Herpetologica, 63(2), 2007, 176–183 E 2007 by The Herpetologists’ League, Inc.
MALE RED-BACKED SALAMANDERS CAN DETERMINE THE REPRODUCTIVE STATUS OF CONSPECIFIC FEMALES THROUGH VOLATILE CHEMICAL SIGNALS BENJAMIN J. DANTZER1,2,3 1
AND
ROBERT G. JAEGER1
Department of Biology, University of Louisiana at Lafayette, Lafayette, LA 70504-2451, USA
ABSTRACT: Previous research suggests that female red-backed salamanders (Plethodon cinereus) in Virginia court biennially whereas males court annually. Therefore, males may face a choice to court either gravid or nongravid females. Because gravid females represent an immediate insemination opportunity (whereas nongravid females do not), male red-backed salamanders may be under selection to be able to distinguish the reproductive status of conspecific females. We conducted an experiment to determine if males could discriminate between gravid and nongravid conspecific females through volatile chemical signals. Focal males were allowed to establish territories in testing arenas for 5 d and then were exposed to three treatments in a randomized order: volatile chemical signals from gravid females, nongravid females, and a control (blank filter paper). Randomization tests revealed that focal males exhibited significantly more aggressive behavior when they were exposed to volatile chemical signals from nongravid females than when they were exposed to those from gravid females and the control. We infer that male red-backed salamanders can determine the reproductive status of conspecific females through volatile chemical signals, which may influence their social associations. Key words: Aggression; Chemoreception; Pheromones; Plethodon cinereus; Randomization statistic; Reproductive status; Social monogamy
PRIOR to the onset of any reproductive act, an organism must determine if the targeted individual is a conspecific and of the opposite sex. In order to maximize their reproductive and/or time investment, males must also determine if a sexually mature conspecific female possesses yolked ova that are fertilizable. Thus, in mammals, males should determine if females are ovulating, whereas in other vertebrates (e.g., amphibians or reptiles), males should determine if females are gravid (i.e., possess yolked ova). Females of many species advertise their reproductive status (i.e., capability to be fertilized) through several signaling mechanisms, including behavioral (e.g., reduced aggression towards males by female pygmy marmosets, Cebuella pygmaea, during the ovulatory period: Converse et al., 1995), chemical (e.g., through pheromone tracks deposited by female red-sided garter snakes, Thamnophis sirtalis parietalis: O’Donnell et al., 2004), acoustic (e.g., changes in vocalizations by Barbary macaques, Macaca sylvanus: Semple and McComb, 2000), and visual 2
CORRESPONDENCE: e-mail,
[email protected] PRESENT ADDRESS: Department of Zoology, Michigan State University, East Lansing, MI 48824, USA. 3
signals (e.g., sexual skin swellings by female nonhuman primates during ovulation: Dixson, 1983). Males appear to use such information to make decisions concerning what females with which to associate. For instance, many laboratory studies using mammals have shown that when given a choice, males will almost always associate with areas saturated with chemical signals from ovulating females over areas saturated with chemical signals from nonovulating females (reviewed by Johnston, 1983). Likely due to the energetic costs associated with yolking ova (sensu Petranka, 1998), female red-backed salamanders (Plethodon cinereus) are thought to court biennially whereas males court annually (Sayler, 1966; Takahashi, 2002). However, the interval between courting periods may vary geographically (Maerz and Madison, 2000). Due to the possibility of a male-biased operational sex ratio, male red-backed salamanders may face a choice to court either gravid (possessing yolked ova) or nongravid (not possessing yolked ova) females. Because gravid females possess yolked ova that are able to be fertilized immediately, courting gravid females is likely more profitable to the male’s immediate reproductive fitness than courting nongravid females. However, the discrimina-
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tory method that males use to make such decisions is currently unknown. Red-backed salamanders are abundant (up to 2.8 salamanders/m2 at our study site in Virginia: Mathis, 1991) in the leaf litter on the forest floors of eastern North America (Conant and Collins, 1998). At our study site in Virginia, both larger males and females appear to defend feeding territories and, possibly in conjunction, mating territories under rocks and logs during both the noncourtship and courtship seasons (Mathis, 1989, 1991). These territories contain a finite reservoir of invertebrates that the salamanders feed upon during dry periods (Jaeger, 1980). Red-backed salamanders appear to use a suite of visual signals (e.g., all-trunk raised threat posture: Jaeger, 1984; Jaeger and