2008 SOUTHEASTERN NATURALIST 7(4):705–716 Life-history Aspects of Stereochilus marginatus, with a Comparison of Larval Development in Syntopic S. marginatus and Pseudotriton montanus (Amphibia: Plethodontidae) Richard C. Bruce* Abstract - The plethodontid salamanders Stereochilus marginatus (Many-lined Salamander) and Pseudotriton montanus (Mud Salamander) have overlapping distributions in the southeastern Atlantic Coastal Plain, where they often co-occur in low, swampy habitats. The main objective of this study was to document life-history traits of S. marginatus at Cool Springs in eastern North Carolina, and compare these findings with life-history parameters of a population surveyed in the late 1960s at a nearby locality (Croatan Forest). A second objective was to compare larval development of S. marginatus and P. montanus at Cool Springs. I found that S. marginatus has a larval period of 13–14 months, hatching in early spring and undergoing metamorphosis late in the second spring. Males may breed initially as early as the autumn following metamorphosis, at 19–21 months of age; females probably require an additional year to attain maturity, ovipositing initially at 3 years. Clutch sizes, based on counts of yolked ovarian follicles of dissected females, ranged from 42 to 60. Compared to S. marginatus, the larval period of P. montanus is slightly longer (14–17 months), extending from hatching in winter to metamorphosis in the second spring. Although larval body sizes of S. marginatus and P. montanus overlap considerably, larvae of the latter species tend to grow larger and metamorphose at slightly larger sizes. The phenologies of the life cycles of both species corroborate earlier studies, both across years and across southeastern localities. However, growth and developmental rates of S. marginatus at Cool Springs appear to be accelerated relative to those reported previously for the Croatan Forest population. Introduction Stereochilus marginatus Hallowell (Many-lined Salamander) is a small, non-descript, lungless salamander endemic to the Atlantic Coastal Plain of the United States. It is highly aquatic, living in and along the margins of swamps, ponds, and sluggish streams. Means (2000) included S. marginatus in an assemblage (assemblage 1) of Coastal Plain plethodontids that inhabit low, swampy habitats, in contrast to a second assemblage (assemblage 2) of Coastal Plain species that are associated with ravines. Various aspects of the life history, feeding ecology, and reproductive biology of S. marginatus have been documented by Bruce (1971), Foard and Auth (1990), Rabb (1956), Schwartz and Etheridge (1954), and Wood and Rageot (1963). In this paper, I report on the life history of S. marginatus in a population inhabiting a swamp ecosystem in the Coastal Plain of eastern North *Department of Biology, Western Carolina University, Cullowhee, NC 28723. Current address - 200 Breeze Way, Aurora, NC 27806; ebruce1563@aol.com. 706 Southeastern Naturalist Vol. 7, No. 4 Carolina. Stereochilus marginatus was the most frequently encountered species of salamander in the swamp. One objective was to compare life-history traits in this population with those of a nearby population that had been studied some years before (Bruce 1971). A second objective was to compare the larval period of S. marginatus with that of Pseudotriton montanus Baird (Mud Salamander), also an assemblage 1 species and the next most frequently encountered species in the swamp. The two species are closely related, and larvae are morphologically similar (Birchfield and Bruce 2000). The life history of P. montanus is known mainly from studies conducted in western North and South Carolina (Bruce 1974, 1975, 1978). Valuable summaries of the natural history of both S. marginatus and P. montanus can be found in Petranka (1998), and in the accounts by Ryan (2005) on S. marginatus and Hunsinger (2005) on P. montanus in Lannoo (2005). Study Area, Materials, and Methods I conducted the study at the Cool Springs Environmental Education Center administered by the Weyerhaeuser Corporation. The Cool Springs tract encompasses about 680 ha adjacent to the confl uence of the Neuse River and Swift Creek in Craven County, NC (35o11.4'N, 77o04.9'W). Elevations are ≤6 m above sea level. Natural habitats include pine-oak sand ridges, mixed pine-hardwood forests, pocosins, and a cypress swamp (Hall 2006). The sampling area in the present study was a small section of the cypress swamp in which Chamaecyparis thyoides L. (Atlantic White Cedar), rather than Taxodium distichum L. (Bald Cypress), was