Authors:
Robin E. Jung, USGS Patuxent Wildlife Research Center, 12100 Beech Forest Rd., Laurel, MD 20708, robin_jung@usgs.gov
Tom Pauley, Department of Biology, Marshall University, Huntington, WV 25755 pauley@marshall.edu

Species list
Description of technique
       Field time components
       Office time
       Equipment
Things that could bias your counts
       Species misidentification
       Leaf litter bag submergence
       Litter bag characteristics
       Weather
       Time of year/time of day
       Observer effects
Advantages and disadvantages
Approaches to analyzing your data
Existing protocols and programs using this technique
Estimates of variation of counts for this technique
Studies that have used this technique
Literature cited
Send a comment on this technique (this takes you to another page)
See existing comments (this takes you to another page)

Desmognathus fuscus, Northern dusky salamander; Desmognathus marmoratus, Shovel-nosed salamander; Desmognathus monticola, Seal salamander; Desmognathus ochrophaeus, Mountain dusky salamander; Desmognathus quadramaculatus, Black-bellied salamander; Eurycea bislineata, Northern two-lined salamander; Eurycea cirrigera, Southern two-lined salamander; Eurycea junaluska, Junaluska salamander; Eurycea longicauda, Long-tailed salamander; Eurycea wilderae, Blue Ridge two-lined salamander; Gyrinophilus porphyriticus, Northern spring salamander; Pseudotriton ruber, Northern red salamander

The leaf litter refugia bag technique was originally designed by Dr. Thomas Pauley (Marshall University, Huntington, West Virginia), as a method to survey larval and juvenile stream salamanders (Pauley 1995, Pauley and Little 1998). Dr. Pauley first conceived of the idea to use leaf litter refugia bags for stream salamanders after working with aquatic invertebrate biologists at the Fernow Experimental Forest Laboratory, who reported that they were finding larval and juvenile salamanders in leaf packs similar to those described by Merritt and Cummins (1996). Leaf litter bags may be attractive to larval and juvenile salamanders because they act as a refuge from cannibalistic adults or predators or they may serve as a substrate for aquatic invertebrate prey. Because of their small size and wet slippery skin, larval and juvenile stream salamanders are more difficult to hand capture than adults, but can be captured using small dip nets in association with survey methods such as transects or quadrats (see Stream Transect and Stream Quadrat sections). Capturing larval and juvenile stream salamanders using transect, quadrat or other survey methods typically involves disturbing habitat and stream channel and bank substrates, because they often hide and escape into rock and gravel interstices in streams. Using leaf litter refugia bags, however, allows the capture of these life stages without disturbing stream habitat.

Leaf litter bags are constructed of plastic netting, either 3 to 4 cm mesh or 1.5 cm mesh (e.g., Bird or Deer Block), which are cut into 50 x 30 cm, 50 x 50 cm, 70 x 70 cm, or 90 x 90 cm pieces (Pauley and Little 1998, Waldron et al., 2002). In the field, approximately 0.23 kg of small rocks, leaves, and moss are placed in the center of the netting, and the sides are gathered and cinched together at the top using a cable tie or twist tie to make a sack. A 12" piece of orange flagging tape is tied through the netting at the top of the leaf litter bag so that they are more visible in the stream. The bags are placed at the edge of the stream partially submerged in the water and surrounded and/or topped with large rocks to keep them in place and to prevent them from being washed downstream in case of high water events. Leaf litter bags are typically placed at intervals (e.g., 5 m) along the edges of streams. Leaf litter bags must first be placed in the stream at some time (e.g., one to two weeks) prior to the first check, so this requires an additional site visit and set up time before the survey can be conducted.

To check leaf litter bags, the rocks are carefully moved away from the bag (noting if any salamanders are under the rocks), and the leaf litter bag is quickly placed into a white plastic dishpan or large dipnet. The leaf litter bag is then shaken (e.g., for 10 seconds) above the dishpan or inside the dipnet so that salamanders fall out of the leaf litter bag through the mesh into the dishpan or dipnet. Pick through leaf debris that falls into the dishpan or dipnet to make sure you don't miss salamanders. Salamanders are then caught and placed into ziploc bags for enumeration and measurements (e.g., snout-vent length, total length). Over time, leaf litter often disintegrates in the bags. Therefore, if leaf litter bags are surveyed repeatedly, it is necessary to replace leaf litter in the bags at least once a month.

Initial setup:

• Travel time to and from stream site
• Time to make and place leaf litter bags in stream
• Cutting out leaf litter bag squares
• Collecting leaf litter, rocks and moss to place in the leaf litter bags
• Placing leaf litter bags in stream

Survey:

• Travel time to and from stream site
• 1 to 2 people to conduct surveys
• Average time = 2.6 hours x 2 people to check 42 bags (Cook et al., in prep.)

