A COMPARISON OF TECHNIQUES TO SAMPLE SALAMANDERS ...

A COMPARISON OF TECHNIQUES TO SAMPLE SALAMANDERS AND OTHER HERPETOFAUNA ALONG SMALL COASTAL PLAIN STREAMS IN MARYLAND

Submitted to: Scott Stranko Maryland Department of Natural Resources Monitoring and Non-tidal Assessment Division Tawes State Office Building C-2 Annapolis, MD 21401

Submitted by: Gabriel F. Strain and Richard L. Raesly Biology Department Frostburg State University Compton Science Center 101 Braddock Road Frostburg, MD 21532

14 November 2006

TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. ii INTRODUCTION .............................................................................................................. 1 STUDY OBJECTIVES....................................................................................................... 3 METHODS ......................................................................................................................... 5 Site selection and experimental design ........................................................................... 5 Visual encounter survey.................................................................................................. 5 Cover board survey ......................................................................................................... 7 Leaf litter bags ................................................................................................................ 7 Drift fences with pitfall and funnel traps ........................................................................ 9 Quadrat leaf litter searches............................................................................................ 11 Statistical analysis ......................................................................................................... 13 Effect of increased effort .............................................................................................. 13 Monitoring microhabitat features ................................................................................. 13 Mud salamander surveys............................................................................................... 14 RESULTS ......................................................................................................................... 15 Comparison of methods ................................................................................................ 21 Comparison of months .................................................................................................. 25 Effect of increased effort .............................................................................................. 25 Monitoring microhabitat features ................................................................................. 28 Mud salamander surveys............................................................................................... 28 DISCUSSION ................................................................................................................... 33 RECOMMENDATIONS .................................................................................................. 37 LITERATURE CITED ..................................................................................................... 39

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ACKNOWLEDMENTS We would like to thank the people who helped make this study possible. We would like to thank S. Stranko for the opportunity to work on this project and for his advice. We thank P. Bright (Mattowoman Natural Environment Area, Smallwood State Park) and Mr. Henderson (Doncaster Demonstration Forest) for permission to use their properties and for their advice. R. Chalmers, M. Southerland, E. Thompson, J. McCann, C. Swarth, R. Hilderbrand, S. Smith, R. Jung, and L. Smith assisted in developing the experimental design for this study. We thank E. Thompson for his assistance in identifying larval salamanders. We would like to thank the field crew, E. McGinley and J. Eells, for their long hours of work. J. Saville assisted with data entry. Finally, we would like to thank R. Hilderbrand for his advice and assistance with statistical analysis of the data.

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INTRODUCTION

Amphibians may be useful indicators of environmental conditions because of their permeable skin, longevity, and intimate association with aquatic systems (Blaustein 1994, Welsh and Ollivier 1998, Jung et al. 2000, Southerland et al. 2004). The dual life cycle of many amphibian species potentially exposes them to both terrestrial and aquatic disturbances and contaminants (Blaustein 1994, Fronzuto and Verrell 2000, Blaustein and Johnson 2003). Larval anurans are sensitive to increased concentrations of heavy metals and reduced pH levels (Jung and Jagoe 1995) and salamander relative abundance is inversely proportional to disturbed habitat (Willson and Dorcas 2003). Since 2000, malformations in at least 57 species of anurans have been reported across 44 states (Meteyer 2000), which may be attributed to anthropogenic causes such as contamination and increased UV-B radiation (Blaustein and Johnson 2003). Indices of biotic integrity (IBIs) using fish are not useful for streams draining catchments of less than 300 acres because fish species richness and abundance numbers are too low (Klauda et al. 1998, Southerland et al. 2000, 2004). Multiple geological and hydrological barriers often prevent fishes from entering these bodies of water (Davic and Welsh 2004). In these smaller, sometimes ephemeral streams, amphibians may assume the role of top predators (Pauley 1995, Southerland et al. 2004) and play an important role in ecosystem processes (Davic and Welsh 2004). ). The total biomass of amphibians in some areas may equal that of small mammals and be twice that of birds (Burton and Likens 1975). In these smaller catchments, therefore, surveys and development of metrics for amphibians may be valuable monitoring tools.

