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Reproductive Life History of Desmognathus folkertsi (Dwarf Black-bellied Salamander)
Carlos D. Camp and Jeremy L. Marshall

Southeastern Naturalist, Volume 5, Number 4 (2006): 669–684

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2006 SOUTHEASTERN NATURALIST 5(4):669–684 Reproductive Life History of Desmognathus folkertsi (Dwarf Black-bellied Salamander) Carlos D. Camp1,* and Jeremy L. Marshall2 Abstract – An earlier collection of monthly samples of presumed Desmognathus quadramaculatus from Union County, GA, revealed the presence a previously unknown, sympatric, sibling species. This new form was recently described as D. folkertsi. In this paper, we report on the reproductive life history of this new species from data taken during (1) a 14-month period in 1989–90 and (2) late spring of 1998. Adult males and females mature at approximately the same size (57–58 mm snout– vent length [SVL]), and males reach larger mean (72 versus 66 mm SVL) and maximum (81 versus 75 mm SVL) sizes. Metamorphosis apparently takes place at approximately two years of age, and reproductive maturity is reached at four or more years of age in both sexes. If number of lobes in the multi-lobed testis indicates age, as has previously been presumed in adult male Desmognathus, then the paucity of singly-lobed, presumably young male D. folkertsi in both sampling periods suggest a declining population. Relative measures of density, however, indicated that the population at the study site was stable for eight years between collections. Therefore, testis-lobe data do not appear to be valid estimators of age in desmognathines. Clutch size, estimated from the number of developing follicles, averages approximately 40 eggs and is positively related to female body size. Ova take more than a single year in which to develop to oviposition size. This does not mean, however, that females follow a strictly biennial reproductive cycle. Introduction “Seek, and ye shall find.” No truer statement has ever been made in depicting the current rate at which new species are being discovered. With the realization of the existence of sibling species (Mayr 1942) and the development of modern techniques of genetic (e.g., Tilley and Mahoney 1996, Titus and Larson 1996) and morphological (e.g., Burbrink 2001) analyses with which to distinguish them, new forms are continually being detected. This is certainly the case within the salamander family Plethodontidae, which is simultaneously characterized by extreme morphological conservatism and broad genetic divergence (Highton 1989, Tilley and Mahoney 1996). Although distinct species are still being found, particularly in the relatively poorly surveyed neotropics (e.g., Wake and Campbell 2001), many have recently been discovered as parapatric or sympatric forms that were previously thought to be conspecific because of their morphological similarities. In the contiguous United States, for example, no fewer than 25 have been described since Petranka (1998) recognized the occurrence of 1Department of Biology, Piedmont College, PO Box 10, Demorest, GA 30535. 2Department of Biology, University of Texas at Arlington, Arlington, TX 76019. *Corresponding author - 670 Southeastern Naturalist Vol. 5, No. 4 86 plethodontid species, an increase of almost 30%. Moreover, this number does not include forms that were previously described as subspecies and recently elevated to species level. Our ecological understanding of these newly described species, however, has not kept pace with their discovery. Each one presumably occupies a niche that is unique (Gause 1934), and yet, little is known about how each of these species functions ecologically. Compounding the problem is that many of these salamander species have very limited geographic ranges and are thus vulnerable to extinction. The result is that land managers, government agencies, and others that are responsible for preserving biodiversity do not have the necessary information with which to make sound decisions regarding the management and protection of these forms. One such species is Desmognathus folkertsi Camp, Tilley, Austin, and Marshall (Dwarf Black-bellied Salamander). This is a newly described species that is sibling to D. quadramaculatus (Holbrook) (Black-bellied Salamander), with which it is sympatric (Camp et al. 2002). Its geographic range, although incompletely known, straddles the Blue