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Reproductive cycle of the golden-striped salamanderChioglossa 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 deVairo, 4485 661 Vairo and Departamento de Zoologia e Antropologia, Faculdade de Cincias, Universidadedo Porto, Praa Gomes Teixeira, 4099 002 Porto, Portugale-mail: fsequeira@mail.icav.up.pt2 Centro de Biologia Ambiental, Faculdade de Cincias, Universidade de Lisboa, Bloco C2, Piso 3 CampoGrande, 1749 116 Lisboa, Portugal

Abstract. The reproductive cycle of the golden-striped salamander, Chioglossa lusitanica, was studied in thevicinity of Porto (northwestern Portugal), in a population that breeds in a mine gallery. Salamanders (67 femalesand 64 males) were collected between January and November of 1998. Both sexes showed a seasonal reproductivecycle. Spermatogenesis and oogenesis took place between winter and late spring and the presence of spermatozoaand mature oocytes were observed mainly between early summer and late autumn. Gonad, liver and tail weightsvaried seasonally in both sexes and appear related to different phases of the sexual cycle. Average potential clutchsize was 17.8 (s Nx D 0:8; range D 9-34). Clutch size was correlated with snout-vent length and apparently notinuenced by tail-length or age.

Introduction

The golden-striped salamander, Chioglossa lusitanica, is the sole member of its genusand 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 signicant data concerningits biology. Examination of reproductivephenology in Northern Spain (Vences, 1990) andin mine galleries in Portugal (Arntzen, 1981; Lima, 1995; Faria et al., 1996) indicateddifferences in the timing of reproductive activity ofC. lusitanica both within and betweenpopulations, but data describing spermatogenetic and vitellogenetic processes are stillunavailable. While the reproductive cycle in urodeles has been extensively studied (see

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

Joly, 1971; Lofts, 1974, 1984) studies correlating male and female reproductive cyclesare scarce. This study provides information on reproductive cycles in both sexes in apopulation from the North of Portugal (Valongo). We describe the testicular and ovariancycles and their relation to seasonal weight variation of the gonads, liver and tail. We alsoanalyse the inuence 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 minegallery of guas Frreas in Serra de Santa Justa, near Valongo, approximately 12 km NE Porto. The Inferno andSimo are permanent watercourses at ca. 100-150 m altitude, with forested valleys ofEucalyptus sp., Pinus sp.and Quercus sp., and an undergrowth ofRubus 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 andautumn (Arntzen, 1981; Faria et al., 1996).

Sampling. Sixty-four males and 67 females were captured in January, March, May, July, September andNovember, 1998. Sex was initially determined by the presence or absence of a swelling on the upper forelimbsand conrmed by dissection.

Sample treatment and histological procedures. Total length (TL), snout-vent length (SVL, tip of snout to mostposterior insertion point of hind legs) and tail length (TAL, dened as TL minus SVL) were recorded to thenearest 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) andthe 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 aqualitative basis. Testes from the 64 males were stored in Bouins solution for 48 h, transferred to 70% ethanol,embedded in parafn, and prepared as 5-m-thick longitudinal sections stained with haemotoxylin and eosin. Weconsidered three tissue types at 40 microscopic magnication (Verrell et al., 1986): immature lobules containingspermatogonia (I and II), spermatocytes (I and II) or spermatids but not spermatozoa; mature lobules containingspermatozoa; and evacuated lobules devoid of spermatozoa. Ovarian conditions were noted immediately afterdissection and the number, diameter (nearest 0.01 mm), colour and presence or absence of pigmentation in oocyteswas 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 oocyteswithout pigmentation 2.2-2.7 mm diameter) and mature (MA; white or ivory oocytes without pigmentation > 2.7mm diameter).

We determined potential fecundity by counting mature or late vitellogenic oocytes (diameter > 2.2 mm) inthe ovaries (clutch size). To determine the relationship between age and clutch size 28 females were examinedby skeletochronological analysis. Humerus and femur were stored in 70% ethanol, decalcied in 5% nitric acidsolution for 20 to 30 min and returned to 70% ethanol until embedding in parafn. Fifteen to 20-m-thick cross-sections were prepared using a rotary microtome, stained with haemotoxylin, and examined at a magnication of40. 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, ageand mass were log-transformed. Analysis of variance (ANOVA) was used for comparison of male and femaleSVL. Regression residuals for gonad, liver and tail weight regressed on SVL were calculated as size-correctedvalues. Analysis of covariance (ANCOVA) was used to examine seasonal changes in gonad, liver and tail weightusing SVL as a covariate. Pearsons correlation coefcient (r/ was employed for correlation analysis. Data onfemale fecundity were analysed by stepwise linear regression, with clutch size (CS) as the dependent variable

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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 assuresignicance. Where appropriate, data are reported as the arithmetic means plus or minus one standard error ( s Nx /.

Results

Dimorphism and sexual maturity

The SVL of the smallest male with spermatozoa was 43.0 mm (average males SVL D45:5 0:2 mm; range D 43.0-48.0 mm; n D 64/. The SVL of the smallest gravid femalewas 43.2 mm (average females SVL D 47:1 0:2 mm; range D 43.2-50.8 mm; n D 67/.Females were signicantly 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 lobulesvaried seasonally (table 1). After the breeding season (November), a progressive decreasein mature and evacuated lobules (g. 1a) and a concomitant increase in immature lobuleswas observed. Between November and January the proportion of males containing sper-matozoa decreased from 90% to 17%, the proportion of immature lobules increased from43% to 97%, and lobulescontainedmainly spermatogoniaand few primary spermatocytes.From March to May the spermatogenetic process was intense, and many cells undergoingmitosis and meiosis were observed (g. 1b). During this period testes containedmany sper-matids 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 toSeptember there was a strong increase in mature and evacuated lobules, and a correspond-ing decline in the proportion of the testis occupied by spermatids and earlier stages in theimmature lobules. By September, all males had spermatozoa (g. 1f). From September toNovember there was a slow decrease in the number of mature and evacuated lobules and anincrease in immature lobules. During this period, three different areas can be recognised:a small area with lobules containing spermatogonia and another with lobules containingsperm. Some evacuated lobules were also present.

Testis weight varied signicantly throughout the year (ANCOVA, F5;57 D 2:39, P