The effects of sex on chemosensory communication in a terrestrial salamander (Plethodon shermani)

Although much evidence reveals sexually dimorphic processing of chemosensory cues by the brain, potential sex differences at more peripheral levels of chemoreception are understudied. In plethodontid salamanders, the volume of the vomeronasal organ (VNO) is almost twice as large in males as compared to females, both in absolute and relative size. To determine whether the structural sexual dimorphism in VNO volume is associated with sex differences in other peripheral aspects of chemosensation, we measured sex differences in chemo-investigation and in responsiveness of the VNO to chemosensory cues. Males and females differed in traits influencing stimulus access to VNO chemosensory neurons. Males chemo-investigated (“nose tapped”) neutral substrates and substrates moistened with female body rinses more than did females. Compared to females, males had larger narial structures (cirri) associated with the transfer of substrate-borne chemical cues to the lumen of the VNO. These sex differences in chemo-investigation and narial morphology likely represent important mechanisms for regulating sex differences in chemical communication. In contrast, males and females did not differ in responsiveness of VNO chemosensory neurons to male mental gland extract or female skin secretions. This important result indicates that although males have a substantially larger VNO compared to females, the male VNO was not more responsive to every chemosensory cue that is detected by the VNO. Future studies will determine whether the male VNO is specialized to detect a subset of chemosensory cues, such as female body rinses or female scent marks.     

Fine-scale population differentiation and gene flow in a terrestrial salamander (Plethodon cinereus) living in continuous habitat

Several recent studies have shown that amphibian populations may exhibit high genetic subdivision in areas with recent fragmentation and urban development. Less is known about the potential for genetic differentiation in continuous habitats. We studied genetic differentiation of red-backed salamanders (Plethodon cinereus) across a 2-km transect through continuous forest in Virginia, USA. Mark-recapture studies suggest very little dispersal for this species, whereas homing experiments and post-Pleistocene range expansion both suggest greater dispersal abilities. We used six microsatellite loci to examine genetic population structure and differentiation between eight subpopulations of red-backed salamanders at distances from 200 m to 2 km. We also used several methods to extrapolate dispersal frequencies and test for sex-biased dispersal. We found small, but detectable differentiation among populations, even at distances as small as 200 m. Differentiation was closely correlated with distance and both Mantel tests and assignment tests were consistent with an isolation-by-distance model for the population. Extrapolations of intergenerational variance in spatial position (sigma(2)<15 m(2)) and pair-wise dispersal frequencies (4 Nm < 25 for plots separated by 300 m) both suggest limited gene flow. Additionally, tests for sex-biased dispersal imply that dispersal frequency is similarly low for both sexes. We suggest that these low levels of gene flow and the infrequent dispersal observed in mark-recapture studies may be reconciled with homing ability and range expansion if dispersing animals rarely succeed in breeding in saturated habitats, if dispersal is flexible depending on the availability of habitat, or if dispersal frequency varies across the geographic range of red-backed salamanders.     

The influence of historical landscape change on genetic variation and population structure of a terrestrial salamander (Plethodon cinereus)

Forest loss and fragmentation is expected to shape the genetic structure of amphibian populations and reduce genetic variation. Another factor widely understood to have impacted these same parameters in North America is the range expansion that occurred following glacial retreat at the end of the Pleistocene. The Eastern Red-Backed Salamander (Plethodon cinereus) has been subjected to both processes. In this context, we investigated the historical events that are likely to have shaped genetic variation in this species using a panel of six microsatellite markers screened on individuals sampled across ten localities in northeastern Indiana, USA. We found low genetic diversity across forest patches and minimal differentiation. We expected population structure associated with forest fragmentation to result from genetic drift in isolation but instead found that a balance between gene flow and drift was ~50 times more likely. Ratios of allele number and range (M), and coalescent modeling of population demography suggested the occurrence of marked historic decline in effective population size across the region. Taken together, the data point to a loss of genetic variation which preceded deforestation over the past 200 years. This result indicates an important role for ancient demographic processes in shaping current genetic variation that may make it difficult to detect the effect of recent habitat fragmentation.     