a co-dominant canopy species, together with Acer rubrum L. (Red Maple), Nyssa aquatica L. (Water Tupelo), Quercus michauxii Nuttall (Swamp Chestnut Oak), and Fraxinus caroliniana Miller (Carolina Ash). This area was fed by a shallow, sluggish stream that discharged into the swamp. The latter consisted of a network of isolated and semi-isolated shallow, mucky pools along the principal, but indistinct, drainage channel. Many of the pools supported mats of Sphagnum spp. I collected salamanders by pulling a dip net along the mucky bottoms of the aquatic habitats. I also raked leaf litter and turned rotting logs along the banks of the stream and pools and in the intervening fl oodplain. A total of 195 S. marginatus (109 larvae, 86 adults) and 75 P. montanus (65 larvae, 10 adults) were captured. Counts of other species of salamanders observed were recorded. For convenience, throughout this paper, I use the term “adult” to refer to fully-metamorphosed individuals, whether immature (juveniles) or sexually mature. Captured S. marginatus and P. montanus were returned to the Cool Springs laboratory, where they were anesthetized in a solution of MS-222 and then measured for standard length (SL), to the nearest 0.1 mm, from the tip of the snout to the posterior end of the cloacal slit. All measurements given herein refer to SL. Most of the salamanders were subsequently revived in swamp water and returned to the site of capture, usually on the same day. 2008 R.C. Bruce 707 Given that the salamanders were not marked, it was unknown how many were subsequently recaptured. However, I assumed that some captures were recaptures, which required a cautious approach to statistical evaluation in order to avoid violations of independence (Scheiner and Gurevitch 2001). In statistical tests, significance was evaluated at α = 0.05. The main set of samples was taken from 21 November 2003 through 29 November 2004. During this period, I attempted to sample twice each month, but in August and September 2004, only single searches were made. Four supplementary samples were taken between 30 March and 25 May 2005, mainly to obtain additional data on early larval development of both species. For the last three collections of 2004 and for those of the spring of 2005, I preserved all adults of S. marginatus (n = 23), in order to dissect the reproductive organs and determine reproductive condition. These specimens were euthanized in MS-222, fixed in 10% formalin, and transferred to 70% ethyl alcohol. Preserved specimens have been deposited in the herpetological collection of the North Carolina State Museum of Natural Sciences (catalog nos. 74251–74273). Results Habitat associations Although I did not attempt to quantify habitat features of the two species, larvae of P. montanus were more abundant in the upper section of the swamp, especially in the small feeder stream and adjacent pools. Most of the firstyear larvae of P. montanus taken in the spring of both 2004 and 2005 were found in a seep adjacent to the stream, at the base of a gentle slope. I assumed that the nesting sites had been in subsurface channels leading into the seep. In contrast, larvae of S. marginatus were more abundant downstream, in lentic habitats, especially shallow pools that were often bordered by Sphagnum mats. However, few larvae were found in Sphagnum; most were taken from decaying vegetation on the bottom of the pools. Notwithstanding these differences, larvae of S. marginatus and P. montanus broadly overlapped throughout the area of the swamp that I searched, and were sometimes taken in the same pool. Most adult S. marginatus were captured in dip-net samples in the same aquatic habitats as larvae. During a dry period in June and July 2004, when pools had little or no water, 19 adults were found by excavating by hand the saturated muck at the bottom of drying pools. In contrast, other than two small, newly transformed juveniles taken in dip-net samples, the adult P. montanus were found on land, under logs or boards on damp soil. I did not make a concerted effort to excavate burrows, a favored habitat for this species elsewhere (Bruce 1975). Other species of salamanders encountered included 43 Desmognathus auriculatus Holbrook (Southern Dusky Salamander), 6 Eurycea chamberlaini Harrison and Guttman (Chamberlain’s Dwarf Salamander), 708 Southeastern Naturalist Vol. 7, No. 4 3 E. cirrigera Green (Southern Two-lined Salamander), 13 Plethodon chlorobryonis Mittleman (Atlantic Coast Slimy Salamander), and 2 juvenile Amphiuma means Garden (Two-toed Amphiuma). Although D. auriculatus may be