Study Summary

For the purpose of unraveling the evolution of the mode of fertilization from external to internal, I examined the breeding ecology and behavioral ecology of the salamander Hynobius nigrescens (Hynobiidae) with external fertilization, in terms of both field observations and various laboratory experiments, and compared them with those of other urodeles accomplishing internal fertilization, which correspond to approximately 90% of the Caudata members composed of more than 700 species (Breeding Pond in March; Breeding Pond in July; Terraria; Voucher Specimens; Aquatic Larvae (Onychodactylus japonicus); 1985-2015 in Iwamuro-mura, Niigata Prefecture, Japan).

The main results were:

(A) I discovered reproductive phenomena, specific to hynobiid salamanders:

(1) the absence of "spermiation" (i.e., sperm release from the testes into the vasa deferentia) during fall, even after the completion of spermiogenesis (Vasa Deferentia Fluctuation 1; in urodeles with internal fertilization, spermiation occurs during both fall and spring);
(2) rapid, exhaustive spermiation for "spawning" (i.e., extrusion of spermatozoa occurs simultaneously with oviposition) during early spring (Male Genital System; in urodeles with internal fertilization, gradual, sporadic spermiation occurs for spermatophore formation);
(3) a single, huge peak in plasma androgen concentration associated with spermiation (in urodeles with internal fertilization, two peaks in plasma androgen concentration are associated with spermiation);
(4) exhaustive secretion by epithelial cells of the vasa deferentia after the completion of spermiation only during the initial stages of breeding, and resultant activation of sperm stocked in the lumina of the vasa (Vasa Deferentia Fluctuation 2; in urodeles with internal fertilization, epithelial secretion by the vasa deferentia occurs throughout the sperm storage in the vasa);
(5) no spermatogenic wave throughout the year, but synchronous spermatogenesis in all the maturing lobules of the testis (Testicular Lobules; in urodeles with internal fertilization, spermatogenic wave is discernible along the entire length of the testis);
(6) sexually active males lack sperm within their testes during spawning (in urodeles with internal fertilization, recovery from sperm depletion depends on testicular sperm); and
(7) a tubercle at the anterior angle of the vent (like a clitoris) is evident from August-April (Sexual Dimorphism in Vents; this structure is unknown in other families).

(1) the absence of a second oogenic phase during early spring (in urodeles with internal fertilization, first and second oogenic phases occur during fall and spring, respectively);
(2) after entering a breeding pond, ovulation is rapid and complete, resulting in the formation of a pair of egg sacs before spawning (Female Body Shape; Ovulation & Egg Sac Formation; Ventral Gland Secretion; in urodeles with internal fertilization, gradual, sporadic ovulation and oviposition occur);
(3) a protrusive, short peak in plasma progesterone concentration associated with ovulation during spring (in urodeles with internal fertilization, several month-long periods of elevated progesterone are associated with ovulation during spring-summer);
(4) August ovarian eggs colored mint green prior to the deposition of cortical melanin pigments (Female Genital System; Color Changes in Ovary; internal-fertilizing urodeles have cream-yellowish eggs with or without melanin, but this feature probably is common to almost every amphibian);
(5) a single peak in plasma estradiol-17 beta concentration during October is associated with the subsequent development of sex accessory structures (in urodeles with internal fertilization, a second peak in plasma estradiol-17 beta concentration during spring coincides both with the development of secondary sexual characteristics and with the second oogenic phase);
(6) terminology of "dilatable ovisacs" where egg sac formation occurs (Ovulation & Egg Sac Formation; this structure is unknown in other families); and
(7) a positive correlation between the increase in gonadotrope cells in the pars distalis and the development of ovaries from July-January, suggesting the absence of a second oogenic phase during early spring (Gonadotropes; there is no comparable study because of using a monoclonal antibody against bullfrog luteinizing hormone beta-subunit).

(B) I pointed out the remarkable swelling of the head of the male during the aquatic phase, being characteristic of hynobiids (Typical Aquatic-Phase Male), which is nothing comparable to other families among urodeles (i.e., they have a seasonally constant head), and proposed a "scramble competition" hypothesis regarding egg sac monopolization for the explanation of increased head widths (Spawning Behavior).

(C) Concerning sperm competition and sexual selection in males, I proposed two hypotheses: a "sperm depletion" hypothesis that a mating ball formed by scramble competitors around a pair of egg sacs is a conditional tactic for depleting sperm of a monopolist; and a "poor mate" hypothesis that the existence of males with disadvantageous behaviors and low fertilization success can explain the absence of positive assortative monopoly of egg sacs by body size during scramble competition.

