Reproductive cycle of the golden-striped salamander Chioglossa lusitanica (Caudata, Salamandridae) in NW Portugal

F. Sequeira1 , N. Ferrand1 , E.G. Crespo2

1 Centro

de Investigao em Biodiversidade e Recursos Genticos (CIBIO/UP), ICETA, Campus Agrrio de Vairo, 4485 661 Vairo and Departamento de Zoologia e Antropologia, Faculdade de Cincias, Universidade do Porto, Praa Gomes Teixeira, 4099 002 Porto, Portugal e-mail: fsequeira@mail.icav.up.pt 2 Centro de Biologia Ambiental, Faculdade de Cincias, Universidade de Lisboa, Bloco C2, Piso 3 Campo Grande, 1749 116 Lisboa, Portugal Abstract. The reproductive cycle of the golden-striped salamander, Chioglossa lusitanica, was studied in the vicinity of Porto (northwestern Portugal), in a population that breeds in a mine gallery. Salamanders (67 females and 64 males) were collected between January and November of 1998. Both sexes showed a seasonal reproductive cycle. Spermatogenesis and oogenesis took place between winter and late spring and the presence of spermatozoa and mature oocytes were observed mainly between early summer and late autumn. Gonad, liver and tail weights varied seasonally in both sexes and appear related to different phases of the sexual cycle. Average potential clutch size was 17.8 (s x D 0:8; range D 9-34). Clutch size was correlated with snout-vent length and apparently not N in uenced by tail-length or age.

Introduction The golden-striped salamander, Chioglossa lusitanica, is the sole member of its genus and has a distribution limited to Northwest Iberia. Studies of C. lusitanica (Gonalves, 1962; Thorn, 1964; Arntzen, 1981, 1994a, b, 1995, 1999; Arnold, 1987; Vences, 1990, 1993; Lima, 1995, 2001; Sequeira et al., 1996, 2001) provide signi cant data concerning its biology. Examination of reproductive phenology in Northern Spain (Vences, 1990) and in mine galleries in Portugal (Arntzen, 1981; Lima, 1995; Faria et al., 1996) indicated differences in the timing of reproductive activity of C. lusitanica both within and between populations, but data describing spermatogenetic and vitellogenetic processes are still unavailable. While the reproductive cycle in urodeles has been extensively studied (see
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F. Sequeira, N. Ferrand, E.G. Crespo

Joly, 1971; Lofts, 1974, 1984) studies correlating male and female reproductive cycles are scarce. This study provides information on reproductive cycles in both sexes in a population from the North of Portugal (Valongo). We describe the testicular and ovarian cycles and their relation to seasonal weight variation of the gonads, liver and tail. We also analyse the in uence of females body size and age on clutch size.