Ridge Divide in northern Georgia, where it is found in and along montane streams (Camp 2004). The purpose of this paper is to present data on its reproductive life history and thereby provide a better understanding of its ecology. Field-site Description This study was conducted at the type locality for D. folkertsi where the West Fork of Wolf Creek winds through the northeast-facing Sosebee Cove (Union County, GA). Second-growth following heavy logging early in the twentieth century has resulted in a forest that is dominated by Liriodendron tulipifera Linnaeus (tulip poplar), although other cove hardwoods such as Quercus rubra Linnaeus (northern red oak) are also present (Wharton 1978). Tsuga canadensis (Linnaeus) (eastern hemlock) and Rhododendron maximum Linnaeus (rosebay rhododendron) are common along streamsides. Wolf Creek flows into the Nottely River and eventually into the Tennessee River by way of the Hiwassee River. The streambed at the study site is a combination of sand and rock with loose, flat rocks scattered throughout. The stream is turbulent, having numerous small waterfalls, with a width usually < 2 m and a depth < 25 cm. All collections were made at elevations ranging 790 to 900 m. The salamander community is diverse and includes, in addition to D. folkertsi, four other species of Desmognathus. These are the D. quadramaculatus, D. monticola Dunn (Seal Salamander ), D. ocoee Nicholls (Ocoee Salamander), and D. aeneus Brown and Bishop (Seepage Salamander ). Gyrinophilus porphyriticus (Green) (Spring Salamander), Eurycea wilderae Dunn (Blue Ridge Two-lined Salamander), and Plethodon Chattahoochee Highton (Chattahoochee Slimy Salamander) are also relatively common. Cryptobranchus alleganiensis (Daudin) (Hellbenders) occur farther downstream. Surveys conducted by the US Forest Service (USFS) 2006 C.D. Camp and J.L. Marshall 671 have found no fish in the stream where our collections were made (M. Cole, USFS, Gainesville, GA, pers. comm.). Methods Collections of what was thought to be a single species (D. quadramaculatus) were initiated at the study site in 1989 as part of a study examining life-history variation among populations occurring at different elevations. Series of metamorphosed salamanders were collected each month from July 1989 through August 1990, with emphasis placed on the collection of mature adults. Collections were made by hand-capturing individuals that had been exposed by turning rocks. In addition, during warm nights, individuals protruding from refugia were caught using a baited hook (Camp and Lovell 1989). Analysis of the data on adult body size revealed a bimodal distribution in both sexes. To determine if this phenomenon represented a stable pattern, a relatively large series was collected in May and June of 1998. In the latter case, equal emphasis was placed on collecting juvenile stages as on adults. Genetic and morphological analyses of salamanders from this and subsequent collections revealed that the two size-differentiated forms represented two distinct species (Camp et al. 2000, 2002). All collected salamanders were killed in chlorotone, preserved in 10% formalin, and then stored in alcohol. Except for those used for genetic analysis, all specimens were later deposited in the Georgia Museum of Natural History (GMNH 47151, 48744–48780, 48807–48810, 48829– 48844, 48854–48888, 48896–48898, 49007–49015, 49063–49087, 49095– 49099, 49128–49134, 49149–49154, 49174–49176, 49192–49198, 49201– 49206, 49399–49401, 49403–49407, 49427–49436, 49440–49449, 49489– 49526, 49569–49570, 49669–49677, 49697–49710). Snout–vent length (SVL) was determined by measuring each specimen from the tip of the snout to the posterior end of the vent. New metamorphs were distinguished from older juveniles and adults by their white bellies (Camp et al. 2002). Appropriate tests were run for normality (comparison to normal curve) and homoscedasticity (variance-ratio F; Sokal and Rohlf 1981). Comparisons of SVL between groups were made using parametric statistics (e.g., student’s t) when the assumptions of normality and homoscedasticity were met; otherwise, non-parametric statistics (e.g., Mann-Whitney U) were used. Specimens were then dissected to determine the state of maturity and reproductive organs. Males were determined to be mature if the testes were enlarged and pigmented and the vasa deferentia were dark in coloration and highly coiled. The number of testis lobes has