(D) I proposed a "wandering-behavior hypothesis" instead of the prolactin-based "water-drive theory" for the explanation of breeding immigration of urodeles (Prolactin & Cutaneous Changes).

(E) I presented a regression equation for estimating dates of oviposition by applying a continuous increase in egg sac mass with increased days after insemination (Egg Sacs; Egg Sac Mass; Roadside Wetland; Fall Pond Basin in Sarukura; Hanging Egg Sacs; Bloody Egg Sacs)

Y =0.002212*EXP(1.825*LN(X))
where X = egg sac mass (g), and Y = days after insemination.
SD =SQRT(0.001296*(0.002212*LN(X))^2+1.513*10^(-7))*EXP(1.825*LN(X)).

If we have an egg sac of 60 g and one of 100 g, we can estimate from this equation that the eggs were deposited 3.89 days before (SD = 0.89) and 9.88 days before (SD = 2.39), respectively.

As a trial for grasping the perspective of aquatic ecology (i.e., reproductive behavior) of the family Hynobiidae, I picked up ones that I could not ignore (as a positive meaning or a negative meaning) from the past related references and discussed what the behavioral science tells us correctly or incorrectly (Hasumi, 2015). Especially, I emphasized that "courtship behavior" and "parental care", whispered plausibly in this family, have not yet been demonstrated. In addition, I pointed out that the American and European researchers have a strong tendency to cite references written in Japanese by reading the English abstract alone in these references, without reading the Japanese text, for their own convenience (without checking the truth of the thing). (doi: 10.1007/s10211-015-0214-z)

I have been working mainly on terrestrial ecology of Salamandrella keyserlingii on and after 1995.

In S. keyserlingii inhabiting Kushiro Marsh, I provided quantitative evidence for fall immigration (during September) toward terrestrial hibernacula near a breeding fen (Study Area (Wetland); Tools for Toe-Clipping), unrelated to mating (their breeding season is April-May; Air Temperatures; for comparison, breeding immigration occurs during fall in other migratory salamanders with internal fertilization, i.e., ambystomatids and salamandrids). I documented seasonal color changes in the throat region of adult males during the terrestrial phase (Terrestrial-Phase Male) from translucent gray (June-August), through translucent-opaque white (September-October), to brilliant or pale yellow (April-May). Moreover, I took censuses for population dynamics using a mark-recapture method and clarified immigratory orientation of each individual, sex ratios during the breeding and nonbreeding seasons, habitat use of dry forest and wet marsh regions, summer home range, and so on (I categorized captured individuals into five classes: adult males, adult females, unsexed individuals, juveniles, and metamorphs). I further demonstrated that relatively large areas (1.5 x 1.5 m), having a water depth of around 40 cm, were utilized as breeding habitats by this species (Fen Topography). Based on an ontogenetic perspective on the evolution of sexual size dimorphism (SSD), I tested whether SSD occurred before sexual maturity also in ectothermic vertebrates (i.e., fishes, amphibians, and reptiles) with indeterminate growth. I determined whether females' larger mean body size was caused by a difference in (1) age at maturity, (2) age-specific size, (3) divergent growth prior to maturity, or (4) selection on post-maturational growth in S. keyserlingii, using 555 independent data points from skeletochronological studies. Femele-larger SSD occurred as a result of females reaching maturity at 3-4 years of age, a year later than males. However, SSD was highly detected before maturity, and females after maturity continued to grow and resulted in larger asymptotic size than males. I therefore concluded that when determining SSD the difference in mean adult-body size results from the difference in age-specific size. Also, I suggested that secondary sexual characteristics (i.e., body mass, head width, and tail height) that were greater in breeding males than in females developed to resolve intersexual ontogenetic conflict by allowing small-sized males to swell their whole body temporarily during the aquatic phase as much as large-sized females. (doi: 10.1007/s11692-010-9080-9)

I determined some refuge characteristics of S. keyserlingii during daytime in summer at Shaamar, Mongolia. Subterranean burrows having mean depth of 15.4 cm were utilized temporarily with proportional habitat use of 0.704. Mean temperature was lower in burrows/under logs (16.22 C) than in ambient air (26.70 C), and mean relative humidity was higher in burrows/under logs (85.54%) than in ambient air (48.33%). Mean illumination intensity was 27.0 lx in burrows/under logs and 17,188.1 lx on the surface out of refugia. Mean soil pH was 7.52 beneath salamanders in refugia. Burrow use was reported for the first time in the family Hynobiidae. (doi: 10.1643/CP-07-237)

