ZOOLOGICAL SCIENCE 4: 159-166 (1987) © 1987 Zoological Society of Japan

Geographic Variation in the Tail of the Japanese Salamander, Hynobius lichenatus, with Special Reference to Taxonomic Bearing

MASATO HASUMI and HISAAKI IWASAWA

Biological Institute, Faculty of Science, Niigata University, Niigata 950-21, Japan

ABSTRACT—Tail vertebrae were observed radiographically in 283 males and 49 females of adult Hynobius lichenatus collected during the breeding season from 19 localities in northeastern Japan. The frequency of specimens with broken and regenerated tails was much higher when the sites of oviposition were in swifter flowing streams. The relative tail length of specimens with normal tails tended to be greater at higher latitude. It is difficult to distinguish externally most regenerated tails from normal tails. The dispersions of relative tail length, however, were remarkably greater in most localities when data from specimens with normal, questionable, and regenerated tails were used. It is, therefore, necessary to take notice of tail regeneration in the measurement of tail length for taxonomic purposes.

INTRODUCTION

Relative tail length, that is tail length/snout-vent length, is one of the diagnostic characters for study of intra- and interspecific variation in urodeles. Since tail-autotomization among salamanders is restricted to most plethodontids and some sala-mandrids [1], tail length of specimens with com­plete and unautotomized tails is used taxonomical-ly in these urodelen species [2, 3]. As far as we know, however, there are no taxonomic papers that take into consideration regeneration following tail breakage in other urodeles.

Hynobius is a genus of the family Hynobiidae that contains the most primitive living salamanders [4]. Hynobius lichenatus is widely distributed in northeastern Honshu, the mainland of Japan. Sato [5] reported that this species had considerable geographic variation in morphology, but he did not provide enough data. We reinvestigated the taxonomic characters of this species in the minutest detail, taking into account the various localities, and drew some new conclusions. Varia­tion in the length of the tail is dealt with in this paper.

MATERIALS AND METHODS

During the breeding seasons of 1983-1985, 283 adult males and 49 adult females of Hynobius lichenatus Boulenger were collected from sites of oviposition in nineteen localities in northeastern Honshu. Sample sites are shown in Table 1. Sample sites 16-19 nearly correspond to the southern limit of distribution for this species. The stream index for sites of oviposition, divided into five types, shows that the stream flows more rapidly at higher type numbers. Additionally, in sample site 8, salamanders were trapped in road­side ditches on their way to the natural site of oviposition.

As soon as possible after collection, animals were anesthetized with 0.01% p-aminobenzoic acid ethyl ester aq. and measured. All measure­ments were made to the nearest 0.1 mm with slide calipers. The specimens were then fixed in 10% formalin. The following morphological data were recorded: snout-vent length, measured from the tip of the snout to the posterior angle of the vent; tail length, from the posterior angle of the vent to the tip of the tail; and axilla-groin length, from the posterior angle of the forelimb to the anterior angle of the hindlimb. Proportions were repre­sented as relative to snout-vent length.

Radiographs of all specimens were taken using Accepted August 1, 1986 Received March 11, 1986

160 M. HASUMI AND H. IWASAWA

TABLE 1. Sample site, site of oviposition, stream index, and number of specimens used in this study

1)

2)

3)

4)

5)

6)

7)

8)

9)

10)

11)

12)

13)

14)

15)

16)

17)

18)

19)

Sample site (Altitude)

Maedanome, Goshogawara-shi Aomori Pref. (320 m)

Kudoji, Hirosaki-shi Aomori Pref. (240 m)

Mt. Ajara-yama, Ohwani-machi Aomori Pref. (340 m)

Mase-keikoku, Hachimori-machi Akita Pref. (50 m)

Natsuzaka, Takko-machi Aomori Pref. (300 m)

Kawamata, Tamayama-mura Iwate Pref. (180 m)

Hirukawa, Ohmagari-shi Akita Pref. (180 m)

Idosawa, Ichinoseki-shi Iwate Pref. (570 m)

Yamanome, Ichinoseki-shi Iwate Pref. (120 m)

Iragawa, Atsumi-machi Yamagata Pref. (100 m)

Hataya, Yamanobe-machi Yamagata Pref. (610 m)

Mt. Ninohji-dake, Shibata-shi Niigata Pref. (720 m)

Hibara, Kitashiobara-mura Fukushima Pref. (820 m)