Schwarz, 1991) and chemical signals (reviewed by Dawley, 1998) in their sociosexual interactions. Chemical signals are used as territorial markers (reviewed by Jaeger, 1986), and also appear to transmit a variety of information between conspecifics (reviewed by Dawley, 1998). These signals may be a singular chemical substance or amalgam of chemical substances that transfers information between conspecifics (sensu Bradbury and Vehrencamp, 1998) and may be contained in fecal pellets, post-cloacal gland secretions (Simons et al., 1994), or other volatile bodily secretions (Dantzer and Jaeger, 2007; Martin et al., 2005). Red-backed salamanders possess two anatomical structures that are used in chemodetection: a well developed vomeronasal organ, which is stimulated by nonvolatile molecules (Dawley and Bass, 1989), and the main olfactory epithelium, which is stimulated primarily by volatile molecules (Dawley, 1992). They stimulate their vomeronasal organ by ‘nose tapping’, which enables nonvolatile molecules to move up the nasolabial grooves to be concentrated in the vomeronasal organ (Dawley and Bass, 1989). Volatile chemical signals are likely detected by rapid oscillations of the buccopharyngeal region, which may increase the rate of air flow (and consequently any volatile chemical signals) over the main olfactory epithelium (Simons et al., 1997; Thurow, 1968). We tested the hypothesis that territorial male red-backed salamanders can discrimi-
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nate between gravid and nongravid conspecific females through volatile chemical signals. In other plethodontid salamanders, the presence of large, yolked ova in females is thought to be correlated with ‘particular integumentary cues’, which act to increase the level of courtship behavior by conspecific males (Houck and Sever, 1994). Because male redbacked salamanders may preferentially court gravid (immediate insemination opportunity) rather than nongravid females, we made the following predictions. (1) Focal males would be more antagonistic (i.e., more time spent in all-trunk raised threat posture: see below for description) when presented with volatile chemical signals from nongravid females compared to gravid females, and a control (blank filter paper). The all-trunk raised threat posture has been shown to be a reliable indicator of the level of aggression by males towards females (e.g., Joseph et al., 2005). (2) Focal males would exhibit significantly more nose taps (chemoinvestigatory behavior) when presented with volatile chemical signals from gravid females relative to nongravid females, and a control treatment. We made this prediction in the context of mate assessment. Nonvolatile chemical cues have higher molecular weights and therefore are thought to contain more information (Alberts, 1992). Thus, after detecting that a female is gravid through volatile chemical signals, we predicted that males would nose tap the substrate to detect nonvolatile chemical cues in an attempt to gain more information about the gravid female (e.g., body size: Mathis, 1990; parasite load: Maksimowich and Mathis, 2001). MATERIALS AND METHODS Collection and Maintenance of Salamanders We collected 60 male, 60 nongravid female, and 60 gravid female red-backed salamanders during the courtship season (Sayler, 1966) in October 2004 from Jefferson National Forest, near Mountain Lake Biological Station (MLBS), Giles County, Virginia. We determined the reproductive status of females (i.e., gravid or nongravid) by checking for the presence or absence of yellowish yolked ova, which are visible through the ventral abdomen. We collected only adult (.32 mm
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snout–vent length [SVL]: Sayler, 1966), nonpaired salamanders (alone under a rock or log: sensu Prosen et al., 2004). To determine the sex of collected salamanders, we placed them individually into plastic bags, held them up to a light source, and checked for the presence or absence of black testes through the semitransparent abdominal wall (Gillette and Peterson, 2001). Due to potential confounding effects (e.g., Wise et al., 2004), we did not collect salamanders with autotomized tails. We placed salamanders individually into plastic jars containing paper towels moistened with spring water, placed the containers in ice chests, and transported them back to our laboratory in Lafayette, Louisiana, U.S.A. We returned salamanders to the collection site upon completion of the experiment. In the laboratory, we measured the SVL of each salamander using digital calipers (VWR, West Chester, Pennsylvania) and excluded from the experiments any juvenile salamanders that were accidentally collected. Salamanders were housed individually in Petri dishes (15 3 2 cm) lined with two moist (spring water) coffee filters. They were fed 15–20 flies (Drosophila melanogaster) weekly and kept at 17 6 2 C with a 12:12 light:dark photoperiod. Until the experiment began, substrates were watered every 3 d with spring water and changed