To test the Bergmann's rule generalized in endotherms (i.e., the body size of animals is predicted to be larger in higher latitudes or cooler environments) in debatable ectotherms, I determined geographic variation in age and body size of S. keyserlingii distributed widely in Eurasia (from 43 to 69oN; mean yearly air temperature: from 8 to -15oC; 27 populations) using skeletochronology. Both sexes had sexual maturity 2-3 years later, lower growth coefficient, and smaller body size in cool Darhadyn than in warm Kushiro. However, constant metamorphic size around 30 mm snout-vent length between populations suggested the existence of genetic determinants. There was an intraspecific tendency to decrease maximum longevity and body size with increased latitude within the geographic range of this species, but the size increased from 57oN and -7oC. This pattern does not follow the intraspecific extension of Bergmann's rule and may follow the converse of Terentjev's optimum rule (i.e., a rule formulated to be an inverted-U shaped curve) between decreased temperature and increased body size. (doi: 10.1007/s13127-012-0091-5)

At Darhadyn Wetland, Mongolia, I searched for individuals of S. keyserlingii hidden under downed logs of a single conifer species (Larix sibirica) and evaluated their microhabitat environments. I then analyzed physical parameters of the downed logs with 2 generalized log-linear models (Poisson regression and negative binomial regression models). I discovered 100% use of downed logs by individuals as refuges during daytime in summer. Only 4 of 125 recapture events showed movements of salamanders between logs (96.8 % had site tenacity to the same downed logs), and numerous individuals were aggregating cohabitually in the refugia (maximum number of 9 was captured per day from the same log). These results suggest that complex sociality may be evolved also in the primitive extant salamander family that practices external fertilization. I provided conservation measures such that total number of individuals captured per log over the course of the study had a positive relationship to log decaying class in ascending order, and thus the retention of more decaying downed logs is important for the conservation of this species. (doi: 10.1007/s00300-013-1443-0)

S. keyserlingii (1995-1997 in Kushiro Marsh (Otanoshike Wetland; Kottaro Marsh), Kushiro-shi, Hokkaido Prefecture, Japan: breeding habitat requirements, phenology of spatial distributions, and demography in conjunction with age estimation by skeletochronology; 2004-2005 in Darhadyn Wetland (Favorite Shot), Mongolia (Russian Jeeps; Rubber Boat; White House Hotel; Screw-propelled Plane; Logs Harboring Siberian Salamanders; Iron Ovens; Camp Site; Sight-seeing Areas of Ulaanbaatar; Pass Ovo (Ovoo, Oboo); Mongolian Nomad; Edelweiss; Box Zartsaa; Deer Paintings; Frost; Edelweiss Hotel; Traffic Circumstances; Natural History Museum; 2005 Membership of Darhadyn Wetland Project; Terrestrial Female; Nylon Mesh Trap & Aquatic Animals; Body Size Measurements; Frost during August; Rainbow; Twilight; Aero Mongolia; Ovoo Worship in 2005; Golio; Camels; Lota lota; Taimen 1; Taimen 2; Yak; Outdoor Water Closet; Ovoo of Fountains; Renchinlhumbe; Sauna; Yurt; Handcrafted Baby Car; New Records; Siberian Salamander (Male); Rings in Pond; Running Horses; Ferris Wheel; Stone Circle; Poster Presentation; Mongolian & Russian Ladies; Japanese, Mongolian & Russian Gentlemen; PIT-tags): microhabitats as a terrestrial environment); and 2005 in Shaamar, Mongolia (Burrow Use by Salamanders; Rudimentary Fifth Toes): burrow use)

I have been working on breeding ecology of Hynobius kimurae since 1986 in Hakuba-mura, Nagano Prefecture, Japan, together with Mr. Masaichi Kakegawa.

In H. kimurae, I investigated breeding ecology, including fall immigration toward aquatic hibernacula and timing of oviposition. This species immigrates from land to water for aquatic hibernation annually during fall (Kakegawa et al., 2017: doi: 10.1016/j.jcz.2017.04.003; Kakegawa and Hasumi, 2017: doi: 10.1002/rra.3162). Body shape of either sex changed firstly during fall after entering the water for aquatic hibernation and secondly during spring for breeding after awaking from overwintering (Kakegawa et al., 2017). This difference suggests that these two-step changes during a prolonged period (fall-spring) are concentrated in a short period of aquatic-breeding activity during spring in other migratory salamanders with terrestrial hibernation. In general, within-year timing of breeding in amphibians is more advanced when water temperatures increase earlier than usual and is more delayed when water temperatures increase later than usual. However, oviposition in H. kimurae was more delayed with earlier exposure to high temperatures during spring. Oviposition was earlier with experiencing higher temperatures during hibernation. Days from submergence to oviposition were sensitive to cumulative temperatures. A rate in increase in days staying in the water was constant within a population. These results suggest that a biological clock exits for days from submergence to oviposition, which could allow survival in situations with either warmer or cooler environments (Kakegawa and Hasumi, 2017).