Materials and methods

Study area. Salamanders were collected in the margins of Inferno brook, Simo river and inside of the mine gallery of guas Frreas in Serra de Santa Justa, near Valongo, approximately 12 km NE Porto. The Inferno and Simo are permanent watercourses at ca. 100-150 m altitude, with forested valleys of Eucalyptus sp., Pinus sp. and Quercus sp., and an undergrowth of Rubus sp., Blechum spicante and Osmunda regalis. The guas Frreas, with the entrance on the left bank of Inferno brook, is a 236 m-long, narrow (ca 1 m), dark and permanently wet mine gallery, with a fairly constant annual air and water temperature (ranging between 15-16 C and 14-15 C, respectively). This mine is used as a reproduction and aestivation site by C. lusitanica, especially in summer and autumn (Arntzen, 1981; Faria et al., 1996). Sampling. Sixty-four males and 67 females were captured in January, March, May, July, September and November, 1998. Sex was initially determined by the presence or absence of a swelling on the upper forelimbs and con rmed by dissection. Sample treatment and histological procedures. Total length (TL), snout-vent length (SVL, tip of snout to most posterior insertion point of hind legs) and tail length (TAL, de ned as TL minus SVL) were recorded to the nearest 0.1 mm. Salamanders were blotted with absorbent paper to remove excess moisture and weighed to the nearest 0.01 g on a Mettler balance. Liver, gonads and tail were removed and weighed (nearest 0.01 g) and the tail was cut at the most posterior point of the cloacal swelling and removed. Due to the small size of C. lusitanica and the anatomical proximity of fat bodies and gonads, fat body development was assessed only on a qualitative basis. Testes from the 64 males were stored in Bouins solution for 48 h, transferred to 70% ethanol, embedded in paraf n, and prepared as 5-m-thick longitudinal sections stained with haemotoxylin and eosin. We considered three tissue types at 40 microscopic magni cation (Verrell et al., 1986): immature lobules containing spermatogonia (I and II), spermatocytes (I and II) or spermatids but not spermatozoa; mature lobules containing spermatozoa; and evacuated lobules devoid of spermatozoa. Ovarian conditions were noted immediately after dissection and the number, diameter (nearest 0.01 mm), colour and presence or absence of pigmentation in oocytes was recorded. We distinguished ve oocyte growth phases: previtellogenic (PV; translucent oocytes with diameter < 0.5 mm), early growth (EV; white oocytes with or without pigmentation 0.5-1.5 mm diameter), mid growth (MV; yellowish oocytes with diffuse pigmentation 1.6-2.1 mm diameter), late growth (LV; yellowish oocytes without pigmentation 2.2-2.7 mm diameter) and mature (MA; white or ivory oocytes without pigmentation > 2.7 mm diameter). We determined potential fecundity by counting mature or late vitellogenic oocytes (diameter > 2.2 mm) in the ovaries (clutch size). To determine the relationship between age and clutch size 28 females were examined by skeletochronological analysis. Humerus and femur were stored in 70% ethanol, decalci ed in 5% nitric acid solution for 20 to 30 min and returned to 70% ethanol until embedding in paraf n. Fifteen to 20-m-thick crosssections were prepared using a rotary microtome, stained with haemotoxylin, and examined at a magni cation of 40. Lines of arrested growth in periosteal bone were interpreted as representing age (Lima et al., 2001). Statistics. In order to normalise the data and minimise effects of allometric growth, body measurements, age and mass were log-transformed. Analysis of variance (ANOVA) was used for comparison of male and female SVL. Regression residuals for gonad, liver and tail weight regressed on SVL were calculated as size-corrected values. Analysis of covariance (ANCOVA) was used to examine seasonal changes in gonad, liver and tail weight using SVL as a covariate. Pearsons correlation coef cient (r/ was employed for correlation analysis. Data on female fecundity were analysed by stepwise linear regression, with clutch size (CS) as the dependent variable

Reproductive cycle of Chioglossa

and SVL, TAL and age as independent variables. All statistical analyses were performed with Statistical/w 4.5 (StatSoft, 1993), following Sokal and Rohlf (1981) and Zar (1996) with an alpha-level of 0.05 selected to assure signi cance. Where appropriate, data are reported as the arithmetic means plus or minus one standard error (s x /. N

Results Dimorphism and sexual maturity The SVL of the smallest male with spermatozoa was 43.0 mm (average males SVL D 45:5 0:2 mm; range D 43.0-48.0 mm; n D 64/. The SVL of the smallest gravid female was 43.2 mm (average females SVL D 47:1 0:2 mm; range D 43.2-50.8 mm; n D 67/. Females were signi cantly larger than males (ANOVA, F1;129 D 59:6, P < 0:05). Reproductive cycles Males. The proportion of the testis occupied by immature, mature and evacuated lobules varied seasonally (table 1). After the breeding season (November), a progressive decrease in mature and evacuated lobules ( g. 1a) and a concomitant increase in immature lobules was observed. Between November and January the proportion of males containing spermatozoa decreased from 90% to 17%, the proportion of immature lobules increased from 43% to 97%, and lobules contained mainly spermatogonia and few primary spermatocytes. From March to May the spermatogenetic process was intense, and many cells undergoing mitosis and meiosis were observed ( g. 1b). During this period testes contained many spermatids and primary and secondary spermatocytes in abundance, but spermatozoa were rare ( gs. 1c, 1d and 1e). By May, only 10% of examined males contained spermatozoa. In July, 50% of testicular lobules were mature, and evacuated lobules were few (1%). From July to September there was a strong increase in mature and evacuated lobules, and a corresponding decline in the proportion of the testis occupied by spermatids and earlier stages in the immature lobules. By September, all males had spermatozoa ( g. 1f). From September to November there was a slow decrease in the number of mature and evacuated lobules and an increase in immature lobules. During this period, three different areas can be recognised: a small area with lobules containing spermatogonia and another with lobules containing sperm. Some evacuated lobules were also present. Testis weight varied signi cantly throughout the year (ANCOVA, F5;57 D 2:39, P < 0:05/, increasing from January to its maximum in May, then decreasing in July, and increasing again in September ( g. 2a). Liver weight (ANCOVA, F5;57 D 9:81, P < 0:05/ and tail weight (ANCOVA, F5;57 D 8:13, P < 0:05/ also varied signi cantly during the year. Highest mean values of liver and tail weight were in May, after which there was progressive decline until November when lowest values were recorded ( gs. 2b and 2c). There was signi cant positive correlation between testis weight and liver weight (r D 0:30, P < 0:05, n D 64/ and tail weight (r D 0:38, P < 0:05, n D 64/. Fat bodies present around gonads were smallest from July to November and largest in spring.