Sperm aggregations in the spermatheca of the red back salamander (Plethodon cinereus)

The spermatheca of Plethodon cinereus is a compound tubular gland that stores sperm from mating in early spring (March–April) to oviposition in summer (June–July). The seasonal variation of sperm storage in this species has previously been studied by light and transmission electron microscopy. In this paper, sperm aggregations, interaction of sperm with the spermathecal epithelium, and spermathecal secretions are studied using scanning electron microscopy. Within spermathecal tubules, relatively small groups of sperm are aligned along their entire lengths in parallel arrays. This pattern is similar to other plethdontids with complex spermathecae. Lumina of spermathecal tubules are filled with secretory material in April prior to the arrival of sperm, and after sperm appear, a coating of secretory material persists on the apices of the spermathecal epithelium. Sperm peripheral to the central luminal mass can become embedded in the secretory matrix or pushed deeper into the spermathecal epithelium. The spermathecal secretions may serve to attract and prolong the viability of sperm, but sperm that become enmeshed in the secretions or epithelium are phagocytized. Sperm and spermathecal secretions are largely absent after ovulation and in summer months, and new secretory vacuoles are formed in fall, although mating does not occur until spring.     

Space Use in Response to Conspecifics by the Red-backed Salamander (Plethodon cinereus, Plethodontidae, Caudata)

The presence of conspecifics, as well as the distribution and quality of resources, can influence habitat settlement decisions of animals. We studied the habitat preference and group size of the red-backed salamander, Plethodon cinereus , from the Upper Peninsula of Michigan. In contrast to previous work with P. cinereus from Virginia, we found groups of P. cinereus within higher quality habitat than solitary individuals and these groups contained both small and large individuals. Under identical conditions in the laboratory, salamanders from Virginia formed a uniform spatial distribution and individuals from northern Michigan displayed an aggregated spatial distribution. Our results suggest that P. cinereus from northern Michigan preferentially settle near conspecifics, while P. cinereus from Virginia avoid them. Our laboratory experiments also suggest that the aggregation behaviour of P. cinereus from northern Michigan may be the result of attraction to conspecifics rather than attraction to habitat features.     

A fresh look at meiosis and centromeric heterochromatin in the red-backed salamander,Plethodon cinereus cinereus (Green)

Male meiosis, with special regard to the centromeric heterochromatin and to centromeric structure, has been studied in the salamander, Plethodon cinereus cinereus. In this salamander, n = 14. Early meiotic prophase proceeds as described by other authors. Pachytene is followed by a diffuse stage in which much of the chromosomal DNA becomes reorganized into fine lateral loops which spring from the bivalent axes. These loops can be seen along the bivalent axes as early as zygotene. Loops are maximally extended in the diffuse stage. The formation of diplotene bivalents involves a return of this extended DNA into the axes of the bivalents. — At leptotone, centromeric heterochromatin is in one or a few large masses. These masses break up during zygotene. At pachytene there is one mass of heterochromatin at the centromeric region of each bivalent. The heterochromatin remains condensed in the diffuse stage. During diplotene, centromeric heterochromatin becomes less conspicuous, and it is possible to see 4 centromere granules in each diplotene bivalent. These observations support the view that centromeres replicate at pre-meiotic S-phase when the associated hetero-chromatin is replicated. In the interphase before the 2nd division, the hetero-chromatin often forms a broken ring corresponding to the positions of the centromeres at the end of anaphase 1. There are 14 masses of heterochromatin in nuclei at prophase of the 2nd division. In spermatids, the heterochromatin appears as a single solid mass or a broken ring.     