the second-most abundant species in the swamp, there were only 6 larvae among the 43 individuals recorded. No larvae of either species of Eurycea were seen, suggesting that neither breeds at the immediate study site. A full account of the amphibians and reptiles of the Cool Springs tract is provided by Hall (2006). Larval development of Stereochilus marginatus The size distribution of larval S. marginatus for the 2003–2004 samples is shown in Figure 1, and that of small larvae taken in the spring of 2005 in Figure 2. The smallest larva (10.5 mm), captured on 15 April 2005, had a belly distended with yolk and thus had likely hatched within the past month. The course of early development in the spring of the successive years was similar; the mean SL of the eight small larvae of 21 May 2004 (mean ± SD = 15.6 ± 1.10 mm) and that of the six first-year larvae of 25 May 2005 (mean ± SD = 16.4 ± 1.16 mm) did not differ significantly (t = 1.35, df = 12, P = 0.203). Among older larvae, there was a trend toward size increase from November to May. The largest larvae, 44–46 mm, were found between 30 January and 29 April. Larger larvae disappeared from the population in May, presumably due to metamorphosis of all 2nd-year larvae. This conclusion is supported by the observation that the smallest adults were captured between Figure 1. Distribution of standard lengths of 100 larval Stereochilus marginatus (Many-lined Salamander) captured between 21 November 2003 (day 1) and 29 November 2004 (day 375). Some symbols in this and other graphs represent more than one individual of equivalent sizes. 2008 R.C. Bruce 709 31 May and 9 July (Fig. 3); five of these, ranging from 38 to 45 mm, showed obvious gill resorption scars, but were otherwise fully metamorphosed. Figure 2. Distribution of standard lengths of first-year larvae of Pseudotriton montanus (Mud Salamander) (triangles, n = 19) and Stereochilus marginatus (Many-lined Salamander) (inverted triangles, n = 8) captured between 30 March and 25 May 2005. The x-axis is indexed in days from 20 November, as in the other graphs. Figure 3. Distribution of standard lengths of 80 adult Stereochilus marginatus (Many-lined Salamander) captured between 21 November 2003 (day 1) and 29 November 2004 (day 375). 710 Southeastern Naturalist Vol. 7, No. 4 Thus, if embryos hatched in early spring, and if most larvae metamorphosed in mid-late spring of their second year, then the larval period was approximately 13–14 months at Cool Springs during the course of this study. Few larvae were found in summer and autumn of 2004; nevertheless, there is a pattern of rapid growth of first-year larvae in the interval from spring to late autumn. Larvae in the final sample, that of 29 November 2004, had attained the same sizes as those taken in the initial sample of 21 November 2003 (2003: n = 12, mean ± SD = 35.4 ± 2.85 mm; 2004: n = 4, mean ± SD = 35.2 ± 3.38 mm; t = 0.121, df = 14, P = 0.905). Reproductive cycles The distribution of body sizes of adult S. marginatus captured in the 2003–2004 samples is shown in Figure 3. The additional six individuals captured in the spring samples of 2005 fell within the same size range. The exceptionally large individual (67 mm) was a male; otherwise, among the 23 individuals that were dissected, maximum sizes of the sexes were similar (male = 56 mm, female = 57 mm). Following Bruce (1971), I have calculated ages from February, the probable median month of entry of individuals into the population as eggs. Females. Of the 17 individuals collected 29 October–29 November 2004 and subsequently dissected, 10 were females. The two smallest (45 and 46 mm) were immature, having narrow, straight oviducts and small, compact ovaries containing small, translucent follicles. In the remaining eight (52–56 mm), vitellogenesis was in progress with yolked follicles ranging from 1.2 to 2.2 mm in diameter. Follicle counts varied from 42 to 60 (mean ± SD = 51.4 ± 5.85). Although the smallest female had the fewest yolked follicles, the slope of the regression of follicle number on SL was non-significant (b = 1.05, t = 1.33, df = 6, P > 0.20; Fig. 4). It is likely that these gravid females would have oviposited in the approaching egg-laying season in mid- to late winter. Figure 4. Counts of yolked ovarian follicles in 8 gravid females of Stereochilus marginatus (Many-lined Salamander) collected in autumn 2004. The slope of the regression line is nonsignificant (see text). 