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Recently, an index was developed and tested for evaluating the effectiveness of a stream salamander IBI for use in Maryland watersheds (Southerland et al. 2004). The index was deemed effective when validated against B-IBIs for the same site, however the average number of species collected at a site was two, which may not be a large enough number “to discern convincing patterns” (Southerland et al. 2000). Southerland et al. (2000) also suggested “more intensive sampling (to identify more species)….would reduce the adverse effects of low metric numbers”. The MBSS currently employs a visual encounter survey (VES) that consists of recording the presence of any herpetofauna at a site along the stream bank and in the channel during electrofishing sessions in the summer sampling period (Kazyak 2001). Uncertainty exists as to whether these methods provide representative data of the herpetofauna at each site (Southerland et al. 2000). Other methods such as cover boards, leaf litter bags, drift fences with pitfall and funnel traps, and quadrat leaf litter searches may be more suitable for determining the herpetofaunal assemblage.

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STUDY OBJECTIVES

The main objective of this study was to determine how representative VES and electrofishing data are by comparing these techniques to four other methods: cover boards, leaf litter bags, drift fences with pitfall and funnel traps, and quadrat leaf litter searches. In addition, peak activity periods for many amphibian species greatly depend on weather (Crump and Scott 1994, Duellman and Trueb 1994), and this may cause variable sampling results from month to month. Another objective of this study, therefore, was to determine the time period that maximizes amphibian captures. Specifically, the following null hypotheses were tested: H01: No significant difference in species richness exists between sampling methods. H02: No significant difference in species richness exists between months sampled. H03: No significant difference in total individuals exists between sampling methods. H04: No significant difference in total individuals exists between months sampled. H05: No significant difference in salamander species richness exists between sampling methods. H06: No significant difference in salamander species richness exists between months sampled. H07: No significant difference in total salamander individuals exists between sampling methods.

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H08: No significant difference in total salamander individuals exists between months sampled.

Secondary Objectives Effect of increased effort - The effect of increasing the time or area searched on species richness was evaluated for most of the methods listed above, particularly the visual encounter survey. Monitoring microhabitat features - At each site, the microhabitat features such as seeps, logs, and rocks were recorded to examine associations between species found and specific features. Testing for sampler biases - The amount of bias caused by multiple samplers was to be determined by having two different samplers visit a site; however due to logistical problems this was unable to be performed for electrofishing, and due to low capture rates for any one species at a site this was unable to be performed for the remaining methods. Mud salamander surveys - The sampling methods were tested at sites that have been specifically identified as potential or actual sites for mud salamander populations.

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METHODS

Site selection and experimental design – Twenty sites (150m in length) were established in small, mostly headwater streams in the Coastal Plain region of Maryland, west of the Chesapeake Bay (Fig. 1). Streams of this region generally have sand or gravel substrates, slow-moving water, low to moderate nitrate concentrations, a largely vegetated riparian zone, and low to moderate levels of dissolved oxygen (Boward et al. 1999). At each site, six treatments were assigned: VES, cover board survey, leaf litter bags, quadrat leaf litter searches, drift fences with pitfall and funnel traps, and electrofishing. The six treatments were ordered randomly within each study site to establish a completely randomized block design, with each study site representing a replicate. Each site was randomly assigned to either the left or the right bank of the stream. The cover boards, drift fence arrays, and leaf litter bags were established in late May/early June 2006. Sampling began in early June 2006 and continued until early August 2006, with three 15-day sampling periods spaced approximately one month apart. All herpetofauna encountered were captured (if possible), identified to species, measured for snout-vent length (SVL), and released. Any anurans heard calling were also recorded. Visual encounter survey – Each VES plot consisted of two parallel transects (Crump and Scott 1994) of 25m x 1m, with one transect running along the wetted width of the stream, and the other 1 m away from the stream margin. The transects were searched slowly, with the observer(s) turning over all natural cover objects within the transect and returning those objects as close as possible to their original position to

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Figure 1. Coastal Plain region of Maryland, east of the Chesapeake Bay, with approximate locations of study sites.

