A robust system of hybridogenesis that increases genetic variability and promotes evolutionary succession in greenlings (Teleostei: Hexagrammidae, genus Hexagrammos): Regeneration of a new hemiclonal lineage.

Unisexual hybrids that reproduce either clonally or hemiclonally are considered to be evolutionarily short-lived as they lack the ability to reduce deleterious mutations and increase genetic diversity. In the greenling (Teleostei: Hexagrammidae, genus Hexagrammos), unisexual hybrids that produce haploid eggs containing only the H. octogrammus (maternal species) genome generate hemiclonal offspring by fertilization with haploid sperm of H. agrammus (paternal species). When hemiclonal hybrids are backcrossed to a male of the maternal species, the offspring (BC-Hoc) are phenotypically similar to the maternal species and produce recombinant gametes through conventional meiosis. BC-Hoc (recombinant generation) individuals referred to as carriers harbor the genetic factor for hybridogenesis, thereby facilitating the production of new hemiclonal lineages through hybridization. Previous studies based on field research have suggested that the carriers produced by two-way backcrossing (mating pattern in which hemiclonal hybrids are backcrossed with both parental species) may overcome the evolutionary dead end imposed by the lack of recombination. The present study verified this hypothesis by regenerating a newly hemiclonal lineage through artificial hybridization. To clarify the genetic mode of hybrids produced by crosses between BC-Hoc and Hag, mature eggs were obtained from 16 individuals and fertilized with either Hag or Hoc sperm. Hybridogenesis was confirmed in one of the 16 individuals. Based on the low occurrence rate, these findings suggest that hemiclonal lineages can be regenerated, and that the hemiclonal factors are likely distributed across multiple genes on different chromosomes. The findings provide important evidence for the retention of a robust system for increasing genetic variability and maintaining evolutionary succession in unisexual hybrids that reproduce hemiclonally.

Fertilization modes and the evolution of sperm characteristics in marine fishes: Paired comparisons of externally and internally fertilizing species.

Fertilization mode may affect sperm characteristics, such as morphology, velocity, and motility. However, there is little information on how fertilization mode affects sperm evolution because several factors (e.g., sperm competition) are intricately intertwined when phylogenetically distant species are compared. Here, we investigated sperm characteristics by comparing seven externally and four internally fertilizing marine fishes from three different groups containing close relatives, considering sperm competition levels. The sperm head was significantly slenderer in internal fertilizers than in external fertilizers, suggesting that a slender head is advantageous for swimming in viscous ovarian fluid or in narrow spaces of the ovary. In addition, sperm motility differed between external and internal fertilizers; sperm of external fertilizers were only motile in seawater, whereas sperm of internal fertilizers were only motile in an isotonic solution. These results suggest that sperm motility was adapted according to fertilization mode. By contrast, total sperm length and sperm velocity were not associated with fertilization mode, perhaps because of the different levels of sperm competition. Relative testis mass (an index of sperm competition level) was positively correlated with sperm velocity and negatively correlated with the ratio of sperm head length to total sperm length. These findings suggest that species with higher levels of sperm competition have faster sperm with longer flagella relative to the head length. These results contradict the previous assumption that the evolution of internal fertilization increases the total sperm length. In addition, copulatory behavior with internal insemination may involve a large genital morphology, but this is not essential in fish, suggesting the existence of various sperm transfer methods. Although the power of our analyses is not strong because of the limited number of species, we propose a new scenario of sperm evolution in which internal fertilization would increase sperm head length, but not total sperm length, and change sperm motility.

The first record of egg masses in tunicates deposited by the snubnose sculpin, Orthonopias triacis, from the Northeastern Pacific: evidence for convergent evolution of an unusual reproductive strategy.

In this study, the authors report the first record of egg masses deposited in solitary tunicates by the snubnose sculpin, Orthonopias triacis, from the Northeastern Pacific. Four egg masses were discovered in the tunicate Ascidia ceratodes that were genetically determined to be O. triacis. Female O. triacis had long ovipositors that allow deposition of their eggs inside the atrium of the tunicates. A comparison of host-tunicate size with ovipositor length of sculpins from the Northwestern Pacific, including the genera Furcina and Pseudoblennius, revealed that O. triacis had shorter ovipositors and spawned in the atrium of smaller species of tunicates. Ancestral state reconstruction of egg deposition in solitary tunicates using 1.86Mbp RNAseq data of 20 sculpin species from Northeastern and Northwestern Pacific revealed that this unusual spawning behaviour may have evolved convergently in different species occurring in the Northeastern vs. the Northwestern Pacific.