The influence of habitat provisioning: use of earthworm burrows by the terrestrial salamander, Plethodon cinereus

Studies examining the influence of habitat provisioning by one species on the behavior of other species can provide key insights regarding impacts of ecosystem engineers on the availability of resources to other species. More specifically, an organism’s use of additional habitat provided by ecosystem engineers may affect the interpretation of observational or demographic data. We chose to examine the possible influence of earthworms, as ecosystem engineers, on the behavior of terrestrial salamanders, common forest vertebrates in North America. We conducted two experiments in microcosms to examine whether the earthworm Lumbricus terrestris acted as an ecosystem engineer by providing additional habitat (burrows) for Plethodon cinereus, a common woodland salamander. We also examined whether the behavioral changes of the non-burrowing, fossorial P. cinereus differed across age classes. Adults and juveniles responded similarly, with both adults and juveniles found under cover objects more often when earthworms were absent and using earthworm burrows when available. Our field data supported our experimental data in that greater numbers of earthworms on field plots were associated with a lower proportion of cover objects being occupied by salamanders (i.e., salamanders were likely underground). We found no differences in the response of adults and juveniles to the presence of earthworms. By providing underground habitat for a common terrestrial amphibian, the presence of earthworms may impact demographic studies on salamanders and conclusions drawn from those studies regarding aboveground faunal communities in eastern North America.     

Sperm storage in the spermatheca of the red-back salamander,Plethodon cinereus (Amphibia: Plethodontidae)

In northern Indiana, the mating season of Plethodon cinereus occurs after hibernation from March until June, when oviposition begins. During the mating season, a female stores sperm in its spermatheca, a compound tubular gland in the roof of the cloaca. The apical cytoplasm of the spermathecal epithelium is filled with large secretory vacuoles whose product is released while sperm are stored. Females induced to oviposit in June and July by injections of human chorionic gonadotropin (hCG) still retain much sperm 1 month after oviposition, but secretory vacuoles are absent in all specimens sacrificed in July and August. Instead, some sperm are embedded in the spermathecal epithelium with resultant spermiophagy involving lysosomes. A female sacrificed in September 2 months after oviposition possesses scant sperm, but spermiophagy alone does not seem extensive enough to account for the decrease in sperm numbers. Females sacrificed in October prior to hibernation lack sperm in their spermathecae; some secretory vacuoles are present, but they are not as numerous or as enlarged as in specimens collected in March and May. Inter- and intrafamilial differences in the cytology of sperm storage may not be phyletically informative at the family level but related to species-specific reproductive adaptations. J. Morphol. 234:131–146, 1997. © 1997 Wiley-Liss, Inc.     

Isolation of dinucleotide microsatellite loci from red‐backed salamander (Plethodon cinereus)

We isolated 13 variable dinucleotide microsatellites from red‐backed salamanders (Plethodon cinereus). After generating fragments using degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR), AC repeats were captured using biotinylated probes and streptavidin‐coated magnetic particles. Captured fragments were cloned into plasmids, screened for microsatellites with a simple PCR reaction, and select plasmids then sequenced. PCR primers were designed and optimized for robust amplification, and nine primers have been further optimized for multiplex reactions with fluorescent primers. These nine loci are variable with an average of 6.11 alleles per locus and an average heterozygosity of 0.61.     

Seasonal variations and inherent variability of selenium in marine biota of a tropical wetland ecosystem: implications for bioindicator species

The present study was designed to study the seasonal variations of selenium in marine biota of different trophic levels collected from Sunderban deltaic complex, northeast India. The primary objective of this work is to provide baseline data for a future environmental quality programme. In general, the sequence of Se accumulation observed in biota is as follows: Bivalve>Zooplankton>Macroalgae>Pisces>Seagrass. An elevated level of Se was recorded during monsoon season. The Se level in zooplankton (4 μg/g) was three to four times higher than seagrass. The content of various soft tissues and shell in bivalves molluscs showed an organ-specific pattern in the following order: gill > visceral MASS = mantle > adductor muscle > podium > shell. Fish contain less Se in comparison with macroalgae, zooplankton and bivalves revealing poor or insignificant trophic transfer of Se in the marine food chain. Pelecyora trigona (Bivalve) seems to be reliable indicator of Se contamination because of its high accumulation capacity. An in-depth monitoring program is recommended in order to clarify the present trend and to establish the studied biota as indicator species.     