• Data entry
• Data quality assurance and control (data proofing)
• Data analysis
• Report writing

• Cable Ties or Twist Ties (to cinch together leaf litter bag at top)
• Data Sheets (on water-resistant paper) and Clipboard
• Flagging Tape (to tie to top of leaf litter bag)
• Leaf Litter (collected from field)
• Netting to make leaf litter bags:
     50 x 30 cm squares with 3-4 cm mesh (Pauley and Little 1998)
     50 x 50 cm squares with 1.5 cm mesh (Jung et al., 2000)
• Pencil or Indelible Ink Pen
• Rocks (collected from field)
• Rubber Boots, Hip Waders, or Chest Waders
• Ruler
• Small Rocks (collected from field, no larger than 10 x 15 cm)
• Thermometer
• Watch
• Water Quality Testing Equipment (e.g., pH meter)

Things that could bias your counts

Certain salamander species larval stages may be difficult to tell apart for beginners as well as experts in the field. Because of the high species diversity in the southern Appalachians, species identification may sometimes be challenging. The benefit of the leaf litter bag technique is that the capture rate is typically 100%. If the species can not be identified in the field, they can be taken back to the laboratory for further assessment.

The depth of leaf litter bag submergence in the water significantly influenced leaf litter bag use by adult and larval salamanders, but had no effect on juvenile salamanders (Waldron et al., in press). Fewer adults but more larvae were captured when leaf litter bags were more fully submerged.

Size of leaf litter bag influences salamander captures. More salamanders were captured in medium (70 x 70 cm) and large bags (90 x 90 cm) compared to small bags (50 x 50 cm) (Waldron et al., in press). It is imperative that litter bag design is standardized and that the same design is used throughout.

Large rainfalls or flood events impacting streams will influence the submergence or placement of leaf litter bags, which could alter the use of leaf litter bags by salamanders (see "Leaf litter bag submergence" section above). Pauley (pers. observation) has found that when ephemeral streams are dry, leaf litter bags still capture a few juveniles, but not larvae. In dry streams, salamander stages seek any available pockets of water. Leaf litter bags that retain some moisture will "fool" some individuals to take refuge in the bags. Herein lies a potential problem with the bags. If left unchecked during dry periods, bags eventually will dry out and may cause death to those juveniles captured in the bags.

Stream salamander species differ in their courtship and breeding seasons, length of larval periods, and time spent within in the stream versus surrounding terrestrial areas. According to Pauley (pers. comm.), June and October in West Virginia are the best months to survey for stream salamanders, as these are the months when the greatest numbers of species are present at streams. In West Virginia, E. cirrigera, E. bislineata, G. porphyriticus, and P. ruber larvae are present year-round, whereas D. fuscus and D. monticola larvae are most abundant from October through June. In Virginia, Desmognathus fuscus and D. monticola females lay eggs from June to August, and eggs hatch in September. Larvae transform approximately 8-10 months later (June-July), so surveys conducted in late summer will miss sampling these Desmognathus larvae (Mitchell 1998a,b). Certain species with short larval periods (e.g., Desmognathus ochrophaeus with a larval period of two weeks; Marcum 1994) will be difficult to survey using this or any method. In the Great Smoky Mountains National Park, Waldron et al. (in press) did not capture larval D. conanti, D. santeetlah or D. monticola in leaf litter bags during surveys from June-November. Waldron et al. (in press) working in the Great Smoky Mountains National Park captured most salamanders in leaf litter bags in June and July. Optimal times of year to survey for stream-dwelling salamanders will vary depending on target species and geographical location.

Stream salamanders are typically active at night when they forage. Leaf litter bags are usually surveyed during the day, because they are easier to find in the streams during the day. As far as we know, no published studies have analyzed whether salamander counts using leaf litter bags differ depending on time of day.

Observer effects are probably not too problematic for this method, as long as observers have been trained to carefully check the rocks holding the bags down for adults and quickly scoop the leaf litter bag up. Additionally, all the salamanders inside the bag are easily captured and are therefore available for careful study.