frequently been used to age individual Desmognathus (e.g., Organ 1961, Tilley 1973a) and, subsequently, to estimate rates of survivorship (Hairston 1986, Organ 1961). The distribution of individuals among testis-lobe categories (those having one, two, three, or four lobes) has also been used to infer relative population stability (Tilley 1973a). Because the number of lobes is usually the same for both the left and right testis (Martof and Rose 1963), testis lobes were counted on the left side for 672 Southeastern Naturalist Vol. 5, No. 4 all specimens. Regression analysis of log-transformed data was used to determine if lobe number depends on SVL. A student’s t -test was used to compare means of the transformed number of testis lobes derived from the two collections, and chi-square analysis was used to determine if there was a difference in the distribution of individuals among testis-lobe categories. Females were determined to be mature if follicles contained yolk and the oviducts were enlarged and convoluted. The number of enlarged follicles has been shown to reasonably estimate actual clutch size in Desmognathus (Danstedt 1975, Harrison 1967, Jones 1986), although there may be a slight overestimation (Tilley 1973b); therefore, we estimated clutch size from follicle number. Because it is difficult to distinguish developing from static follicles at diameters < 1.0 mm (Martof 1962, Tilley 1968), only follicles that were clearly enlarging (> 1.5 mm in diameter) were counted to estimate clutch size. Using log-transformed data, these estimates were regressed against SVL to determine if clutch size was dependent on body size as in other species of Desmognathus (Tilley 1968). One female in the 1989–90 sample had a single enlarging follicle in each ovary. Because the next smallest number of enlarging follicles in this sample was 23, we deemed this individual to be abnormal and excluded it from analyses dealing with ova. The number of 1998 females with enlarging ova (N = 6) was too small for these analyses to be meaningful. Therefore, most analyses involving ova did not include data from the 1998 sample. To detect seasonal changes in ova size, the diameter of five representative follicles from each ovary was measured in all mature females. Mean diameter was then plotted against month. Two distinct groups of ova were evident, having parallel developmental trajectories and separated by approximately one year. Shifting the upper group and plotting it 12 months later gave a decidedly linear pattern of increase. Linear regression was used, therefore, to generate a model (mean ova diameter = dependent variable, projected month = independent variable) from which the number of months necessary for ova to reach oviposition size could be estimated. “0 months” was set where the line crossed 1.0 mm on the y-axis. Because nests were not found, egg size at oviposition had to be estimated. Gravid female Desmognathus move to secretive nesting sites several weeks prior to oviposition (Forester 1981), making their collection during normal sampling unlikely. Ova development continues during this time, and the largest ova measured from follicles are usually considerably smaller than the mean size of eggs that are actually laid (e.g., 3.8 versus 4.1 mm in D. marmoratus [Martof 1962], 4.0 versus 5.1 mm in D. quadramaculatus [Austin 1993]). Therefore, we estimated oviposition size of eggs by adding an arbitrary correction factor of 0.5 mm to the size of the largest follicles measured. Results In the 1989–90 collections, adult males ranged 58.4–79.5 (SD = 5.29) mm and averaged 71.2 mm in SVL. Similar sizes were recorded in 1998, 2006 C.D. Camp and J.L. Marshall 673 with mature males ranging 60.0 to 80.7 (SD = 4.72) mm and averaging 72.7 mm (Fig. 1). There was no difference between the two years in mean size (t = 1.463, df = 96, P = 0.147). Adult-female SVL ranged 56.0–74.5 (SD = 4.00) mm and averaged 65.1 mm in 1989–90 (Fig. 1). In 1998, females Figure 1. Body sizes of adult D. folkertsi collected in 1989–90 and 1998. 