is implied in nature because F1 hybrid adults do not occur. Our study addresses limitations of previous studies and supports new criteria for inferring sympatric speciation.

Internal Gametic Association and External Fertilization in the Elkhorn Sculpin, Alcichthys alcicornis

Morphological studies have demonstrated that fertilization in the copulating cottid species, the elkhorn sculpin (Alcichthys alcicornis), in which fertilization previously has been assumed to occur within the females genital tract, occurs externally. In the ovarian cavity of impregnated female fish, a number of spermatozoa were found to have entered the micropyle and a fertilizing spermatozoon had reached the lower end of the micropylar canal, but penetration of the spermatozoon into the ooplasm did not occur until after exposure to seawater. Based on these results and past studies, it is concluded that other cottids now regarded as internally-fertilizing species may also exhibit external fertilization with internal insemination.

Effects of extracellular environments on the motility of spermatozoa in several marine sculpins with internal gametic association

Abstract 1. 1. The motility of spermatozoa in several marine sculpins, which exhibit a special reproductive manner of the internal gametic association, was measured in various artificial solutions, ovarian fluid and seminal plasma. 2. 2. In the elkhorn sculpin, Alcichthys alcicornis, spermatozoa showed high motility in solutions of 300 to 400 mOsm/kg, containing sodium ion, with pH higher than 7.5, which coincided with the nature of ovarian fluid of the fish. 3. 3. Spermatozoa of sunrise sculpin, Pseudoblennius cottoides, and elegant sculpin, Bero elegans, were motile at osmolalities isotonic to the ovarian fluid but not at osmolalities higher than 500 and 800 mOsm/kg, respectively, indicating that the gametic association in these fish is carried out exclusively in their ovaries. 4. 4. Spermatozoa of littledragon sculpin, Blepsias cirrhosus, were motile at osmolalities higher than 300 mOsm/kg, but not in sea water, suggesting an internal gametic association to occur in this species of sculpins. 5. 5. The results indicate that spermatozoa of the marine sculpins with the internal gametic association show their motility in environmental conditions appropriate to respective reproductive modes.

Evidence for divergent natural selection of a Lake Tanganyika cichlid inferred from repeated radiations in body size

Divergent natural selection is thought to play a vital role in speciation, but clear, measurable examples from nature are still few. Among the many possible sources of divergent natural selection, predation pressure may be important because predators are ubiquitous in food webs. Here, we show evidence for divergent natural selection in a Lake Tanganyika cichlid, Telmatochromis temporalis, which uses burrows under stones or empty snail shells as shelters. This species contains normal and dwarf morphs at several localities. The normal morph inhabits rocky shorelines, whereas the dwarf morph invariably inhabits shell beds, where empty snail shells densely cover the lake bottom. Genetic evidence suggested that the dwarf morph evolved independently from the normal morph at two areas, and morphological analysis and evaluation of habitat structure revealed that the body sizes of morphs closely matched the available shelter sizes in their habitats. These findings suggest that the two morphs repeatedly evolved through divergent natural selection associated with the strategy for sheltering from predators.

Sperm transfer during copulation in the marine sculpin Hemitripterus villosus (Pisces : Scorpaeniformes) by means of a retractable genital duct and ovarian secretion in females

Among the diverse repertoire of reproductive behaviors shown by sculpins, copulating behavior is known to occur in some species (Breder and Rosen, 1966). Most of these copulating sculpins transfer sperm by using a huge flexible penis (Morris, 1952; Ragland and Fischer, 1987; Munehara, 1988). However, the little dragon sculpin Blepsias cirrhosus, whose penis is too diminutive to be noticed, and the Japanese sea raven Hemitripterus villosus, which does not possess such a functional genital organ, also have been inferred to copulate on the basis of histological observations of eggs collected from, their ovarian cavities (Munehara et al., 1991). It is not clear, however, by what means the sperm of these fishes are actually transferred from the male into the females ovarian tract. The present study, therefore, was undertaken to clarify this issue by observing copulating and spawning behavior of H. villosus in an aquarium.

Microsatellite markers and multiple paternity in a paternal care fish, Hexagrammos otakii

Abstract The fertilization success of sneaking behavior in a paternal care fish, Hexagrammos otakii, was estimated using microsatellites as genetic markers. Sneaking behavior was observed using a video camera placed near the nest of a breeding territorial male. Two egg masses fertilized by different patterns of sperm emission by the territorial male and sneaker males were examined for paternity discrimination. When a sneaker emitted a large amount of sperm after sperm emission by the territorial male, the sneaker fertilized more eggs than the territorial male. When the territorial male and a sneaker alternately emitted semen, the territorial male fertilized the most eggs. These results suggest that both the amount of emitted sperm and the timing of sperm emission are important factors in sperm competition.