Nitrogen fixation in hardwood forests of the northeastern United States

Summary Nitrogen-fixing activity in hardwood forests of the northeastern United States occurred in wood litter, greater than 2 cm in diameter. Activity in large dead wood was independent of species, in the case of deciduous wood litter, but was restricted to partially decayed wood with a high moisture content. Maximum rates of activity were observed in the summer months, minimum rates in the winter. Evidence from six stands of varying ages showed that fixation in large wood litter occurred in only 25% of the samples assayed.Fixation was highest in the youngest, 4 years, and oldest, over 200 years, stands; being about 2 kg/ha/yr. The quantity of nitrogen fixed appears to be related to the biomass of dead wood. Large amounts of wood litter in the youngest stands were from slash left after cutting. As the supply of slash is exhausted by decay, nitrogen fixation decreases, with a low around year 20. Fixation then gradually increases as natural thinning adds wood to the litter compartment.Apparently, the amount of nitrogen fixed in dead wood the first 20 years following clearcutting can only replace a modest fraction of the amount lost as a result of the cutting and product removal. Finally, the results indicate that nitrogen fixation in wood litter does not equal nitrogen fixation in a northern hardwood forest calculated using a mass balance approach, suggesting that additional nitrogen inputs exist.     

Differences in pigment-containing organelles between color forms of the red-backed salamander,Plethodon cinereus

Summary Chromatophores of two color forms of the salamander, Plethodon cinereus were studied by electron microscopy. Erythrophores found in the red-backed form possess pterinosomes, some with regions of high electron density. In addition, a few melanosome-like organelles are present. On the other hand, the lead-backed form displays no visible erythrophores but instead melanophores with melanosomes being the most prevalent organelle and a few pterinosomes. The possibility that this represents a kind of hybrid chromatophore with intermediate stages in melanosome-pterinosome interconversion is discussed.This study was supported in part by GB-8347 from the National Science Foundation.Contribution No. 255, Department of Biology, Wayne State University.     

Structural heterogeneity in the basal regions of the teeth of the red-backed salamander,Plethodon cinereus (Amphibia,Plethodontidae)

Ultrastructural and histochemical features of marginal (monostichous) teeth associated with the jawbones are compared with those of palatal (polystichous) teeth that compose two patches in the roof of the mouth. The apices and uncalcified regions are similar in both kinds of teeth, but the basal regions display distinctive differences. While bases (pedestals) of marginal teeth are essentially hollow cylinders that attach to the jawbones by their labial faces, bases of teeth in palatal patches are fused to form two horizontal plates which lack direct attachment to underlying bone. The plates are separated from each other by a pulp-filled space containing fibroblasts, blood vessels, and vertically oriented elements resembling bony spicules. Cylindrical pedestals like those of marginal teeth project from the ventral plate. While the identity of the material composing the basal regions remains controversial, the following evidence suggests that it is similar to “bone of attachment” (Tomes, '23): most of it, unlike dentin, does not develop in direct association with an enamel organ; alcian blue stains the bases of developing teeth but stains dentin, developing dentin, enamel, or mature bone very weakly (if at all); bases of teeth in palatal patches develop in isolation from the parasphenoid bone and thus cannot be considered extensions of it; and marginal teeth attach directly to the jawbones, but the material composing their bases does not blend with the bone. Structural heterogeneity of the basal regions appears to be linked to functional differences exhibited by these two types of teeth.     

Structure of the radially asymmetrical uncalcified region of the teeth of the red-backed salamander,Plethodon cinereus (Amphibia,Plethodontidae)

The teeth of the adult plethodontid salamander, Plethodon cinereus, were examined by light and electron microscopy with emphasis on the ringlike zone of uncalcified dentin that divides the calcified portion of each tooth into a proximal pedestal and a distal apex. The uncalcified region displays radial asymmetry, forming an integral part of the posterior wall of the tooth but bulging into the pulp cavity anteriorly, thus forming a hingelike structure. All portions of the dentin, including the uncalcified region, are composed predominantly of collagenous fibers but lack elastin. In scanning electron micrographs of teeth from which the oral mucosa has been removed, the location of the anterior uncalcified hinge is marked externally by a notch-like articulation of the apex and pedestal. Sites of transition between calcified and uncalcified areas of the dentin show no special modifications in transmission electron micrographs, but collagenous fibers in calcified portions are associated with more electron-dense amorphous material than are those in the uncalcified region. Odontoblasts associated with the uncalcified region possess ultrastructural features closely resembling those of odontoblasts found in calcified areas. The uncalcified region seems to afford the teeth a certain degree of flexibility, and the asymmetry of the region appears to allow the teeth to flex only in a posterior direction, thus facilitating the entry of living prey but hindering its escape. The uncalcified region also seems to permit the apex of a tooth to break away from its pedestal without damage to underlying bone.     