674 Southeastern Naturalist Vol. 5, No. 4 averaged 66.6 mm while ranging 57.3–73.4 (SD = 4.14) mm (Fig. 1). Similar to males, there was not a significant difference in mean female SVL between the two collections (t = 1.358, df = 61, P = 0.179). There was a greater variance in male SVL than in female for 1989–90 (F63, 39 = 2.007; P = 0.018) but not in 1998 (F33, 21 = 1.144; P = 0.735), although this latter result may have been due to smaller samples in 1998. Adult males had larger mean sizes than females in both 1989–90 (Mann-Whitney U = 431, U' = 2129; P < 0.001) and in 1998 (t = 4.865; df = 54; P < 0.001). Two newly metamorphosed juveniles were found in 1989–90 (a specimen with an SVL = 34.1 mm in September and a 39.0 mm specimen in January). Six new metamorphs were found in 1998, all in July. This latter group averaged 36.18 mm in SVL, ranging from 33.7–37.4 (SD = 1.31) mm. The smallest salamander with signs of pigmentation on the belly was 36.5 mm in SVL in 1989–90 and 40.2 mm in SVL in 1998. The largest immature specimens (both females) had completely black venters and measured 56.2 mm and 58.4 mm in SVL , respectively, in the 1989–90 and 1998 samples (Fig. 2). The largest immature male was 57.7 mm in SVL and was collected in 1998. The smallest mature males and females were 58.4 and 56.0 mm in SVL, respectively, in 1989–90, and 60.0 and 57.3 mm, respectively, in 1998. A significantly greater number of males (N = 64) than females (N = 40) was collected in 1989–90 (􀁲2 = 5.538, df = 1, P = 0.020). Although more males (N = 34) were collected than females (N = 22) in 1998, the ratio between them was not significantly different from 1:1 (􀁲2 = 2.570, df = 1, P = 0.10). The number of mature D. folkertsi (N = 104 for 1989–90 and 56 for 1998) equaled the number of mature D. quadramaculatus (N = 114 for 1989–90 and 57 for 1998) in both 1989–90 (􀁲2 = 0.459, df = 1, P = 0.50) and 1998 (􀁲2 = 0.004, df = 1, P > 0.90). On the other hand, immature D. folkertsi (N = 20 for 1989–90 and 24 for 1998) were greatly outnumbered by immature D. quadramaculatus (N = 71 and 129 for 1989–90 and 1998, respectively) during both samples. Mature males had 1–4 lobes per testis and averaged 2.03 (SD = 0.883) and 1.97 (SD = 0.717) in 1989–90 and 1998, respectively. There was not a significant difference in the mean log-transformed number of testis lobes between the two collections (t = 0.072, df = 96, P = 0.943). Moreover, the distribution of the number of testis lobes in 1998 did not differ from 1989– 90 (􀁲2 = 6.792, df = 3, P = 0.075). For both years, there were fewer mature males having single-lobed testes than with two-lobed testes (Fig. 3). There was a significant dependence of testis-lobe number on male SVL for both 1989–90 (F1, 62 = 30.512, P < 0.001, r2 = 0.330; ln testis lobes = -14.09 + 3.45 x ln SVL) and 1998 (F1, 32 = 27.053, P < 0.001, r2 = 0.458; ln testis lobes = -16.24 + 3.93 x ln SVL). The coefficient of variation (r2), however, indicated that < 50% of the variance in testis-lobe number could be explained by variation in SVL. Excluding the individual with only two developing ova, mean estimated clutch size for 1989–90 was 40.76 (range = 23–62, SD = 9.41). Although 2006 C.D. Camp and J.L. Marshall 675 only six specimens were collected with enlarging follicles in 1998, the mean clutch size was similar (38.0, range = 28–44, SD = 6.10). Clutch size estimated from the 1989–90 sample was significantly dependent on female SVL (F1, 27 = 16.738, P = 0.001). The coefficient of variation (r2) was 0.383, indicating that 38% of the variation in clutch size was explained by variation in SVL (Fig. 4). The regression equation was: ln clutch size = -6.40 + 2.42 x ln SVL. Ova diameter showed a distinct seasonal pattern of development (Fig. 5A). The largest follicles measured were 4.0 mm in diameter and occurred in April. Therefore, oviposition likely occurred during May or June, with egg size at oviposition estimated to be 4.5 mm. When the large size class of ova was plotted 12 months later and the data were analyzed by regression, the following model equation was generated: ova diameter = 1.0 + 0.21 x month (r2 = 0.721). According to this model, 16.7 months is required for ova to reach 4.5 mm (Fig. 5B). Figure 2. Body sizes of metamorphosed, immature D. folkertsi collected in 1989–90 and 1998. 676 Southeastern Naturalist Vol. 5, No. 4 Discussion The reproductive life history of D. folkertsi is largely typical of dusky salamanders. Like other members of the genus, both oviposition (Austin 1993, Bruce 1988, Organ 1961) and metamorphosis (Austin and Camp 1992, Beachy and Bruce 2003, Bernardo 2000, Bruce 1989) tends to occur during warmer months. The single new metamorph collected in January, however, suggests that a few individuals may be asynchronous in this respect. Size classes have been shown to accurately estimate pre-maturation ages in Desmognathus (Austin and Camp 1992, Beachy and Bruce 2003, Bruce 1989, Castanet et al. 1996). Camp et al. (2002), therefore, used size classes to estimate a larval period of two years. This estimate was based on the presence of four size classes of larvae (presumably a mixture of both D. folkertsi and D. quadramaculatus) in an April sample and the fact that SVL’s of new metamorphs of D. folkertsi and D. quadramaculatus from Figure 3. Distribution of adult male D. folkertsi sorted by testis-lobe number. 