F. Sequeira, N. Ferrand, E.G. Crespo

Figure 1. Longitudinal section of a Chioglossa lusitanica testis: a) testis in November showing mature (MA) and evacuated (EV) lobules (Bar D 400 m); b) testis from a male collected in March, among which primary spermatocytes (SPC I) can be observed (Bar D 25 m); c) testis in May showing primary and secondary spermatocytes (SPCI, SPC II) (Bar D 100 m).

Reproductive cycle of Chioglossa

Figure 1. (Continued): d) testis from a male collected in May showing spermatids (SPM) (Bar D 50 m); e) testis from a male collected in July, in which spermatids (SPM) and spermatozoa (SPZ) can be observed. Bar D 25 m; and, f) testis from a male collected in September, in which spermatozoa (SPZ) can be observed (Bar D 100 m).

F. Sequeira, N. Ferrand, E.G. Crespo

Table 1. Average (%) standard error of immature lobules (IL), mature lobules (ML) and evacuated lobules (EL) for males, and pre-vitellogenic (PV), early vitellogenic (EV), mid vitellogenic (MV), late vitellogenic (LV) and mature oocytes (M) for females. Proportion (P) of males and females that presented mature gametes and the number of individuals (n/ sampled. Month n January March May July September November 12 10 10 12 10 10 IL 97:4 2:5 100 99:9 0:1 50:2 10:5 11:3 4:4 42:5 10:4 Males ML 1:3 1:3 0 0:1 0:1 48:6 10:2 81:7 4:7 53:1 10:4 EL 1:3: 1:3 0 0 1:2 0:6 7:0 1:5 4:4 1:5 Females n January March May July September November 12 10 10 14 11 10 PV 65:9 2:5 61:4 2:8 62:6 3:6 45:8 3:4 60:4 3:8 55:5 2:1 EV 18:5 1:4 21:8 1:8 21:5 2:2 29:8 1:8 22:4 2:3 36:5 3:3 MV 15:6 2:4 6:2 2:1 2:3 0:9 0:7 0:4 0 1:6 0:8 LV 0 10:6 3:1 5:7 2:2 0:9 0:7 0 0 M 0 0 7:9 3:0 22:8 3:1 17:2 3:4 6:4 3:3 P 0 0 50 93 82 40 P 17 0 10 75 100 90