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minimize bias due to habitat alteration (Robin Jung, personal communication). Cover board survey – Cover boards consisted of pine boards of approximately 30 x 30 x 5cm (Fellers and Drost 1994, Marsh and Goicochea 2003). Leaf litter and debris was cleared away so that the boards were placed upon bare dirt (Fellers and Drost 1994, Hyde and Simons 2001, Marsh and Goicochea 2003). Each plot contained 12 cover boards in a six x two pattern (Fig. 2), with one row of six boards placed 2m away from the stream margin, and one row of six boards placed 4m away from the stream margin. Sampling consisted of the observer(s) walking a set path along the cover objects and lifting up the boards to check for amphibians. All amphibians were then released at the edge of the cover board to minimize exposure and desiccation (Fellers and Drost 1994). Leaf litter bags – Leaf litter bags were constructed of 61 x 40cm plastic netting with 2.5cm mesh, similar to the various sizes suggested by Jung and Pauley (2003). Each bag was filled with leaf litter debris and several small rocks and the ends of the netting were drawn together and secured with a cable tie to form a bag (Jung and Pauley 2003, Pauley and Little 1998). The bags were then placed in the stream. Each site contained five bags, with one bag every 5m (Jung and Pauley 2003). Yellow flagging was tied to each bag for greater visibility, and rocks were placed around or on top of each bag to prevent them from washing away in case of high water events (Pauley and Little 1998). In some cases rocks were not available, so sticks were pushed through the bags and into the substrate to hold them in place. To check each bag, the observer(s) quickly pulled it out of the water and placed it in a white pan, shaking it to dislodge all salamanders

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Figure 2. Cover board array, showing direction of observer movement (adapted from Reading 1997).

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(Pauley and Little 1998). Because leaf litter debris will deteriorate (Jung and Pauley 2003), debris was replaced before returning the bag to the stream. Drift fences with pitfall and funnel traps – One drift fence array was set up at each site. The drift fences consisted of 50cm high aluminum flashing which was buried in the ground to a depth of 12-15cm, yielding a 35-38cm high barrier (Enge 1997). Two 5mlong fences were used for each array (Corn 1994), with one approximately 1m away from the wetted width of the stream running parallel with the stream and the other running perpendicular to the stream forming an “L” shape (Fig. 3). A third 1m-long fence was placed running perpendicular to the stream on the other side of the parallel fence as close to the stream margin as possible. Pitfall traps were constructed of 1.75 gallon restaurant buckets, approximately 21cm in diameter and 19.5cm deep, with holes drilled in the bottom for drainage (Enge 1997). A wetted sponge was placed in each trap to prevent desiccation (Enge 1997). One trap was buried at the end of the 5m arms of the array, and one trap was buried at the junction of the three arms (Fig. 3). The traps were buried flush with the ground, and a piece of masonite with 4-6cm wooden legs attached was placed over each trap to provide shade and prevent rain from entering the trap (Corn 1994). Funnel traps were constructed of aluminum window screening and based on the design for double-ended funnel traps described by Enge (1997). Two wetted sponges were placed in each trap to prevent desiccation (Enge 1997). Four funnel traps were used in each array, with one trap placed on each side at roughly the midpoint of the two 5m fence arms (Fig. 3). Squares of 41 x 41 cm masonite were leaned against the fence over

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Figure 3. Drift fence array.

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each funnel trap to provide shade (Enge 1997). Leaf litter was pushed up into the mouth of each funnel to form a ramp that guided animals into the trap. The traps were opened for two nights and checked the following day. To prevent accidental captures when the traps were not in use, the pitfall traps were sealed tightly with plastic lids, and the sponges in the funnel traps were placed in the mouth of each funnel (Enge 1997). Quadrat leaf litter searches – The 25m x 3m section of each site receiving this treatment was divided into 75 1m x 1m quadrats numbered consecutively (Fig. 4). For each site, six quadrats were randomly selected each month using a random numbers table, and quadrats were selected without replacement to minimize bias due to habitat alteration (Jaeger and Inger 1994). A 1m x 1m frame constructed of PVC was placed over each selected quadrat and the leaf litter within the frame searched thoroughly down to bare soil. Captured herpetofauna were released in an adjacent quadrat to prevent recaptures during the search. Electrofishing – Electrofishing was performed with a backpack unit (Smith-Root 12-B electrofisher), with one observer electrofishing and one netting. Each section was sampled thoroughly and continuously (as opposed to intermittently) from bank to bank, including backwater areas, sloughs, and shallows (Kazyak 2001). Originally a standard 1/8th inch mesh dipnet was used, but some salamander larvae (most notably smaller Eurycea bislineata individuals) were able to escape through the holes of the net. Therefore a dipnet with smaller mesh (Coleman insect net, approx. 1/16th inch or 2mm mesh) was used.