2006 C.D. Camp and J.L. Marshall 677 Figure 4. Relationship between clutch size and female body size in D. folkertsi. Statistical analysis was performed using log-transformed data. multi-year, pooled samples from July correspond to the second and fourth size class, respectively (Fig. 6). The apparent loss in body-size variance in the third-year larval class is consistent with metamorphosing D. folkertsi having left the larval population prior to three years of age. Too few immature individuals were collected to estimate the length of the juvenile period, although the 1998 sample suggests the possible occurrence of at least three size classes (Fig. 2). The 1989–90 sample also appears to be distributed among three size classes. This, however, is an aberration because these specimens were collected over the course of 14 months. It seems likely, however, that, like other members of the genus, at least two years elapse between metamorphosis and sexual maturity (Bruce et al. 2002, Castanet et al. 1996). Therefore, reproductive maturity in D. folkertsi is reached in 4+ years. In other Desmognathus, males mature earlier at smaller sizes than females and then grow to reach larger sizes (Bruce 1993, Bruce et al. 2002, Castanet et al. 1996). The result is that, at least in many populations, male and female mean body sizes are approximately equal (Austin 1993, Bruce et al. 2002, Castanet et al. 1996). Because male and female Desmognathus have similar pre-maturation growth rates (Austin and Camp 1992, Beachy and Bruce 2003, Bruce et al. 2002, Castanet et al. 1996), male and female D. folkertsi appear to mature at the same age. This is suggested by both the largest immatures of both sexes and the smallest matures of both sexes having similar SVLs. Male D. folkertsi do reach larger maximum sizes than females and apparently exhibit greater post-maturation growth than 678 Southeastern Naturalist Vol. 5, No. 4 Figure 5. Seasonal variation in mean follicle diameter in adult female D. folkertsi. (A) Actual variation in follicle size in females collected from July 1989 through August 1990. (B) Regression of mean follicle diameter over time used to predict time necessary for follicles to reach oviposition size. Open circles represent data points shifted 12 mo; see text for explanation. Regression equation: ova diameter = 1.0 + 0.21 x month (r2 = 0.721). 2006 C.D. Camp and J.L. Marshall 679 females, both situations being typical of the genus (Bruce 1993, Bruce et al. 2002, Castanet et al. 1996, Tilley 1980). The phenomenon of females maturing at an unusually small size relative to adult-male size, however, may have led to males having larger average, not just maximum, sizes than females, unlike many other species of Desmognathus. The paucity of immature individuals in our samples indicates that our sampling techniques were biased towards adults. The 1:1 ratio between Figure 6. Body sizes of a sample of presumed larval D. folkertsi and D. quadramaculatus collected in April 1990 and new metamorphs collected in July pooled across several years. Figure is taken from Camp et al. (2000). 680 Southeastern Naturalist Vol. 5, No. 4 mature D. folkertsi and D. quadramaculatus in both samples, taken eight years apart, suggests that D. folkertsi was equally dense both years and that the population was probably stable during and between the times when the two collections were made. Moreover, the relatively large number of immature D. quadramaculatus collected at the same time using the same techniques shows that our techniques did not overly favor adult individuals of that species, particularly during the 1998 sample. Similar to our collections of D. quadramaculatus, immature individuals have outnumbered adults in most studies of large desmognathines (Austin 1993; Beachy and Bruce 2003; Bruce 1988, 1989, 1995). These observations raise the possibility that young D. folkertsi are more secretive and/or they