when soiled (approximately every 7 d). Salamanders were given two months to acclimate to these conditions before behavioral trials began (sensu Joseph et al., 2005). Description of Treatments and Sources of Chemical Cues We used males as the focal salamanders (i.e., subjected to experimental manipulations), and gravid and nongravid females as source salamanders (i.e., chemical cue donors). To reduce potential kinship or familiarity, we collected focal and source salamanders from two collection sites separated by at least 1 km. We randomly chose 30 focal males from the total pool of males and randomly paired each one to one gravid source female and one nongravid source female, which were both from the geographically distant site. Focal male salamanders and their paired chemical cue donors were housed individually and were
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never in contact in the laboratory. We also size-matched gravid and nongravid source females to the nearest 2 mm. To determine if male red-backed salamanders can determine the reproductive status of females through volatile chemical signals, during March 2005, we exposed all focal males to two experimental treatments and one control treatment in a randomized order. Treatment 1 was termed ‘Gravid female pheromones’ and Treatment 2 was termed ‘Nongravid female pheromones’, which were filter papers that had lined the Petri dishes of source gravid and nongravid females respectively for 5 d. Treatment 3 (control) was termed ‘Blank filter paper’, which were filter papers that had lined Petri dishes that did not contain salamanders. Experimental Protocol On day 1 of each testing cycle, we placed focal male salamanders (n 5 30) into testing arenas (24 3 24 3 2 cm, Nunc bioassay dishes: Cole-Parmer, Vernon Hills, Illinois) lined with moistened paper towels, fed them 15–20 flies (D. melanogaster), and allowed them to establish territories for 5 d (sensu da Silva Nunes and Jaeger, 1989). In the center of each testing arena, we placed a piece of opaque PVC plastic tubing (10 3 1 cm) that was capped with patches of vinyl screening secured with MIG welding wire. These pieces of plastic tubing later held the sources of chemical cues. On the same day, we placed female source salamanders into clean Petri dishes (15 3 2 cm) that were lined with 1 moistened coffee filter and 1 smaller piece of filter paper (9 3 9 cm), with the latter being used as the source of chemical cues to which focal salamanders were subjected. For control treatment 3 (blank), we prepared Petri dishes in the same manner described above except that they did not contain salamanders. On day 6 of each testing cycle, we conducted the observational trials of focal males. Immediately prior to testing, we misted the testing arenas with spring water and placed each focal male under a habituation cup (9 3 1.5 cm inverted Petri dish) in a randomly determined corner of the bioassay dish. Using a clean pair of forceps for each trial, we rolled the filter papers from the
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source and control Petri dishes into cylinders (9 3 0.5 cm) so that any salamander secretions were on the outside, and individually placed them into the plastic tubing. To prevent the focal males from physically accessing the filter papers, we recapped the tubes with the patches of vinyl screening. Thus, the focal salamanders were unable to contact the soiled filter papers, which ensured that they only responded to volatile chemicals emitted from them. We then replaced the tube (containing filter paper) into the center of the bioassay dish. This process was repeated for each focal salamander during each testing day. Following a 15-min habituation period, we recorded two behavioral patterns of focal males continuously for 15 min under dim lighting (i.e., 45-W incandescent light bulb pointed towards the white ceiling): (1) time (in seconds) spent in the all-trunk raised threat postures ATR2–ATR5, where the limbs are extended downward and at least the head and posterior thoracic region are off the substrate (Jaeger and Schwarz, 1991) and (2) the number of nose taps to the substrate by focal males. Behavioral observations occurred from 0900–1800 h. One focal male escaped, and thus the sample size was reduced to n 5 29. Statistical Analyses Because the data were right skewed, somewhat heteroscedastic, and not normally distributed, conventional parametric statistics were inappropriate. Rather than conduct traditional nonparametric statistics, which may result in a loss of information due to the use of ranks (Adams and Anthony, 1996), we used a randomization procedure (Manly, 1997). Randomization tests are considered to be more powerful than some nonparametric (i.e., Kruskal-Wallis test: Adams and Anthony, 1996; Manly, 1997) and parametric statistical tests (Efron and Tibshirani, 1993). Although randomization tests relax the assumption of normality (Manly, 1997), they do not relax the assumption of homoscedasticity (Hayes, 2000); therefore, we square-root transformed all of the raw data so that the variances among the groups were more similar. We conducted two randomization tests: (1) time spent in the ATR threat postures and (2)