Advantages:

• Easy to deploy leaf litterbags and to identify sampling locations (Chalmers and Droege 2002; Waldron et al., in press)
• Inter-observer variation is reduced because observers check bags rather than searching the stream channel, which is more subject to observer bias (e.g., differences among observers in search image and experience) (Chalmers and Droege 2002).
• Non-destructive methodology with minimal or no disturbance to habitat (Pauley and Little 1998; Waldron et al., in press).
• With the exception of Eurycea bislineata larvae found primarily in plunge pools, leaf litter bags were just as effective a technique as turning over cover objects and capturing salamanders with aquarium nets or searching the bottom of streams at night with flashlights (Pauley and Little 1998).
• Comparing leaf litter bags, quadrat (1 m2), transect (50 x 1 m) and electrofishing along 100 m of stream segments, Cook et al. (in prep.) found that leaf litter bags provided the highest total larval counts and densities (assuming a leaf litter bag covers a 0.25 m2 area) and was the most precise and efficient method for Eurycea bislineata larvae. Compared to other methods, leaf litter bags yielded the highest capture rates for Pseudotriton ruber and Gyrinophilus porphyriticus larvae (Cook et al, in prep; Waldron et al., in press).
• Hamilton (2002) found that leaf litter bags were more effective at catching salamanders than visual encounter surveys (turning over rocks) and rock on rock arrangements (putting a flat rock on top of a larger flat rock at the water's edge) at sites with few natural cover objects. However, where cover objects were abundant, visual encounter surveys were more productive than leaf litter bags.
• Useful for determining salamander presence during inventory programs (Waldron et al., in press).

Disadvantages:

• Life stage bias. Leaf litter bags preferentially capture larval and juvenile salamanders rather than adults (Pauley and Little 1998).
• Misses Desmognathus larvae because they have shorter larval periods and tend to stay in the water film on the undersides of rocks or the interstices of the substrate in riffles rather than seek refuge in leaf litter (Marcum 1994).
• If small mesh leaf litter bags are used, technique may potentially miss species with larger-bodied larval stages that can't fit through the mesh, such as Gyrinophilus and Pseudotriton, although Cook et al. (in prep.) showed high capture rates for these species' larval stages. Leaf litter bags did not capture Pseudotriton montanus and Desmognathus aeneus at stream sites where these species were known to occur in the Great Smoky Mountains National Park (Waldron et al., in press).
• Entanglement hazard which can cause mortality of some amphibians and reptiles. Species that died in leaf litter bags in streams in Shenandoah National Park include Rana clamitans and Nerodia sipedon (Jung, pers. obs.; see also Stuart et al., 2001).
• Non-biodegradable material present in the environment. Sometimes leaf litter bags become dislodged when stream flows become heavy. When leaf litter bags get washed downstream, they are sometimes difficult to find and retrieve.
• Stream width may bias salamander captures. More salamanders and species were captured in small streams (mean width = 3.6 m) compared to medium (6.7 m) and large (14.9 m) streams (Waldron et al., in press).
• Leaf litter bag may not be useful and could possibly be detrimental for salamanders in ephemeral streams that dry up (Pauley, pers. observation).
• Leaf litterbags showed variation in the numbers of individuals captured and counts may not relate to the actual abundance of salamanders in the stream, making the technique questionable for use in monitoring population trends (Waldron et al., in press; Chalmers and Droege, 2002).

Data are typically recorded as the number of salamanders of each species' life stage per leaf litter bag. Data from leaf litter bags provide indices (counts) of relative abundance of species' life stages among stream sites when compared under similar environmental conditions and sampling periods. Calculating salamander densities (numbers of animals per unit area) in leaf litter bags may not be appropriate, because it is difficult to define the area sampled by a leaf litter bag. Chalmers and Droege (2002) found that in most cases, numbers of Eurycea bislineata larvae in leaf litter bags was not related to the actual number of larvae present in experimental enclosures where the leaf litter bags were housed (Chalmers and Droege 2002). Because of this, these authors did not recommend using leaf litter bags to index populations of this species for population monitoring purposes. Leaf litter bags can be used in inventories to document presence of salamander species in streams. Surveying leaf litter bags repeatedly (multiple visits) at a number of sites could be used to calculate species detection rates and to estimate the percent of streams occupied by the species (Chalmers and Droege 2002, MacKenzie et al. 2002).

• Tom Pauley, Marshall University, Huntington, WV
• Jayme Waldron and Ken Dodd, Great Smoky Mountains National Park, TN and NC
• Jennifer Mravintz Williams, Herpetological Populations in a Reclaimed Mountaintop Mine Landscape, West Virginia University, Morgantown, WV.

Cook et al. (in prep.) reports coefficients of variation for leaf litter bag counts of various species' life stages over time. Two streams were surveyed repeatedly (6 to 9 times total) every two weeks in the summer of 1998. They found low CVs in counts for Eurycea bislineata larvae (0.26 and 0.34), but higher CVs for other species' life stages, ranging from 0.68 (E. bislineata adults) to 3.00 (Gyrinophilus porphyriticus larvae, Desmognathus fuscus larvae). Chalmers and Droege (2002) reported the following within-year coefficients of variation for Eurycea bislineata larval counts (5 daily) for sets of 8 leaf litter bags: 0.62 for Hunter's Brook, 1.36 for Richardsons Brook, and 0.51 for Breakneck Stream.