exploit different microhabitats than adults. Humphrey (1922) suggested that the number of testis lobes can be used to age male Desmognathus, with one lobe representing two consecutive reproductive years. Following this hypothesis, several researchers attempted to age male Desmognathus and use these ages to estimate rates of survivorship (e.g., Danstedt 1975; Organ 1961; Tilley 1973a, 1974). Hairston (1980, 1986) argued that small Desmognathus evolved to become more terrestrial in response to predation by large, aquatic congeners. He based this hypothesis, in part, on Organ’s (1961) survivorship estimates derived form testis-lobe data. Tilley (1977), however, pointed out that the relationship between the number of testis lobes and ages may not be exact, a fact confirmed through studies of spermatogenesis (Sipe 1973) and aging using skeletochronological techniques (Bruce et al. 2002, Castanet et al. 1996). We found a significant, though rather weak, relationship between the number of testis lobes and male body size. We also found that fewer one-lobed (“younger”) than two-lobed (“older”) individuals were present in both the 1989–90 and the 1998 samples. If testis lobes indicate age in adult male Desmognathus, then the relatively few one-lobed, presumably younger individuals would necessarily indicate an unstable or declining population (Tilley 1973a). The apparent stability of our sampled population from 1989 to 1990 indicates that testis-lobe number cannot be used to estimate age or survivorship, and the reliance on such estimates (e.g., Hairston 1980, 1986) may yield fallacious conclusions. Clutch size was estimated to be approximately 40 and to significantly depend on female body size. The dependence of clutch size on body size is a general trend in Desmognathus (Bruce 1996; Csanady 1978; Harrison 1967; Hom 1987; Jones 1986; Tilley 1968, 1973a, 1974; Trauth et al. 1990), although there are exceptions (Bruce 1996, Bruce and Hairston 1990, Tilley 1968). The study population of D. folkertsi is both seasonal and synchronous in the rate of ova development. Organ (1961) hypothesized that female Desmognathus of all five species he studied (D. quadramaculatus, D. monticola, D. fuscus, D. orestes, D. wrighti) lay eggs every other year, as did Martof (1962) for D. marmoratus. The proposed biennial reproductive cycle in female Desmognathus has been challenged (Tilley 1977), and 2006 C.D. Camp and J.L. Marshall 681 certainly smaller members of the genus often lay annually (Fitzpatrick 1973, Forester 1977, Harrison 1967, Huheey and Brandon 1973). We cannot conclude that D. folkertsi has a truly biennial cycle in the sense that each female oviposits every other year. Nevertheless, the 16-month estimate for ova development clearly indicates that annual oviposition is not possible (Fig. 5B). Even if we did not add the additional correction in diameter of 0.5 mm to allow for secretive, pre-oviposition development, and presumed that eggs are laid at 4.0 mm, this would still require 14 months of development. Egg size is directly related to body size in plethodontids (Bernardo 1994, Bruce 1990, Salthe 1969). Eggs that are too large simply require too much time to complete vitellogenesis within the constraints of an annual cycle. It is likely, then, that larger species are characterized by female cycles that require at least two years between oviposition events. Calling such a cycle biennial is probably convenient. However, as Tilley (1977) pointed out, the possibility of irregular cycles in which females skip one or more years cannot be discounted. Although the reproductive biology of D. folkertsi is typical of that for large species of Desmognathus, other aspects of its ecology have yet to be studied in detail. Camp et al. (2002) indicated that it appears to have the same microhabitat preferences as the larger D. quadramaculatus, with which it is sympatric. Camp (1997) showed that D. folkertsi is not a significant prey item of D. quadramaculatus, although how the former species interacts with any of its sympatric congeners is not known. Even so, the data we have presented gives a clearer picture of the ecological niche occupied by D. folkertsi. Acknowledgments We thank Rick Austin, Paul Hotchkin, Will Bush, and Marcelo Saldivia for help in collecting specimens and D. Huestis for critical review of the manuscript. This manuscript was prepared while J.L. 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