Dispersion and settlement of two sympatric sculpins of the genus Gymnocanthus.

Larval dispersion rather than adult migration generally leads to the worldwide expansion of fishes. Species of the genus Gymnocanthus have expanded geographically while undergoing allopatric speciation. Of this genus, while Gymnocanthus tricuspis inhabits the Arctic Ocean and surrounding area, G. herzensteini and G. intermedius occur around northern Japan. Larval early life histories of G. herzensteini and G. intermedius from northern Japan and G. tricuspis from Unalaska Island were investigated to estimate their dispersal potential during larval stages. The larval and juvenile abundances of G. herzensteini and G. intermedius were highest in May in shallow sandy bottoms below 7 m in depth, and the body sizes were 9.7-34.6 mm notochord length (NL) and/or standard length (SL) in G. herzensteini and 8.4-46.7 mm NL and/or SL in G. intermedius. Two egg masses of G. tricuspis (1.92 ± 0.08 mm in diameter) and hatched larvae (6.20 ± 0.19 mm NL) were collected in March. Compared with other sculpins in previous studies, the body sizes of G. herzensteini and G. intermedius at hatch are large and at settlement are small, while both hatch and settlement sizes of G. tricuspis are much larger. Counting micro-increments between the hatch check and settlement marks in G. herzensteini and G. intermedius demonstrated that the pelagic larval durations for 2 weeks with an immature body suggests that these species cannot disperse widely during the pelagic phase, while pelagic larvae of Arctic species such as G. tricuspis with long pelagic larval durations could disperse.

Unisexual hybrids break through an evolutionary dead end by two-way backcrossing.

Unisexual vertebrates (i.e., those produced through clonal or hemiclonal reproduction) are typically incapable of purging deleterious mutations, and, as a result, are considered short-lived in evolutionary terms. In hemiclonal reproduction (hybridogenesis), one parental genome is eliminated during oogenesis, producing haploid eggs containing the genome of a single parent. Hemiclonal hybrids are usually produced by backcrossing hemiclonal hybrids with males of the paternal species. When hemiclonal hybrids from a genus of greenlings (Hexagrammos) are crossed with males of the maternal species, the progeny are phenotypically similar to the maternal species and produce recombinant gametes by regular meiosis. The present study was conducted to determine if the hemiclonal genome is returned to the gene pool of the maternal species in the wild. Using a specific cytogenetic marker to discriminate between such progeny and the maternal species, we observed that Hexagrammos hybrids mated with maternal and paternal ancestors at the same frequency. This two-way backcrossing in which clonal genomes are returned to the gene pool where they can undergo recombination plays an important role in increasing the genetic variability of the hemiclonal genome and reducing the extinction risk. In this way, hybrid lineages may have survived longer than predicted through occasional recombinant generation.

Freeze Tolerance in Sculpins (Pisces; Cottoidea) Inhabiting North Pacific and Arctic Oceans: Antifreeze Activity and Gene Sequences of the Antifreeze Protein.

Many marine species inhabiting icy seawater produce antifreeze proteins (AFPs) to prevent their body fluids from freezing. The sculpin species of the superfamily Cottoidea are widely found from the Arctic to southern hemisphere, some of which are known to express AFP. Here we clarified DNA sequence encoding type I AFP for 3 species of 2 families (Cottidae and Agonidae) belonging to Cottoidea. We also examined antifreeze activity for 3 families and 32 species of Cottoidea (Cottidae, Agonidae, and Rhamphocottidae). These fishes were collected in 2013⁻2015 from the Arctic Ocean, Alaska, Japan. We could identify 8 distinct DNA sequences exhibiting a high similarity to those reported for Myoxocephalus species, suggesting that Cottidae and Agonidae share the same DNA sequence encoding type I AFP. Among the 3 families, Rhamphocottidae that experience a warm current did not show antifreeze activity. The species inhabiting the Arctic Ocean and Northern Japan that often covered with ice floe showed high activity, while those inhabiting Alaska, Southern Japan with a warm current showed low/no activity. These results suggest that Cottoidea acquires type I AFP gene before dividing into Cottidae and Agonidae, and have adapted to each location with optimal antifreeze activity level.

Karyological evidence of hybridogenesis in Greenlings (Teleostei: Hexagrammidae).