Photographs related to H. kimurae: Egg Sacs; Two Pairs of Egg Sacs; Development of Embryos in Egg Sacs; Development of Embryos in Egg Sacs 2; Terrestrial Male; Postbreeding Male; Oviposition Site & Egg Sacs; Overwintering Male in Torrent; Breeding Torrent; Overwintering Stream; Slopes for Migration & Wandering; Animal Tracks to Site; Fall Surveys; Juvenile & Snail; Descending Immigrant & Slope; Juvenile Toad; Orange Toad (Throat Region); Orange Toad (Whole Body); Overwintering Locations; Compact pH-meter; Egg Sac Mass in Torrent; Overwintering Larvae

I conducted other studies in the following six species:

(1) Hynobius lichenatus (1983-1985 in 19 localities (Roadside Sites of Oviposition; Pond-Type Sites of Oviposition; Major Breeding Population; Egg Sacs) within its geographic range, Tohoku and Kita-Kanto districts, Japan; Body Sizes of Sympatric Species; morphocline and geographic variation in taxonomic characters such as external features (Tail Vertebrae; Trunk Vertebrae), teeth (Shapes of Prevomerine Teeth), pedes (Pes Composition; Pes Variation), skulls (Skull Variation), and so on),

(2) Hynobius hidamontanus (Forest Floor (Habitat); New Oviposition Site; Breeding Habitat & Egg Sacs; Oviposition Site; Major Breeding Habitat; Major Breeding Habitat 2; Snow in Hakuba Area; Female Toad Preyed; Wetland (Extinct Population?); Breeding Fountain; Swamp (Wetland); Wetland above Swamp; Breeding Pond in Wetland; Breeding Male & Egg Sacs; Aquatic-Phase Male & Female; Throat White Patch; Aquatic-Phase Female; Female Cloacal Opening; Rudimentary Fifth Toes; Egg Sacs; Egg Sacs with Striae; Egg Sacs at Open Area; Egg Sacs in Rapid Stream; Open Field for Breeding; Newt Defensive Posture; Overwintering Larva; Swamp (Wetland) with Two Hynobiids Mingled; Terrestrial Male (Onychodactylus japonicus); Communal Breeding Locations; 1990-Present in Hakuba-mura, Nagano Prefecture, Japan; growth, sexual maturity, and reproductive cycle in juvenile, subadult, and adult individuals; distributional survey on occasion),

(3) Rana pirica (1997 in Bekambeushi Marsh (Bear's Tracks), Akkeshi-cho, Hokkaido Prefecture, Japan; amphibian fauna and Habitats for breeding and larval growth), and

(4-6) Pseudepidalea raddei (formerly Bufo), Hyla japonica, and Rana amurensis (2005 in Shaamar, Selenge Province, Mongolia (Shaamar Members & Russian Jeep; Pond in Shaamar; Camp Site in Shaamar; Male Toad; Nylon Mesh Trap & Metamorphosing Juvenile; Examination Tools; Examination Tools 2; Hedgehog; Campfire; Log House in Shaamar; Ovoo near Shaamar); amphibian and other aquatic animal species diversity). (doi: 10.1007/s10201-010-0319-z)

When I initiated to study the breeding ecology of the salamander Hynobius nigrescens (around 1985), no basic data existed on the reproduction of hynobiid salamanders (at that time, one of my study goals was catching up on the Japanese herpetology). Up to date, considerable amounts of data on this matter have been accumulated (its basis has already been established!!). I affirm now is a time for developing any studies concerning hynobiids.

*Handling of Salamandrella keyserlingii is regulated by the Government of Kushiro-shi, Hokkaido Prefecture, Japan. Handling of Hynobius hidamontanus is also regulated by the Government of Hakuba-mura, Nagano Prefecture, Japan, and recently by the Government of Nagano Prefecture, Japan (by means of the Regulations of Conservation of Rare Wildlife, issued on 22 March 2005). All studies on the two hynobiids were conducted under the permission authorized by these governments.
Copyright 2002-2017 Masato Hasumi, Dr. Sci. All rights reserved.
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