Females. Oocyte types varied seasonally (table 1). In November the proportion of gravid females (females with mature oocytes) was 40%, with ovaries containing 6% mature oocytes, 56% previtellogenic oocytes, 37% early-stage vitellogenic oocytes and 2% midstage vitellogenic oocytes. From November to January mature oocytes decreased from 6% to 0%; no gravid females were found in January. In contrast, mid-stage vitellogenic oocytes increased from 2% to 16% while early-stage oocytes decreased from 37% to 19%, clearly marking the onset of a new vitellogenic cycle. In March, we found 11% late-stage vitellogenic oocytes and a concomitant decrease in mid-stage oocytes. In May, mature oocytes were apparent and 50% of examined females had mature oocytes; the total proportion of mid (2%) and late (6%) stage vitellogenic oocytes decreased signi cantly. In July, the proportion of mature oocytes was 23%, the proportion of both mid- and late vitellogenicoocytes was less than 1%, and 93% of females examined were gravid. Ovarian composition in September was similar to that in July, while from September to November the proportion of mature oocytes and gravid females decreases. Ovary mass varied signi cantly during the year (ANCOVA, F5;60 D 5:89, P < 0:05/, increasing from January to September when the highest value was recorded ( g. 3a). Liver and tail mass also varied signi cantly during the year (ANCOVA, F5;60 D 9:00, P < 0:05; F5;60 D 5:04, P < 0:05, respectively).The highest value for each structure was recorded in May, but the lowest value for liver mass was recorded in September, and tail mass reached its minimum in November ( gs. 3b and 3c). There was no signi cant correlation between

Reproductive cycle of Chioglossa

Figure 2. Seasonal changes in the testis (a), liver (b) and tail (c) weights of male Chioglossa lusitanica. The vertical line represents standard deviations, the box two standard errors and the square, the mean. Abscises show the regression residuals between each variable and the snout-vent length, SVL (both log-transformed).

F. Sequeira, N. Ferrand, E.G. Crespo

liver, tail and gonad mass. Fat bodies were small in summer/autumn months and more conspicuous in spring. Fecundity Average clutch size was 17:8 0:8 (range 9 to 34; n D 35/. SVL, TAL and age ranged from 43.2 to 50.8 mm, 65.7 to 112.7 mm and 4 to 8 years, respectively. Only SVL was signi cantly correlated with clutch size (Log CS D 8:38 C 5:73 log SVL; r D 0:71, P < 0:05, n D 28) ( g. 4). Discussion Chioglossa lusitanica exhibits a seasonal reproductive cycle. In males, there is a slow proliferation of spermatogonia immediately following sperm evacuation (between summer and early winter). The process is arrested or delayed during autumn/early winter before the intense germ cell proliferation that occurs between late winter and spring. Spermiogenesis begins in late spring/early summer, continues over the summer and appears complete by early autumn. This testicular cycle is approximately coincident with the basic pattern found in temperate-zone urodeles (Joly, 1971; Lofts, 1974, 1984). In females, yolk deposition requires approximately ve or six months. After oviposition, females begin vitellogenesis from a limited stock of pre- and early vitellogenic oocytes that remains practically constant throughout the year. The process is arrested initially before intense yolk deposition occurs namely between winter and late spring. Females with mature oocytes appear in late spring and continue over the summer until late autumn. Winter/spring vitellogenic development in C. lusitanica contrasts with the pattern shown by most urodeles from temperate areas in which vitellogenic growth begins in summer, continuesthroughoutautumn-winter and is usually complete by late winter and early spring (Lofts, 1984). Male and female reproductive cycles in C. lusitanica are largely synchronic, a pattern not generally observed in temperate-zone urodeles, in which gametogenesis follows a slightly different course between the sexes (Lofts, 1984; Verrell et al., 1986; Guarino et al., 1992). The occurrence of gravid females and the pattern of spermatozoa abundance and spermiation are in agreement with the local breeding periods reported by Lima (1995), Faria et al. (1996), and with results from a recent monitoring program of mine galleries between 1999 and 2001 (unpublished data), but not with those reported for mid summer/autumn of 1977 (Arntzen, 1981). Our results are also congruent with reproductiveseasons reported by Vences (1990) for populations at Monte Pindo and El Morazzo, but not for Northern populations in La Corua province (Caaveiro and Valxestoso) where the season extends from December to July. While there are no clear explanations for variation within C. lusitanica, it is well documented that timing and pattern of amphibian reproductive cycles can vary widely within species (see Lofts, 1974, 1984; Paniagua et al., 1990). This temporal and

Reproductive cycle of Chioglossa

Figure 3. Seasonal changes in the ovary (a), liver (b) and tail (c) weights of female Chioglossa lusitanica. The vertical line represents standard deviations, the box two standard errors and the square, the mean. Abscises show the regression residuals between each variable and the snout-vent length, SVL (both log-transformed).