Yutagami, Tagami-machi Niigata Pref. (80 m)

Kamijoh, Kamo-shi Niigata Pref. (40 m)

Tanne, Kashiwazaki-shi Niigata Pref. (160 m)

Mt. Atema-yama, Tohkamachi-shi Niigata Pref. (800 m)

Okushiobara, Shiobara-machi Tochigi Pref. (930 m)

Fujiwara, Minakami-machi Gunma Pref. (740 m)

Oviposition site and stream index

fountain, 2

fountain, 2

fountain, 2

roadside pool, 1

roadside fountain, 2

roadside stream, 3

fountain-flowing pond, 2

*

pool, 1

torrent, 5

fountain, 2

swamp stream, 4

fountain-flowing ditch, 3

torrent, 5

fountain-flowing pool, 2

torrent, 5

brook, 4

brook,4

fountain-flowing ditch, 2

No. of specimens

Male

2

11

3

12

15

18

34

13

5

7

5

12

9

44

17

10

9

9

48

Female

0

0

0

0

0

0

4

12

0

0

0

3

4

10

1

0

0

3

12

* See text.

SOFRON equipment (TYPE SRO-M50, SOKEN

CO., LTD., Tokyo). The tail vertebrae observed

were divided into four types as follows: a. exter­

nally broken tails, including obviously regenerated

tails, with tail vertebrae half-cut the same as the

broken parts; b. regenerated tails not distinguish­

able from normal tails externally but with partial

or incomplete tail vertebrae (Fig. IB, C); c.

questionable tails with tail vertebrae somewhat

abnormal but not partial; and d. normal tails with

tail vertebrae normal and complete (Fig. 1A).

The presence of correlation between two vari­

ables was tested with Spearman's rank correlation

coefficient. For statistical analysis of relative tail

length, data from the specimens having normal

tails (type d) were used. The significance between

161 Salamander Tail

two sample means was tested with the median test. For histological observations, regenerated tails

were embedded in paraplast, sectioned serially at 8 µm, and stained with Delafield's hematoxylin and eosin.

RESULTS

Interpopulation variation was found in the ratios of the four types of tail vertebrae, and sexual difference in the ratios was also recognized in the specimens collected at sample sites 7, 12, and 14 (Fig. 2). The frequency of specimens with exter­nally broken and regenerated tails was much higher where the sites of oviposition were the swifter flowing streams (Table 1 and Fig. 2). Animals with newly cut tails were not found among specimens having broken tails. No signi­ficant inverse correlation was found between the frequency of male specimens with broken and regenerated tails and the altitude (r s= -0.0725, a =0.05; Table 1 and Fig. 2).

The mean relative tail length did not differ significantly (α = 0.05) between males and females in sample site 7, but was significantly greater in males than in females in sample sites 8 (P<0.05), 12 (P<0.05), 13 (P<0.05), 14 (P<0.02), and 19 (P<0.005) (Table2). The relative tail length of

this species tended to be greater at higher latitude (Tables 2 and 3). The mean relative tail length of the males in sample site 15 was significantly less than the means in other sites, except for sample sites 9 and 10 (Table 3). The absolute value of the relative tail length of the specimens collected in sample site 5 was the greatest of all (Tables 2 and 3). When data from specimens with normal, questionable, and regenerated tails were com­bined, the maximum and minimum relative tail lengths were 0.911 and 0.463, respectively, and these values were both found in males with regenerated tails (compare with the values shown in Table 2). The relative axilla-groin length was significantly (P<0.001) greater in females (0.495 + 0.014, mean±SD) than in males (0.466 + 0.014).

In the cross sections of the regenerated tails having no vertebrae, cartilage was observed to surround the spinal cord (Fig. 3A), while in the cross sections of the regenerated tails with frag­mentary vertebrae, chondral cartilage or fragmen­tary bone tissue was seen (Fig. 3B).

DISCUSSION

1. The tail as a taxonomic character

Most salamanders seem to regenerate complete tails after suffering tail breakage, so it may be difficult to distinguish externally regenerated tails from normal tails. However, only herpetologists studying tail-autotomizable salamanders have pointed out the above-mentioned phenomenon [1, 6, 7]. In amputation-regeneration experiments on the tails of Ambystoma larvae, Holtzer et al. [8] showed that larvae regenerated all parts of the tail, but the lengths of the regenerated tails ranged from 20% to 35% below those of the controls.