Two types of natural hybrids were discovered in populations of three Hexagrammos species (Teleostei: Hexagrammidae) distributed off the southern coast of Hokkaido in the North Pacific Ocean. Both hybrids reproduce by hybridogenesis, in which the maternal haploid genome is transmitted to offspring without recombination and the paternal haploid genome is eliminated during gametogenesis. While natural hybrids are unisexual and reproduce hemiclonally by backcrossing with the paternal species (BC-P), artificial F1-hybrids between the pure species produce recombinant gametes. Thus, despite having the same genome composition, the natural hybrids and the F1-hybrids are not genetically identical. Here, to clarify the differences between both hybrids, we examined the karyotypes of the three Hexagrammos species, their natural hybrids, the artificial F1-hybrids, and several backcrosses. Artificial F1-hybrids have karyotypes and chromosome numbers that are intermediate between those of the parental species. Conversely, the natural hybrids differed from F1-hybrids by having several large metacentric chromosomes and microchromosomes. Since the entire maternal haploid genome is inherited by the natural hybrids, maternal backcrosses (BC-M) between natural hybrids and males of the maternal species (H. octogrammus; Hoc) have a hemiclonal Hoc genome with large chromosomes from the mother and a normal Hoc genome from the father. However, the large chromosomes disappear in offspring of BC-M, probably due to fissuring during gametogenesis. Similarly, microsatellite DNA analysis revealed that chromosomes of BC-M undergo recombination. These findings suggest that genetic factors associated with hemiclonal reproduction may be located on the large metacentric chromosomes of natural hybrids.

Origins of two hemiclonal hybrids among three Hexagrammos species (Teleostei: Hexagrammidae): genetic diversification through host switching.

Two natural, hemiclonal hybrid strains were discovered in three Hexagrammos species. The natural hybrids, all of which were females that produced haploid eggs containing only the Hexagrammos octogrammus genome (maternal ancestor; hereafter Hoc), generated F1 hybrid-type offspring by fertilization with haploid sperm of Hexagrammos agrammus or Hexagrammos otakii (paternal species; Hag and Hot, respectively). This study was performed to clarify the extent of diversification between the two hybrids and the maternal ancestor. Genealogical analysis using mtDNA revealed that all 38 Hoc/Hot hybrids formed a branch (Branch I) with 18 of the 33 Hoc/Hag hybrids. No haplotype sharing was observed with the maternal ancestor. Further, microsatellite DNA analysis suggested that the members of Branch I shared the same hemiclonal genome set. The results suggested that Hoc/Hot hybrids originated by anomalous hybridization, or "host switching," between Hoc/Hag and Hot, and not from interspecific hybridization between Hoc and Hot. The remaining 9 of 11 Hoc/Hag haplotypes and all of the 27 Hoc haplotypes were mixed within the genealogical tree, as if they had originated from multiple mutations. However, Hoc/Hag could also mate with Hoc. Although offspring from this host switch (Backcross-Hoc) have the same genome as normal Hoc, a part of their genome retains genetic factors capable of producing hemiclones. Consequently, when a descendant of a BC-Hoc hybrid mates with Hag males, a new hemiclone lineage will arise. Multiple haplotype revival through host switching from a single mutation in hybrids is another possible hypothesis for the observed mixing of Hoc/Hag haplotypes within the mtDNA genealogical tree.

Complete mitochondrial genome of the Antlered sculpin Enophrys diceraus (Scorpaeniformes, Cottidae).

The complete mitochondrial genome (mitogenome) sequence of Enophrys diceraus (Cottidae) was determined for the first time by primer walking methods. It was 16,976 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region (CR). Within the CR, the extended termination-associated sequence, the central conserved sequence block (CSB-F, CSB-E, CSB-D, CSB-C, CSB-B, CSB-A) and the conserved sequence block (CSB-1, CSB-2, CSB-3) were identified. This first mitogenome information among marine sculpins is expected to enrich the knowledge on phylogenetic studies of this taxon.

Spermatangium formation and sperm discharge in the Japanese pygmy squid Idiosepius paradoxus.