Effects of androgens on behavioral and vomeronasal responses to chemosensory cues in male terrestrial salamanders (Plethodon shermani)

Chemosensory stimuli and sex steroid hormones are both required for the full expression of social behaviors in many species. The terrestrial salamander, Plethodon shermani, is an emerging nonmammalian system for investigating the nature and evolution of pheromonal communication, yet little is known regarding the role of sex steroid hormones. We hypothesized that increased circulating androgen levels in male P. shermani enhance chemoreception through morphological, behavioral, and physiological mechanisms. Experimental elevation of plasma androgens increased development of cirri, morphological structures thought to enhance the transfer of chemosensory cues from the substrate to the vomeronasal organ (VNO). Elevated plasma androgens also increased expression of a chemo-investigatory behavior (nose tapping) and increased preference for some female-derived chemosensory cues. Male-produced courtship pheromones activated a large number of cells in the VNO as measured by the method of agmatine uptake. However, androgen levels did not affect the total number of vomeronasal cells activated by male-produced courtship pheromones. Future studies will determine whether androgens potentially modulate responsiveness of the VNO to female-derived (as opposed to male-derived) chemosensory cues.     

白鹭作为无锡太湖地区环境污染指示生物的研究

2000年4—6月对分布在无锡太湖地区的4种要鸟中的白鹭(Egretta garzetta)行为生态学及其生境污染状况进行了调查研究．调查地白鹭主要栖息树种包括马尾松(Pnus masson±ana)、香樟树(C±nnamomum hupehanum)、麻砾树(Quercus acut±ssema)、榆树(Ulmus prm±la)和杨梅树（Myr±ca rubra)．栖息地总面积为7hm^2,白鹭总巢数为4200个,每棵树平均0．38个．白鹭的孵化期为19—21d,卵重23．9±4．0g(n=41),平均卵大小(44．5±4．1)mm×(32．6±4．9)mm(n＝41),平均窝卵数5．02(2—8)枚,平均窝雏数3．86(2—7)只,孵化成功率为84．25％．对白鹭雏鸟左右跗Zhe的测量分析表明不对称性不显著(P>0．05)．白鹭雏鸟食物较小,繁殖期间主要以小型鱼类和虾类为食．污染物分析结果表明,白鹭卵中除例外,六氯苯、六六六、环二烯类、DDTs、DDE、PCBs、Cr、Hg和Pb均检测到．食物链(底泥-食物-白鹭雏鸟)中,有机杀虫剂和重金属都逐级富集．通过与相对无污染的鄱阳湖地区比较,太湖地区和鄱阳湖地区卵样品中的DDs(包括DDT、DDE和DDD)浓度最高(超过2μg·g^1),其它污染物浓度均较小．太湖地区白鹭卵中的DDTs、Cd和Cr浓度低于鄱阳湖地区,其它污染物浓度鄱阳湖地区均低于太湖地区．经比较分析,两地样品中有机氯杀虫剂、PCBs和各种重金属浓度都未达到影响白鹭繁殖成功率的阈值．     

Histology and ultrastructure of the caudal courtship glands of the red‐backed salamander,Plethodon cinereus (Amphibia: Plethodontidae)