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Figure 4. Arrangement and numbering of quadrats.

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Statistical analysis – Because each site was re-sampled each month, an uncertain amount of non-independence was inherent in the experimental design. Therefore, program PROC MIXED (SAS version 8e for Windows) was used to perform a repeated measures analysis. AIC values were used to determine the best covariance structure. One-way ANOVA was used to test for differences between months. A significance level of 0.05 was used in testing all hypotheses. The means of differences under each hypothesis were compared using Tukey’s multiple range test.

Effect of Increased Effort Some visual encounter surveys and electrofishing sessions and all quadrat leaf litter searches were extended to examine the effect of an increase in sampling effort. Occasionally visual encounter surveys and electrofishing sessions were extended beyond the 25m section to sample 50m and 75m sections. All quadrat leaf litter searches used six quadrats to examine differences between yields of the first three and yields of all six.

Monitoring Microhabitat Features As salamanders were encountered at each site, the specific microhabitat features in which the individual was found were recorded. On the streambank these included substrate type and natural cover objects, and in the stream these included flow, water depth, and substrate type.

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Mud Salamander Surveys Sites determined to be suitable for mud salamanders and some sites where populations of mud salamanders are known to exist were sampled with all six sampling methods to determine the best method by which to sample this rare species.

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RESULTS

During the three sampling periods in June, July, and August of 2006, 28 species of amphibians and reptiles totaling 632 individuals were captured or encountered (Table 1). Rana clamitans, Eurycea bislineata, and larvae of the genus Pseudotriton were the three most common taxa caught or encountered by all sampling methods, and accounted for 72.4% of the total (Fig. 5). Salamander species encountered included Ambystoma maculatum, Ambystoma opacum, Desmognathus fuscus, E. bislineata, Notophthalmus viridescens, Plethodon cinereus, Pseudotriton montanus, and Pseudotriton ruber. These species accounted for 26.5% of the total. Separated by sampling method, the three most common taxa captured during electrofishing were R. clamitans, E. bislineata, and Pseudotriton larvae, which accounted for 81.0% of the total (Fig. 6). Eurycea bislineata, Pseudotriton larvae, and Bufo fowleri were the only species captured in leaf litter bags, and therefore comprised 100% of the total (Fig. 7). The three most common species caught during drift fence surveys were R. clamitans, Bufo americanus, and B. fowleri, which accounted for 68.0% of the total (Fig. 8). Rana clamitans, Rana sphenocephala, and Rana palustris comprised 80.2% of VES species (Fig. 9). Eurycea bislineata and R. clamitans were the only two common species encountered during quadrat leaf litter searches and accounted for 72.7% of the total (Fig. 10). Similarly, cover board searches yielded only two common species, E. bislineata and R. sphenocephala, which made up 71.4% of the total (Fig. 11).

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Table 1. Number of individuals of each taxon encountered during each sampling period. Taxa

June

Acris crepitans Ambystoma maculatum Ambystoma opacum Bufo americanus Bufo fowleri Chelydra serpentina Chrysemys picta Coluber constrictor Desmognathus fuscus Elaphe obsoleta Eumeces fasciatus Eurycea bislineata Hyla cinerea Nerodia sipedon Notophthalmus viridescens Plethodon cinereus Pseudacris crucifer Pseudotriton montanus Psuedotriton ruber Psuedotriton spp. larvae Rana catesbeiana Rana clamitans Rana palustris Rana sphenocephala Rana sylvatica Scincella lateralis Terrapene carolina Thamnophis sirtalis

9 27 -5 5 --1 1 --39 2 -----4 17 2 109 9 13 1 1 ---

Sampling Period July August 2 --4 4 1 ----1 26 ----2 --18 5 123 4 17 2 -1 1

4 -2 5 1 1 1 --2 -16 -1 1 1 -2 4 10 5 100 10 8 --2 --

Total 15 27 2 14 10 2 1 1 1 2 1 81 2 1 1 1 2 2 8 45 12 332 23 38 3 1 3 1

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25 OTHER SPECIES 28%

RACL 52% PSEUDOTRITON LARVAE 7% EUBI 13%

Figure 5. Relative abundances of the three most common taxa encountered with all methods (EUBI=Eurycea bislineata, RACL=Rana clamitans). Relative abundance figures adapted from Foley and Smith 1999.