2008 R.C. Bruce 711 If the two small, immature females, which overlapped in size with the three largest larvae, had metamorphosed in the previous spring, it seems likely that they had been scheduled to yolk their initial clutch the next autumn (i.e., 2005) and oviposit initially at an age of 3 years. Based on the limited data available, this is the best estimate of age at first reproduction of females in this population. The four females dissected from the April–May 2005 samples were 51–57 mm SL. All four appeared mature, and three were in an obvious spent condition, showing convoluted oviducts and loosely organized ovaries having a scattering of small, translucent follicles. The ovaries of one individual had three orange-pigmented, atretic follicles. Although adults captured in the earlier samples of 2003 and 2004 were not dissected, I did identify gravid females by the presence of yolky ovarian follicles visible through the body wall of the distended abdomen. Eight such females were found between 21 November and 29 February, but none thereafter until the next autumn. These females were similar in size (n = 8, mean ± SD = 51.9 ± 1.76 mm) to the gravid females dissected in the late 2004 samples (n = 8, mean ± SD = 53.2 ± 2.66 mm), and the difference was non-significant (t = 1.22, df = 14, P = 0.243). Males. Seven males (46–67 mm) were collected and dissected in the period 29 October–29 November 2004. Although the testes varied in size and shape, all seven had dark, coiled vasa deferentia, and were thereby scored as mature. In five of these males, including the smallest (46 mm), a cord of spermatozoa was detected in the vasa. Thus, it is likely that many males breed initially in the autumn following metamorphosis, at approximate ages of 19–21 months. Only two males were dissected from the spring 2005 samples, both collected on 25 May. The smaller, 46 mm, possibly a recent metamorph, was immature; the larger, 50 mm, was mature based on the presence of black, coiled vasa, but was not in breeding condition, inasmuch as the testes were thin and the vasa were empty. Larval development of Pseudotriton montanus During the principal sampling period in 2003–2004, 42 larvae and eight adults of P. montanus were captured. Most larvae were taken during the earlier phase of the sampling interval (Fig. 5). The smallest were collected on 19 April and, from that date to mid-June, two distinct size classes of larvae were present, presumably representing the cohorts of 2003 and 2004. In the former, a slight trend of increasing size was evident in the samples taken from November to mid-June. The loss of this cohort in late June was, in part at least, an effect of metamorphosis in the spring months. A 46-mm larva collected on 24 March metamorphosed in the laboratory on 19 April. Two fully metamorphosed individuals (44 and 49 mm), which retained larval pigmentation (brown), were found on 31 May and 15 June, respectively; on the latter date, a metamorphosing larva (41 mm) was taken as well. The supplementary samples of spring 2005 included two adults, four large larvae (33–46 mm), and 19 1st-year larvae; the size distribution of the 712 Southeastern Naturalist Vol. 7, No. 4 latter (Fig. 2) was similar to that of 1st-year larvae taken the previous year (Fig. 5). The mean sizes in two pairs of samples of 1st-year larvae captured on nearly the same dates of the two years were not significantly different at α = 0.05/2 (Bonferroni adjustment) (19 April 2004 vs. 15 April 2005: t = 0.116, df = 10, P = 0.910; 29 April 2004 vs. 30 April 2005: t = 1.394, df = 8, P = 0.201). Given that the smallest larvae of P. montanus, already ≥15 mm (n = 6, mean ± SD = 15.8 ± 0.61 mm), were found on 30 March 2005, it is likely they had hatched 1–2 months earlier in both 2004 and 2005, in mid-winter, as at other localities (Bruce 1974). Most larvae at Cool Springs probably metamorphose in the spring of their second year, after a larval period of 14–17 months. Discussion In an earlier study of the life history of S. marginatus in Croatan National Forest, NC (a locality 32 km south of Cool Springs), I estimated that the larval period was usually 25–28 months, but that some larvae transformed at 13–16 months (Bruce 1971). Although based on smaller samples, the Cool Springs data nonetheless suggest strongly that most larvae metamorphose in their second year, 13–14 months after hatching, at an approximate age of 15 months. In the Croatan study, I estimated that Figure 5. Distribution of standard lengths of 42 larvae and 2 recent metamorphs (31 May: 43.9 mm; 15 June: 48.9 mm) of Pseudotriton montanus (Mud Salamander) captured between 21 November 2003 (day 1) and 14 October 2004 (day 329). 