• Pauley 1995
• Pauley and Little 1998
• Jung et al., 2001
• Chalmers and Droege 2002
• Cook et al., in prep.
• Waldron et al., in press

Boward, D. M., P. F. Kazyak, S. A. Stranko, M. K. Hurd, and T. P. Prochaska. 1999. From the Mountains to the Sea: The State of Maryland's Freshwater Streams. EPA 903-R-99-023. Maryland Department of Natural Resources, Monitoring and Non-tidal Assessment Division, Annapolis, Maryland, USA.

Chalmers, R. J., and S. Droege. 2002. Leaf litter bags as an index to populations of northern two-lined salamanders (Eurycea bislineata). Wildlife Society Bulletin 30:71-74.

Cook, K. L., R. E. Jung, S. Droege, J. R. Sauer, J. C. Mitchell, M. Crossland, C. J. Leary, M. Larson, and E. Chattin. In prep. Comparison of quantitative methods to survey streamside salamanders in Blue Ridge mountain streams. Submit to Herpetologica.

Corn, P. S., and R. P. Bury. 1989. Logging in western Oregon: responses of headwater habitats and stream amphibians. Forest Ecology and Management 29:39-57.

Hamilton, M.S. 2002. Effects of developmental activities on streamside salamander communities in Boone County, West Virginia. M.S. Thesis, Marshall University, Huntington, WV.

Jung, R. E., S. Droege, J. R. Sauer, and R. B. Landy. 2000. Evaluation of terrestrial and streamside salamander monitoring techniques at Shenandoah National Park. Environmental Monitoring and Assessment 63:65-79.

Marcum, C., Jr. 1994. Ecology and natural history of four plethodontid species in the Fernow Experimental Forest, Tucker County, West Virginia. Master's thesis, Marshall University, Huntington, WV. 254 pp.

MacKenzie, D. I., J. D. Nichols, G. B. Lachman, S. Droege, J. A. Royle, and C. A. Langtimm. 2002. Estimating site occupancy rates when detection probabilities are less than one. Ecology 83:2248-2255.

Merritt, R. W., and K. W. Cummins. Editors. 1996. An Introduction to the Aquatic Insects of North America. Third Edition. Kendall Hunt Publishing Co., Dubuque, Iowa. 862 pp.

Middlekoop, M. J., T. Watts, and M. Schorr. 1999. Acid mine drainage and its effects on physicochemical conditions and salamander populations in a Cumberland Plateau stream. Journal of the Tennessee Academy of Science 73:36.

Mitchell, J. C. 1998a. Amphibian Decline in the Mid?Atlantic region: Monitoring and Management of a Sensitive resource. Final Report, Legacy Resource Management Program, U.S. Department of Defense, Alexandria, VA.

______. 1998b. Guide to Inventory and Monitoring of Streamside Salamanders in Shenandoah National Park. Supplement No. 2 to Amphibian Decline in the Mid-Atlantic Region: Monitoring and Management of a Sensitive Resource. Final Report, Legacy Resource Management Program, U.S. Department of Defense, Alexandria, VA.

Orser, P. N., and D. J. Shure. 1972. Effects of urbanization on the salamander Desmognathus fuscus fuscus. Ecology 53:1148-1154.

Pauley, T. K. 1995. Aquatic salamanders. Pp. 15-22 in: Reardon, R. C. (Ed.). Effects of diflubenzuron on non-target organisms in broadleaf forested watersheds in the northeast. USDA Forest Service. National Center of Forest Health Management. FHM-NC-05-95.

Pauley, T. K., and M. Little. 1998. A new technique to monitor larval and juvenile salamanders in stream habitats. Banisteria 12:32-36.
Petranka, J. W., M. E. Etheridge, and K. E. Haley. 1993. Effects of timber harvesting on southern Appalachian salamanders. Conservation Biology 7:363-370.

Rocco, G. L., and R. P. Brooks. 2000. Abundance and Distribution of a Stream Plethodontid Salamander Assemblage in 14 Ecologically Dissimilar Watersheds in the Pennsylvania Central Appalachians. Final Report. Report No. 2000-4. Penn. State Cooperative Wetlands Center, Forest Resources Laboratory, Pennsylvania State University, University Park, PA, USA.

Stuart, J. N., M. L. Watson, T. L. Brown, and C. Eustice. 2001. Plastic netting: An entanglement hazard to snakes and other wildlife. Herpetological Review 32:162-164.

Waldron, J. L., C. K. Dodd, and J. D. Corser. In press. Leaf litterbags: Factors affecting capture of stream-dwelling salamanders. Applied Herpetology.

Welsh, H. H., Jr., and L. M. Ollivier. 1998. Stream amphibians as indicators of ecosystem stress: A case study from California's Redwoods. Ecological Applications 8:1118-1132.