Caudal courtship glands (CCGs) are sexually dimorphic glands described in the skin of the dorsal tail base of some male salamanders in the genera Desmognathus, Eurycea, and Plethodon in the family Plethodontidae. These glands are believed to deliver pheromones to females during courtship, when the female rests her chin on the dorsal tail base during the stereotypic tail straddling walk unique to plethodontids. Although CCGs have been studied histologically, no investigations of their ultrastructure have been made. This article presents the first study on the fine structure and seasonal variation of CCGs, using the plethodontid Plethodon cinereus. The CCGs vary seasonally in height and secretory activity. The mature secretory granules observed in males collected in October and April consist of oval, biphasic granules that are eosinophilic and give positive reactions to periodic acid‐Schiff for neutral carbohydrates but do not stain for acidic mucosusbtances or proteins with alcian blue and bromphenol blue, respectively. Granular glands, some of which contain mucous demilunes, are twice as large as CCGs, are syncytial (unlike CCGs), and stain for proteins. Mucous glands are similar in size to CCGs, but are basophilic, show no seasonal variation in secretory activity, and stain positive for acidic mucosubstances. CCGs do not resemble cytologically the sexually dimorphic mental glands of some plethodontids, which contain round or oval granules filled with an electron‐dense amorphous substance. The CCGs are similar histologically to sexually dimorphic skin glands described in some anurans, but more comparative work is needed. J. Morphol. 276:319–330, 2015. © 2014 Wiley Periodicals, Inc.     

The effect of sampling density and study area size on landscape genetics inferences for the Mississippi slimy salamander (Plethodon mississippi)

In landscape genetics, it is largely unknown how choices regarding sampling density and study area size impact inferences upon which habitat features impede vs. facilitate gene flow. While it is recommended that sampling locations be spaced no further apart than the average individual''s dispersal distance, for low‐mobility species, this could lead to a challenging number of sampling locations, or an unrepresentative study area. We assessed the effects of sampling density and study area size on landscape genetic inferences for a dispersal‐limited amphibian, Plethodon mississippi, via analysis of nested datasets. Microsatellite‐based genetic distances among individuals were divided into three datasets representing sparse sampling across a large study area, dense sampling across a small study area, or sparse sampling across the same small study area. These datasets were a proxy for gene flow (i.e., the response variable) in maximum‐likelihood population effects models that assessed the nature and strength of their relationship with each of five land‐use classes (i.e., potential predictor variables). Comparisons of outcomes were based on the rank order of effect, sign of effect (i.e., gene flow resistance vs. facilitation), spatial scale of effect, and functional relationship with gene flow. The best‐fit model for each dataset had the same sign of effect for hardwood forests, manmade structures, and pine forests, indicating the impacts of these land‐use classes on dispersal and gene flow in P. mississippi are robust to sampling scheme. Contrasting sampling densities led to a different inferred functional relationship between agricultural areas and gene flow. Study area size appeared to influence the scale of effect of manmade structures and the sign of effect of pine forests. Our findings provided evidence for an influence of sampling density, study area size, and sampling effort upon inferences. Accordingly, we recommend iterative subsampling of empirical datasets and continued investigation into the sensitivities of landscape genetic analyses using simulations.     

Assessment of soil calcium status in red spruce forests in the northeastern United States

Long-term changes in concentrations of available Ca in soils of redspruce forests have been documented, but remaining questions aboutthe magnitude and regional extent of these changes have precluded anassessment of the current and future status of soil Ca. To addressthis problem, soil samples were collected in 1992—93from 12 sites in New York, Vermont, New Hampshire, and Maine toprovide additional data necessary to synthesize all availableresearch results on soil Ca in red spruce forests. Sites werechosen to encompass the range of environmental conditionsexperienced by red spruce. Concentrations of exchangeableCa ranged from 2.13 to 21.6 cmol_ckg^–1 in the Oa horizon, and from 0.11 to 0.68cmol_c kg^–1 in the upper 10 cm of theB horizon. These measurements expanded the range of exchangeable Ca reported in the literature for both horizons in northeastern redspruce forests. Exchangeable Ca was the largest Ca fraction in theforest floor at most sites (92% ofacid-extractableCa), but mineral Ca was the largest fraction at the three sites that also had the highest mineral-matter concentrations. Theprimary factor causing variability in Ca concentrations among siteswas the mineralogy of parent material, but exchangeable concentrationsin the B horizon of all sites were probably reduced by acidicdeposition. Because the majority of Ca in the forest floor isin a readily leachable form, and Ca inputs to the forest floor from the mineral soil and atmospheric deposition have beendecreasing in recent decades, the previously documented decreasesin Ca concentrations in the forest floor over previous decades mayextend into the future.