2008 R.C. Bruce 713 oviposition occurred in winter, with hatching following in late March– early April; this is supported by the newer data from Cool Springs. Thus, the presence of gravid females in the population in autumn and winter—as late as late February—indicates that the oviposition season may extend to late winter. The two tiny larvae (10, 11 mm) taken in mid- and late April at Cool Springs were similar in size to hatchlings found at Croatan in April and May (taking into account the difference in methodology of measuring body length). At Croatan, I found that metamorphosis occurred in late spring and summer, similar to the findings at Cool Springs. Thus, whereas larval developmental rates appear to differ between the two populations (studied 35 years apart), the phenologies of the larval phases were very similar. Perhaps climatological changes during the interval and/or habitat differences between the sites contributed to the observed differences. I was unable to conduct parallel studies of the Cool Springs and Croatan populations in 2003–2005 because of changes in the habitat and severe reduction of the S. marginatus population at the latter site. The course of the larval period of P. montanus at Cool Springs was similar to that of S. marginatus; however, hatching occurred earlier in the year in the former species, probably indicating an earlier oviposition season, in late autumn–early winter, as at other localities (Brimley 1944, Fowler 1946, Goin 1947). Inasmuch as the smallest larvae in 2004 and 2005 were taken in late March 2005, and had already grown to 15 mm and larger, I presumed that they had hatched at least 1–2 months earlier and had undergone early development in the subsurface nesting sites. Given that metamorphosis occurred in the spring and presumably included all (or nearly all) 2nd-year larvae, the estimated duration of the larval period is 14–17 months. This estimate is similar to estimates for populations in the Piedmont of western South Carolina (Bruce 1974, 1978). In a higher-elevation population in the Blue Ridge of North Carolina, the usual larval period was found to be a year longer (Bruce 1978). At Cool Springs, the earlier oviposition season of P. montanus versus S. marginatus gave the former an advantage in growth during the first year. Although larger larvae of the two species overlapped broadly in size, those of P. montanus tended to grow larger and metamorphose at slightly larger sizes (≈41–50 mm) than those of S. marginatus (≈38–45 mm). Moreover, as larvae of S. marginatus have a more slender habitus than those of P. montanus (Birchfield and Bruce 2000), metamorphs of the latter probably have even greater masses than those of the former species. Data on the reproductive cycles of male and female S. marginatus at Cool Springs are incomplete, given the necessity of dissection to determine reproductive status of both sexes. However, the data obtained on specimens taken in late autumn 2004 and spring 2005 agree well with the more comprehensive picture of reproduction at Croatan Forest in 1968–69 (Bruce 1971). In the latter population, males were in breeding condition, with vasa 714 Southeastern Naturalist Vol. 7, No. 4 deferentia packed with sperm, by 1 November; similarly, for the October– November 2004 samples from Cool Springs, all seven males were in breeding condition, including the smallest (46 mm). The two males dissected from the spring samples of 2005 were likewise similar to their spring counterparts at Croatan Forest. The estimated age at first reproduction of 19–21 months at Cool Springs represents the lower limit of the earlier estimate (21–45 months) for the Croatan Forest population. I contend that most males in both populations were initiating spermatogenesis immediately following metamorphosis; as such, the earlier age of reproduction at Cool Springs likely refl ects the earlier age at metamorphosis. The dissection data suggest that an additional year may be required for development to sexual maturity in females versus males. Thus, age at first reproduction (i.e., oviposition) is estimated as 3 years in female S. marginatus at Cool Springs, a year earlier than in the Croatan Forest population (Bruce 1971). As in males, the difference reflects the difference in age at metamorphosis. At Croatan Forest, counts of follicle numbers in the ovaries of gravid females were obtained for only three individuals; the values were lower (n = 22–29) than for the eight gravid females at Cool Springs (n = 42–60). No deposited eggs clutches were found at Croatan earlier or at Cool Springs recently, but have been found by