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F. Sequeira, N. Ferrand, E.G. Crespo

Figure 4. Regression between the clutch size and snout-vent length in female Chioglossa lusitanica.

geographic variation found in reproductive activity of C. lusitanica may suggest a potentially continuous cycle in this species, mostly dependent upon environmental variables, which differs from species exhibiting a reproductive cycle controlled by an endogenous rhythm determined genetically and much less dependent of climatic conditions (see Witschi, 1924; Van Oordt, 1960; Paniagua et al., 1990). In some urodeles showing discontinuous reproductive cycles, gonad weight is an appropriate indicator of gametogenesis activity (e.g. Verrell et al., 1986; Guarino et al., 1992). In C. lusitanica seasonal variation in ovary mass seems to be related to the different phases of the sexual cycle. Increase in ovary size appears associated with vitellogenesis, attaining its maximum during summer months when the highest proportion of mature oocytes was recorded. In contrast, the relation between testes mass and the sexual cycle was not so clear. The increase in testes size from winter to spring (prior to the breeding period) corresponds to the development of the seminiferous lobules, but irregular variation in testes mass was found during the breeding period. Dispersion in testes size during these months may be attributed to asynchronous spermatogenesis among individuals. In both sexes C. lusitanica, liver and tail mass also varied seasonally, with the lowest values recorded between July and November, corresponding to the breeding period. The liver is an important organ for fat and glycogen storage (e.g. Bush, 1963; Seymour, 1973) and plays an important role in the formation of vitellogenin, a precursor of yolk (Lofts, 1984). Thorn (1968) speculated that the tail in C. lusitanica served as an energy storage reserve, an hypothesis later supported by Arntzen (1981) who suggested that autotomy had a negative effect on reproductive capacity, since females with autotomised tails were less likely to be gravid. Thus, we suggest that the observed decrease in liver and tail size of C. lusitanica over the breeding period may be due to mobilisation of stored reserves in support of the energetic demands of reproduction. Clutch size in C. lusitanica ranges from 10 to 17 based on ovarian egg counts (Gonalves 1962; Busack, 1976; Arntzen, 1979). Our data indicate an average clutch size of 17:8 0:8

Reproductive cycle of Chioglossa

11

(range 9-34). The age of gravid females scored here varied between four and eight years, and is in accord with the age at maturity (4 years) and longevity (8 years) mentioned by Lima et al. (2001) for two C. lusitanica populations. This study showed a positive correlation between clutch size and SVL, as previously suggested by Arntzen (1981), but an in uence of age and TAL were not found. Thus, the absence of a clear relation between SVL and age in adult females of C. lusitanica reported by Lima et al. (2001) together with our results suggest the possibility of selection on female SVL to increase fecundity. Our results, together with a description of deposition sites (Arntzen, 1981; Sequeira et al., 2001) indicate that C. lusitanica follows a reproductive mode typi ed by the deposition of few eggs in restricted areas beneath stones or other objects in lotic habitats or in moist places such as mine galleries or underground watercourses. Considering the similar reproductive mode observed in some nearctic streamside plethodontids (Tilley, 1968; Bruce, 1969; Semlitsch and McMilan, 1980; Hairston, 1987), as well as in the closely related Mertensiella caucasica (Tarkhnishvili and Serbinova, 1993; Tarkhnishvili, 1994; Titus and Larson, 1995; Veith et al., 1998) body shape and ecological niche probably constitute the most important factors in uencing the reproductive mode of C. lusitanica.

Acknowledgements. We thank Rui Rebelo for his assistance in the interpretation of the skeletochronological data, Alexandra Rema for technical assistance in the laboratory, and Steven Weiss, M.A. Carretero and J.W. Arntzen for critical comments on previous versions of the manuscript. Permission to collect was provided by the Institute for Nature Conservation (ICN) Lisboa. Portugal. F. Sequeira was supported by a PhD grant (SFRH/BD/3365/2000) from Fundao para a Cincia e a Tecnologia (FCT).

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Received: April 3, 2002. Accepted: July 4, 2002.