It seems, however, that tail regeneration has not been taken into consideration in tail-unau-totomizable salamanders, so that the lengths of tails including regenerated ones have been used for taxonomic comparison. Therefore, in most papers [9-14] in this field the relative tail length in each urodeien species shows a remarkable variation in comparison with other taxonomic characters, such as relative head length, trunk length, and axilla-groin length. In fact, when data from specimens

FIG. 1. Radiographs of tails: A. normal vertebrae; B. fragmentary vertebvrae; C. no vertebrae. Scale: 5 mm.

162 M. HASUMI AND H. IWASAWA

with normal, questionable, and regenerated tails are combined, the dispersions of relative tail length are greater and the means of relative tail length are less in most localities (Fig. 4).

The main reasons why relative tail length is significantly greater in males than in females may

be that relative axilla-groin length is significantly greater in females than in males, and that breeding males have knife-edged tails so that the tails are longer. The relative lengths of many regenerated tails were less than those of normal tails, but the relative lengths of a few were greater. In addition,

FIG. 2. The ratios of the four types of tail vertebrae in each sample site. The size of circles reflects sample size (See Table 1).

163 Salamander Tail

TABLE 2. Geographic variation of relative tail length of specimens with normal tails

Sex

Male

Female

Sample site

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19

7 8

12 13 14 15 18 19

No.

0 6 2

10 9 8

21 6 3 1 3 5 5

13 4 2 0 1

29

4 6 3 3 6 0 1 7

Mean snout-vent length + 2SE (range) (mm)

67.4 ± 2.4(63.9-71,6) 59.6 (58.2-61.0) 61.9 ± 0.7(60.0-63.6) 60.6 ± 1.8(56.7-66.1) 67.9 ± 2.7(63.7-74.8) 68.7 ± 1.6(63.0-76.1) 58.8 ± 2.4(54.5-63.6) 59.4 ± 2.9(56.8-61.7) 67.8 63.5 ± 5.5(60.0-69.0) 68.1 ± 4.3(63.0-73.8) 57.4 ± 1.9(54.4-60.2) 68.0 ± 2.3(61.8-75.2) 67.6 ± 2.2(64.3-69.1) 73.8 (73.0-74.6)

59.4 66.0±1.3(58.1-74.1)

67.0 ± 3.2(65.2-71.7) 60.1 ± 1.8(57.6-62.8) 68.9 ± 5.4(64.1-73.5) 60.4 ± 3.3(58.7-63.7) 69.6 ± 3.7(65.0-77.0)

63.1 69.4 ± 2.2(65.9-73.9)

Mean relative tail length ± 2SE (range)

.804±.020 (.770-.844)

.781 (.780-.782)

.782±.018 (.742-.827)

.827± .029 (.765-.890)

.786±.037 (.700-.844)

.795± .021 (.715-.877)

.763± .014 (.750-.785)

.724±. 107 (.618-.793)

.701

.764±.032 (.732-.781)

.802±.013 (.790-.828)

.751± .017 (.728-.771)

.766±.028 (.680-.846)

.633 ± .034(.607-.682)

.786 (.764-.808)

.795

.760±.012 (.678-.820)

.747± .050 (.715-.822)

.724±.019 (.688-.759)

.663 ± .032 (.637-.693)

.672±.060 (.612-.705)

.717±.022(.678-.748)

.658

.695± .024 (.655-.738)

TABLE 3. Median test for the mean relative tail length (x) of specimens with normal tails

Sample site of males

Sample site of females

x min.

15

(18)

(10)

12

9

13

13

19

19

14

8

8

11

7

14 (3) 4 (16) 6 (18) 7

x max.

12 2 5

Each category is arranged in linear as less value comes left. Each bar indicates the groups that the difference is not statistically significant (α =0.05). However, the mean values of males in sample sites 19 and 4 are significantly less than the means in sample sites 4 and 2, respectively. Sample sites with no adequate number of specimens having normal tails are shown in parentheses.

the relative tail length showed a definite latitudinal variation, so it cannot always be determined from external observations that shorter tails must be regenerated ones.

In conclusion, it is necessary to take notice of tail regeneration, sexual difference, and latitudinal variation when cosidering tail length for taxonomic purposes.