other investigators. Whereas Schwartz and Etheridge (1954) and Rabb (1956) provided data on single deposited egg clutches, the most comprehensive report on nesting of S. marginatus was provided by Wood and Rageot (1963), who discovered 43 clutches between late winter and early spring of 1954 and 1955. Number of eggs per clutch ranged widely (6–92), and attending females were largely absent. Thus, it is possible that the smaller clutches had been reduced by predation and that the larger were products of more than one parent. However, Noble and Richards (1932) reported a mean clutch size of 57 in 19 females of S. marginatus following implantation of frog pituitaries; subsequent dissection of the females showed that the total ovarian complements of “mature” eggs had averaged 70, with a range of 16–121. From this series of studies, including the present report, it appears that clutch size in S. marginatus is highly variable; whether there is a tendency of clutch size to increase with body size, as in its closest relatives in the genera Gyrinophilus and Pseudotriton (Bruce 1969), is uncertain. High residual variance obscures such a correlation in small samples in species where the range of female body size is narrow, as in S. marginatus. It would be of interest to examine interactions between S. marginatus and P. montanus in habitats like that of the Cool Springs swamp, where both species are relatively abundant. A working hypothesis is that interactions, especially interspecific competition, are stronger between larvae of the two species, which are morphologically similar (Birchfield and Bruce 2000) and overlap in habitat use, than between adults, which show greater divergence in size, morphology, and habitat associations. However, it is possible that 2008 R.C. Bruce 715 adult P. montanus prey on S. marginatus, given Brimley’s (1944) observations of predation by the former species on Eurycea cirrigera. The swamp habitat is a difficult one for conducting natural manipulative experiments, but laboratory and mesocosm experiments could prove fruitful. Acknowledgments I thank Jeff Hall, former manager of the Cool Springs Environmental Education Center, for providing access to the site; he also assisted in the field work. Scientific collecting licenses were issued by the North Carolina Wildlife Resources Commission. Literature Cited Birchfield, G.L., and R.C. Bruce. 2000. Morphometric variation among larvae of four species of lungless salamanders (Caudata: Plethodontidae). Herpetologica 56:332–342. Brimley, C.S. 1944. Amphibians and Reptiles of North Carolina. Carolina Biological Supply Co., Elon College, NC. 63 pp. Bruce, R.C. 1969. Fecundity in primitive plethodontid salamanders. Evolution 23:50–54. Bruce, R.C. 1971. Life cycle and population structure of the salamander Stereochilus marginatus in North Carolina. Copeia 1971:234–246. Bruce, R.C. 1974. Larval development of the salamanders Pseudotriton montanus and P. ruber. American Midland Naturalist 92:173–190. Bruce, R.C. 1975. Reproductive biology of the Mud Salamander, Pseudotriton montanus, in western South Carolina. Copeia 1975:129–137. Bruce, R.C. 1978. A comparison of the larval periods of Blue Ridge and Piedmont Mud Salamanders (Pseudotriton montanus). Herpetologica 34:325–332. Foard, T., and D.L. Auth. 1990. Food habits and gut parasites of the salamander Stereochilus marginatus. Journal of Herpetology 24:428–431. Fowler, J.A. 1946. The eggs of Pseudotriton montanus montanus. Copeia 1946:105. Goin, C.J. 1947. Notes on the eggs and early larvae of three Florida salamanders. Chicago Academy of Sciences, Natural History Miscellanea 10:1–4. Hall, J.G. 2006. Herpetological sampling in the North Carolina Coastal Plain: A comparison between techniques across habitats. M.Sc. Thesis. East Carolina University, Greenville, NC. 173 pp. Hunsinger, T.W. 2005. Pseudotriton montanus Baird, 1849. Mud Salamander. Pp. 858–860, In M. Lannoo (Ed.). Amphibian Declines: The Conservation Status of United States Species. University of California Press, Berkeley, CA. Lannoo, M. (Ed.). 2005. 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University of California Press, Berkeley, CA. Scheiner, S.M., and J. Gurevitch (Eds.). 2001. Design and Analysis of Ecological Experiments. Oxford University Press, Oxford, UK. 415 pp. Schwartz, A., and R. Etheridge. 1954. New and additional herpetological records from the North Carolina Coastal Plain. Herpetologica 10:167–171. Wood, J.T., and R.H. Rageot. 1963. The nesting of the Many-lined Salamander in the Dismal Swamp. Virginia Journal of Science 14:121–125.