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number of nose taps to the substrate. If significant differences were detected from these overall tests (a 5 0.05), we then conducted the following pairwise comparisons also using randomization tests: (1) Gravid female pheromones versus Nongravid female pheromones, (2) Gravid female pheromones versus Blank filter paper, and (3) Nongravid female pheromones versus Blank filter paper. To reduce the probability of Type 1 errors, the alpha levels of the pairwise comparisons were adjusted using a standard Bonferroni adjustment (i.e., a 5 0.0167) (Sokal and Rohlf, 1995). All P-values reported here are two-tailed. For all randomization tests, we used the following procedure. First, using a two-way analysis of variance (ANOVA) with treatment and individual blocks as the two factors, we calculated the Type III sums-of-squares treatments (SStreat) among the three groups (for the two initial randomization tests for the overall effects) or among the two groups (for any pairwise comparisons). Second, using a program that was written in SAS (SAS Institute Inc., Version 9.1, 2004), the data were randomly reshuffled without replacement within each individual block. The program then used a two-way ANOVA to calculate the Type III SStreat from the reshuffled data set. This process was repeated to create 6600 new reshuffled data sets, which was more than the minimum number of replications recommended by Adams and Anthony (1996). Last, the SStreat from the actual data was compared to the frequency distribution of the SStreat generated from all 6600 reshuffled data sets. We calculated Pvalues by dividing the number of times the SStreat from the reshuffled data sets exceeded the SStreat from the actual data by the total number of reshuffled data sets (6600). Thus, P-values reported here are analogous to the proportion of the SStreat from all 6600 reshuffled data sets that exceeded the SStreat from the actual data. Using these randomization tests, we tested the null hypothesis that the observed patterns in the data were simply due to chance alone and that therefore the observed pattern in the data would be a typical result of randomly reshuffling the data among the individual blocks. Copies of the SAS
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program used to conduct the randomization tests are available from the senior author upon request. RESULTS The initial randomization test for overall effects detected significant differences for the time focal male salamanders spent in the ATR threat postures among the three treatments (Fig. 1A). Focal males spent significantly more time in the ATR threat postures when exposed to volatile chemical signals from nongravid females than when they were exposed to those from gravid females (P 5 0.010) and the control (P 5 0.0001). The second randomization test for overall effects did not detect significant differences for the number of nose taps to the substrate by focal males among the three treatments (Fig. 1B). However, because the P-value was relatively small (P 5 0.066, but a 5 0.05), we also conducted pairwise comparisons for this response variable. The general trend in the data indicated that focal male salamanders nose tapped the substrate more often when they were exposed to volatile chemical signals from gravid females or nongravid females than when they were exposed to the control (P 5 0.031 and P 5 0.028, respectively) (Fig. 1B). However, none of the pairwise comparisons for this response variable were statistically significant (a 5 0.0167). DISCUSSION Previous studies have shown that territorial red-backed salamanders are more aggressive toward conspecific intruders that are a greater ‘threat’. For example, resident males are more antagonistic towards male intruders than towards female intruders, likely because the former are competitors for both mates and resources, whereas the latter are only competitors for resources (Jaeger, 1984). Furthermore, red-backed salamanders demonstrate such a pattern when exposed to both nonvolatile (Mathis, 1990; Page and Jaeger, 2004) and volatile (Dantzer and Jaeger, 2007) chemical signals from same-sex and opposite-sex conspecifics (i.e., they exhibit significantly more aggression when exposed to chemical cues from same-sex conspecifics