164 M. HASUMI AND H. IWASAWA

2. Causes of tail breakage

The breakage of tails in tail-autotomizable salamanders was the result of traumatic events, such as attacks by predators, rock falls, entrap­

ment, and so on [1]. Predators Previous investigators thought

that tail autotomy in tail-autotomizable salaman­ders reflects predation pressure from any predator so long as the tail is attacked at least at some time [7, 15, 16]. The tails of tail-unautotomizable salamanders, too, may be injured by potential predators. So differential predation pressure ought to contribute to the differences in the ratios of the four types of tail vertebrae in Hynobius lichenatus. Shaffer [7] reported that snake densi­ties (potential predators) were found to decrease with elevation, and a significant inverse correlation was found between tail loss and elevation; that is, tail loss and the regenerated tails were equivalent expressions. However, this correlation was not found in the present study.

Biting Kusano [17] suggested based on his observations on Hynobius nebulosus tokyoensis during the breeding season that tail breakage was

FIG. 3. Cross sections of regenerated tails: A. no vertebrae; B. fragmentary vertebrae, s: spinal cord, c: cartilage, cc: chondral cartilage, b: bone tissue, m: muscle, d: dermal gland, v: blood vessel. Scale: 0.5 mm.

FIG. 4. Comparison of relative tail length. Left vertical lines indicate mean±2SE when data from specimens with normal, questionable, and regenerated tails are combined. Whereas right vertical lines indicate mean±2SE when only data from specimens having normal tails are used. Open circle: normal tail, semisolid circle: questionable tail, solid circle: re­generated tail.

165 Salamander Tail

the result of biting between breeding males, because the percentage of animals with cuts in the tail was higher in males than in females, and some breeding males which appeared in the pond were seen to lose their tail tips during their stay in the pond. It seems, however, that only tail tips or small parts of caudal fins would be lost in such cases.

Larval cannibalism Cannibalism during the larval period easily happens when larval density is high. It seems unlikely, however, that differential tail breakage resulted from cannibalism in the sites of oviposition, because the ratios of the four types of tail vertebrae differed according to sex.

Rapid stream The present results suggest that tail breakage in Hynobius lichenatus is due to rapid stream flow in most cases. During early spring thaw streams often overflow so rapidly that breeding adults may be carried away and injured because they are unable to resist the current. Moreover, the salamanders with just broken tails are even less able to resist and are carried away anew. Breeding males have knife-edged thin tails and stay longer in the stream, so they may be injured more frequently than females. It seems, however, that larvae are not so strongly influenced by the streams even in these sites, because the streams flow more slowly after the breeding season, except when there is occasional heavy rain. If tail breakage happens in the larval period, no difference according to sex would be found in the ratios of the four types of tail vertebrae. It is conceivable, therefore, that the breakage of tails usually happens to adults rather than to larvae.

At any rate, the causes of tail breakage in this species seem to be very complex.

3. Supplementary comments on the causes of tail breakage in three sample sites

Sample site 8 Roadside ditches in a forest were dug a year before the present collection, so tail breakage is not due falling into these ditches. A torrent was found near this site. Since the incidence of regenerated tails in the specimens collected at this site was quite high, the torrent may be the natural site of oviposition. However, because little difference according to sex was found in the ratios of the four types of tail

vertebrae, it is also possible that the tails were injured during the construction of the road.

Sample site 15 This site is on either side of a railroad line and is almost a static body of water into which water from fountains flows. On both sides of the railroad line are vertical precipices 5 m high faced with blocks of stone. Thawing water cascades down from parts of the precipices during the breeding season, so it is conceivable that some salamaders fall down the precipices when they come to the site of oviposition, and some may fall down with the cascading water. These may be the causes of a higher frequency of specimens with regenerated tails in this site.

Sample site 19 This site is a roadside ditch 20 cm wide, and a body of water about 30 m long remains throughout the year because fountains flow into the ditch. More than 100 pairs of egg sacs were found in this ditch, and larval density was remarkably high. There was little difference according to sex in the ratios of the four types of tail vertebrae, and few specimens had severely broken tail vertebrae. It seems, therefore, that most tail breakage at this site is due to cannibalism of the tail in the larval period.

ACKNOWLEDGMENTS

We are grateful to Professor N. Nara of Hirosaki University for giving us information on the sites of oviposition. We are also grateful to Professor K. Kobayashi and Dr. I. Sasagawa of Nippon Dental Uni­versity for the use of SOFRON equipment. We wish to express our gratitude to Professor K. Takata of Niigata University for his advice on statistical analysis.

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166 M. HASUMI AND H. IWASAWA

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