Spawning behavior and interspecific breeding in three japanese greenlings (hexagrammidae)

The spawing behavior ofHexagrammos otakii. H. octogrammus andH. agrammus was observed in two different regions of northern Japan using underwater video cameras placed near nests guarded by males. The spawning behavior of the three species consisted of similar patterns, although body size and nuptial coloration and nest location of territorial maleH. otakii differed from those of the other twoHexagrammos species. The courtship display of territorial males in each species involved rushing, butting and undulation of the trunk. When a female entered the nest, the male leaned his head on the future spawning bed in the nest and spasmodically undulated his trunk. The female that responded to the courtship laid her eggs within the seaweed bed. The territorial male then passed over the eggs, touching his genital pore to the egg mass, and released sperm. Sneaking by other males was frequently observed following the sperm emission. In both regions, females ofH. octogrammus andH. agrammus commonly responded to courtship of maleH. otakii and mated with them, but not vice versa. Possble reasons for the natural occurrence of such unidirectional hybridization are discussed.

Fertilization environment of the non-copulating marine sculpin, Hemilepidotus gilberti

To clarify the extracellular environment for external fertilization in the non-copulating marine sculpin Hemilepidotus gilberti, sperm motility was measured in NaCl, KCl, mannitol solutions, seawater, and ovarian fluid. Spermatozoa of H. gilberti actively moved in seminal plasma the moment they were removed from the genital papilla. Spermatozoa showed higher motility in NaCl solution at osmolalities between 300–400 mOsmol kg-1. In KCl and in mannitol solutions, spermatozoa actively moved at osmolalities between 500 and 800 mOsmol kg-1, and at osmolality 300 mOsmol kg -1, respectively. The ovarian fluid was a transparent and viscous gelatinous material, rich in sodium with an osmolality of 340 mOsmol kg-1. Sperm motility in the ovarian fluid lasted more than 90 min, which was six times longer than in seawater. This sperm motility under conditions isotonic to body fluid is similar to that of copulating marine sculpins rather than to other non-copulating marine fishes. In addition, eggs of H. gilberti could be fertilized in the ovarian fluid. This suggests that external fertilization takes place under physiological conditions similar to the internal conditions of the ovary provided by the ovarian fluid, which isolates the eggs from sea water for several hours after spawning. This manner of fertilization is thought to be one of the evolutionary pre-adaptations allowing copulation among marine sculpins.

Non-copulatory spawning and female participation during early egg care in a marine sculpinHemilepidotus gilberti

The reproductive behavior of a marine sculpin,Hemilepidotus gilberti, was observed in the aquarium. The behavioral sequence comprised fight for terriotory, courtship and fight for females, spawning and caring for the egg mass. Females very slowly deposited eggs enclosed with viscous ovarian fluid onto the substrate. Almost all the eggs contained in the ovary were released in a single spawning, its completion requiring 21–22 min. Territorial males emitted of spawning. Sneaking by neighboring males followed subsequently. This was the first observation of non-copulatory spawning among marine sculpins. Females cared for the eggs for severl days after spawning, and then male alone care continued until hatching (47–49 days after spawning). Participation by females in egg caring is likely to be caused by their spawning only once during the spawning season and the consequent lack of urgency for post-spawning feeding.

Living on the wedge: female control of paternity in a cooperatively polyandrous cichlid

Theories suggest that, in cooperatively breeding species, female control over paternity and reproductive output may affect male reproductive skew and group stability. Female paternity control may come about through cryptic female choice or female reproductive behaviour, but experimental studies are scarce. Here, we show a new form of female paternity control in a cooperatively polyandrous cichlid fish (Julidochromis transcriptus), in which females prefer wedge-shaped nesting sites. Wedge-shaped sites allowed females to manipulate the siring success of the group member males by spawning the clutch at the spot where the large males were just able to enter and fertilize the outer part of the clutch. Small males fertilized the inner part of the clutch, protected from the large aggressive males, leading to low male reproductive skew. Small males provided more brood care than large males. Multiple paternity induced both males to provide brood care and reduced female brood care accordingly. This is, to our knowledge, the first documented case in a species with external fertilization showing female mating behaviour leading to multiple male paternity and increased male brood care as a result.