In cephalopods, sperm discharge is an important event not only for sperm transfer but also influencing sperm storage capacity of attached spermatangia (everted spermatophores). To investigate sperm discharge from spermatangia and the condition of naturally attached spermatangia in Japanese pygmy squid (Idiosepius paradoxus) we (i) investigated the morphology of spermatophores and spermatangia, and the process of spermatophore evagination and sperm discharge from spermatangia obtained in vitro; (ii) observed spermatangia that were naturally attached to female squids at 6, 12, 18, 24 and 48 h after copulation to investigate alterations in naturally attached spermatangia with time. The spermatophore of I. paradoxus is slender and cylindrical and consists of a sperm mass, a cement body and an ejaculatory apparatus, which is similar to those of loliginid squids. The spermatangium is fishhook-shaped, its distal end being open and narrow. After the spermatangium is formed, the sperm mass gradually moves to the open end of the spermatangium, from where sperm are released. Sperm discharge is a rapid process immediately after the beginning of sperm release, but within 5 min changes to an intermittent release of sperm. Although the volume of residual spermatozoa differed among spermatangia that were naturally attached to a single individual, the probability that spermatangia would be empty increased with time. Most naturally attached spermatangia discharged almost all of their spermatozoa within 24h after copulation, and no spermatangia were attached to females 48 h after copulation. These results suggest that sperm transfer from the spermatangium to the seminal receptacle must occur within 24h, and that the spermatangium functions as a transient sperm storage organ in pygmy squids.

Accumulation of organotins in wharf roach (Ligia exotica Roux) and its ability to serve as a biomonitoring species for coastal pollution.

In this study, we measured the accumulation of tributyltin (TBT) in wharf roach (Ligia exotica Roux) and examined the species' ability to be used for TBT biomonitoring in coastal environments. In an exposure test, wharf roach were exposed to TBT via diet for 2d. TBT was accumulated in wharf roach, and its metabolite dibutyltin was detected. The concentrations of these compounds gradually decreased during the depuration period, but they were still detected 12d after exposure ceased (TBT 290±140ng/g; dibutyltin 1280±430ng/g). The biological half-life of TBT in wharf roach was estimated to be about 4d. In a field study conducted in 2011-2012, wharf roach were collected from 15 coastal sites in Japan and 3 sites in Manado, Indonesia. TBT was detected in both Japanese and Indonesian samples. The highest concentration of TBT was found in wharf roach collected at Bitung ferry port, Manado (57.9±16.5ng/g), which is close to a shipyard, and the highest concentration at a Japanese site was 12.3±6.2ng/g. Thus, we were able to detect organotins in the coastal environments by testing wharf roach, suggesting that L. exotica might serve as a good bioindicator for monitoring organotin pollution.

Two patterns of parasitic male mating behaviors and their reproductive success in Japanese medaka, Oryzias latipes.

We found two patterns of parasitic mating behaviors by male in Japanese medaka, Oryzias latipes, in the spawning by one female and two males in the aquarium condition. In the first type of parasitic mating behavior, the unpaired male would rush toward the closely adhering female and paired male, and simultaneously perform emission behavior with close adhesion to the female (simultaneous emission). The second type of parasitic mating behavior was that the unpaired male would rush toward the female with spawned eggs hanging down from the genital pore several seconds after pair-spawning, perform emission behavior with close adhesion to the female without any prespawning behavior (post-spawn emission). The frequencies of "simultaneous emission" and "post-spawn emission" were 20 (19.6%) and 18 cases (17.7%), respectively, in 102 trials, and the average reproductive successes about 41% and 20%, respectively. The reproductive success of simultaneous emission was not correlated with the timing and duration of spawning behaviors of the paired/ unpaired male, while the reproductive success of post-spawn emission significantly decreased as the length of time during which the paired male adhered to female increased. Observations of two consecutive spawning behaviors using same combination of two males revealed that paired males always tend to become paired males. However, the role of both males was occasionally reversed, indicating flexibility in mating tactics.

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 occurs frequently with an allopatric congener in an area of slight distributional overlap, we found no F(1) 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 F(1) or backcross generation. No intrinsic fitness effects are observed in F(1) hybrids from sympatric species pairs, however these treatments exhibited reduced fertilization success and complete pre-mating isolation is implied in nature because F(1) hybrid adults do not occur. Our study addresses limitations of previous studies and supports new criteria for inferring sympatric speciation.

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.

Alternative male mating tactics of the substrate brooding cichlid Telmatochromis temporalis in Lake Tanganyika.

Telmatochromis temporalis is a bi-parental substrate brooding cichlid endemic to Lake Tanganyika. Paired males were always larger than their mates and had territories around nests against conspecific males. However, males smaller than the paired females were found in 18% of the nests. Here we report a reproductive tactic of these small males. The small males had as heavy gonads as paired males, and the gonad somatic index (GSI) of the small males was much higher than that of the latter. The examinations of the paternity and maternity using microsatellite-DNA as a genetic marker revealed that the small males were not genetically related to the pair members, and sired some young in 3 of 5 nests. These small males did not guard the broods, suggesting that they are likely to perform reproductive parasitism as sneakers. Paired males could not enter their spawning nests due to their large size, which made it difficult to chase out sneakers once they entered the nest. Some males as small as the sneakers were found outside the territories of paired males, and their gonads were quite small. Circumstantial evidence suggests that small males have two alternative investment patterns: investing in gonad to be sneakers, and investing in growth to probably be territorial males.