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compared to those from opposite-sex conspecifics). In our experiment, resident males spent significantly more time in the ATR threat posture in the presence of volatile chemical signals from nongravid females than they did when exposed to volatile chemical signals from gravid females and the control (blank filter paper). Thus, male red-backed salamanders identified nongravid females as a greater threat (or of less value) than gravid females and therefore may repel nongravid females that they encounter. Similar previous experiments have yielded differing results. Horne (1988) found that nongravid females were more submissive toward male intruders than were gravid females. Horne (1988) advocated that these data suggested that males are more antagonistic towards nongravid females compared to gravid females. Further, Horne (1988) found that male intruders were more submissive toward gravid female residents than they were to nongravid female residents. Intruding males, she argued, are more submissive because gravid females are potential mates while nongravid females (in their current state) are not. Conversely, Thomas et al. (1989) found that the agonistic behavioral patterns of male residents did not differ when presented with gravid and nongravid female intruders. Our data support the suppositions of Horne (1988) but not those of Thomas et al. (1989); i.e., resident males treated volatile pheromones from nongravid females with more hostility than those from gravid females. Some red-backed salamanders at our study site in Virginia are thought to engage in socially monogamous behavior (i.e., reciprocal preferential behaviors between two conspecifics of the opposite sex: sensu Gillette et al., 2000; but not always genetic monogamy: Liebgold et al., 2006). Through socially monogamous associations, females and males are thought to co-defend territories (Lang and Jaeger, 2000) and gain access to opposite-sex conspecifics for matings. Because female redbacked salamanders in Virginia are thought to court biennially, males may form such associations with either nongravid or gravid females. We suggest that the ability to discriminate between gravid and nongravid females may influence the socially monoga-
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FIG. 1.—Responses of male red-backed salamanders (n 5 29) to the control and two experimental treatments. (A) A randomization test (6600 iterations) indicated that significant differences (a 5 0.05) existed among the data for the time focal males spent in the ATR threat posture (P 5 0.0007). (B) A second randomization test (6600 iterations) indicated that significant differences did not exist among the data for the number of nose taps to the substrate by focal males (P 5 0.066). Different letters above the box and whisker plots indicate significant differences (a 5 0.0167) for pairwise comparisons that were detected using randomization tests. Raw data are presented in the box and whisker plots, but all statistical analyses were conducted using the square-root transformed data. In the box and whisker plots, the boxes represent the interquartile range, whiskers represent the range, the horizontal black bar represents the median, and the triangle represents the mean.
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mous associations that males form (i.e., preferentially form such associations with gravid females rather than nongravid females). However, both Hom et al. (1997) and Thomas et al. (1989) argued that males that associate with nongravid females may increase their probability of obtaining future matings when such females become gravid. Further, in a field survey during the noncourtship season (summer), Jaeger et al. (1995) found 47 malenongravid female pairs (out of a total of 48 pairs), which may suggest that males and nongravid females may also form socially monogamous associations outside of the courtship season. Because males appear to be able to determine the reproductive status of conspecific females through volatile chemical signals, they may exert more aggressive behavior towards nongravid females to ‘drive them off’ because such females do not currently possess fertilizable ova. On the other hand, if males detect that an encountered female is gravid, they may exhibit less aggressive behavior towards her to lessen the chance of ‘driving her off’. We conclude then that male red-backed salamanders can discriminate between gravid and nongravid conspecific females through volatile chemical signals, and that the acquisition of such information influences their behavior toward gravid versus nongravid females and possibly their social associations. Acknowledgments.—We thank H. M. Wilbur and E. S. Nagy for permission to conduct research at MLBS, N. R. Kohn, D. McCauley, and M. M. Wilcox for field assistance, D. C. Adams, S. Mopper, and especially P. L. Leberg for statistical advice, and E. B. Liebgold, B. R. Moon, and two anonymous reviewers who provided critical reviews of an earlier draft of this manuscript. This research was partially funded by the Graduate Student Organization of The University of Louisiana at Lafayette and a University Master’s Fellowship to B. J. Dantzer. Salamanders were collected under permit 020999 from the Virginia Department of Game and Inland Fisheries, and were maintained in accordance with IACUC protocol number 2004-8717-079, which was approved by The University of Louisiana at Lafayette Animal Care and Use Committee.
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