Ultrastructural observations of eu- and paraspermiogenesis in the cottid fish Hemilepidotus gilberti (Teleostei: Scorpaeniformes: Cottidae).

The developmental process of eu- and paraspermatozoa in the cottid fish, Hemilepidotus gilberti, was observed by electron microscopy. Euspermatozoa of H. gilberti consist of a thin disk-like sperm head (about 3 microm in length), a short middle piece, and a long flagellum, but lack an acrosome. On the other hand, during spermiogenesis, aberrant spermatids, rich in cytoplasm and possessing binuclei, develop into cysts containing spermatids. The developing aberrant spermatids connect with normal spermatids and euspermatozoa by intercellular bridges. The early phase of chromatin condensation in aberrant spermatids is almost identical to that in normal spermatids, but the nuclei in the later phase develop into a mass of highly electron-dense globules. Since the aberrant spermatids complete karyokinesis but not cytokinesis at telophase of the second meiotic division, they are considered to develop into hyperpyrenic cells due to incomplete cytokinesis of the second meiotic division. These spermatids are oval in shape (5-7 microm in diameter) and lack a flagellum. The aberrant spermatids of H. gilberti are shed along with euspermatozoa and amount to about 50% of semen in volume. Judging from their form and developmental process, aberrant spermatids produced in H. gilberti are considered hyperpyrenic paraspermatozoa.

Sperm storage and motility in the ovary of the marine sculpin Alcichthys alcicornis (Teleostei: Scorpaeniformes), with internal gametic association.

Elkhorn sculpin, Alcichthys alcicornis, is a marine teleost with a unique reproductive mode called "internal gametic association," in which sperm introduced into the ovary by copulation enter the micropylar canal of ovulated eggs in the ovarian cavity, but actual sperm-egg fusion does not occur until the eggs have been released into sea water. It is also known that this fish is a multiple spawner, which spawns at intervals of a few days for one month, and the sperm introduced into the ovary at the beginning of the spawning season retain their fertilizability for the entire period. To clarify how the fertilizability of sperm is maintained internally, the ultrastructure of sperm, the morphological characteristics related to sperm storage in the ovary, and the characteristics of sperm motility were investigated. Mature sperm generally have the normal form of teleost sperm, devoid of acrosomal structures. However, it was found that the midpiece is comparatively elongated and has a compact aggregation of many small-size mitochondria. The intraovarian sperm remained floating in the ovarian fluid throughout the spawning season. The sperm showed high motility in isotonic and weak alkaline solution, containing sodium ions, which was similar to the ovarian fluid of this fish. Sperm continued to move in artificial ovarian fluid for 7-14 days. Considering these results together, it is thought that the intraovarian sperm move throughout the spawning season due to the plentiful energy generated by the many mitochondria.

Sperm storage and degradation in the ovary of a marine copulating sculpin, Alcichthys alcicornis (Teleostei: Scorpaeniformes): Role of intercellular junctions between inner ovarian epithelial cells.

The morphology of female sperm storage during the spawning period and the morphology of sperm degradation after the spawning period were investigated by electron microscopy in a copulating teleost, Alcichthys alcicornis. The spermatozoa were maintained in the ovarian cavity, floating in the ovarian fluid during the spawning period. The spermatozoa then degenerated and were phagocytized by macrophages invading the ovarian cavity after the spawning period. In the ovary during the spawning period, horseradish peroxidase used as a tracer revealed tight junctional complexes connecting adjacent cells of the inner ovarian epithelia (ovarian wall epithelium and ovigerous lamella epithelium). This indicates that a compartmentalization of the ovarian cavity occurs during the spawning period. The junctional complexes were breached after the spawning period, as shown by the fact that horseradish peroxidase penetrated the ovarian cavity via the intercellular space between the adjoining ovigerous lamella epithelia. These results suggest that the spermatozoa in the ovarian cavity are isolated from the maternal immune system by the tight junctional complexes between the adjoining inner ovarian epithelia during the spawning period, and then are eliminated by immune cells following the breakdown of the junctional complexes after the spawning period. J. Morphol. 233:153-163, 1997. © 1997 Wiley-Liss, Inc.