8 OTHER SPECIES 19%

PSEUDOTRITON LARVAE 13%

RACL 52%

EUBI 16%

Figure 6. Relative abundances of the three most common taxa captured with electrofishing (RACL=Rana clamitans, EUBI=Eurycea bislineata).

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BUFO 6% PSEUDOTRITON LARVAE 35%

EUBI 59%

Figure 7. Relative abundances of the three taxa captured in leaf litter bags (BUFO=Bufo fowleri, EUBI=Eurycea bislineata).


RACL 49%

BUFO BUAM 8% 1%

Figure 8. Relative abundances of the three most common taxa captured with drift fence surveys (BUAM=Bufo americanus, BUFO=Bufo fowleri, RACL=Rana clamitans).

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RAPA 7% RASP 10%

RACL 63%

Figure 9. Relative abundances of the three most common taxa encountered with VES (RACL=Rana clamitans, RAPA=Rana palustris, RASP=Rana sphenocephala).

3 OTHER SPECIES 27% RACL 37%

EUBI 36%

Figure 10. Relative abundances of the two most common taxa encountered with quadrat leaf litter searches (EUBI=Eurycea bislineata, RACL=Rana clamitans).

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4 OTHER SPECIES 29%

EUBI 50%

RASP 21%

Figure 11. Relative abundances of the two most common taxa encountered with cover board surveys (EUBI=Eurycea bislineata, RASP=Rana spenocephala).

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Comparison of Methods Species Considering only salamander species, in June a significant difference existed only between drift fence surveys, which captured zero species, and electrofishing, which captured a mean of 0.63 species (Fig. 12). The lack of significant differences may be accounted for by low overall captures and relatively high standard error (Table 2). Electrofishing produced a significantly greater number of species than all other methods in July (Fig. 12). No significant differences between methods were detected in August, which again may be due to low number of captures and high standard error. If all herpetofauna species encountered are considered in the analysis, VES and electrofishing in both June and July yielded significantly more species than cover board surveys, quadrat leaf litter searches, and leaf litter bags (Fig. 13, Table 3). In August, VES produced significantly more species than cover boards, quadrat leaf litter searches, and leaf litter bags, whereas electrofishing produced significantly more species than only quadrat searches and leaf litter bags (Fig. 13).

Total Individuals No significant differences existed between methods for total salamander individuals in June and August. However, electrofishing in July yielded significantly more individuals than cover boards, drift fence surveys, quadrat searches, and visual encounter surveys (Fig. 14). The lack of significant differences may be accounted for by low overall captures and relatively high standard error (Table 4).

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0.7 0.6

Number of species

0.5 VES CB

0.4

LLB QU

0.3

DF EL

0.2 0.1 0 June

July

August

Month (2006)

Figure 12. Mean number of salamander species encountered with each technique during the three sampling periods (VES=visual encounter survey, CB=cover board survey, LLB=leaf litter bags, QU=quadrat leaf litter search, DF=drift fence survey, EL=electrofishing).

Table 2. Salamander species minimum, maximum, mean, and standard error values for each technique for each month. Sampling Method

Month

Minimum

Maximum

Mean

SE

Drift fence Leaf litter bag Cover board survey Quadrat search Electrofishing Visual encounter survey Drift fence Leaf litter bag Cover board survey Quadrat search Electrofishing Visual encounter survey Drift fence Leaf litter bag Cover board survey Quadrat search Electrofishing Visual encounter survey

June June June June June June July July July July July July August August August August August August

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 1 1 2 2 2 1 1 1 1 2 1 2 1 2 1 2 1

0 0.42 0.05 0.16 0.63 0.37 0.05 0.11 0.05 0.05 0.58 0.05 0.26 0.11 0.21 0.05 0.31 0.21

0.00 0.12 0.05 0.11 0.17 0.14 0.05 0.07 0.05 0.05 0.18 0.05 0.13 0.07 0.12 0.05 0.13 0.10

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2 1.8 1.6

Number of Species

1.4

VES

1.2

CB LLB

1

QU

0.8

DF EL

0.6 0.4 0.2 0 JUNE

JULY

AUGUST

Month (2006)

Figure 13. Mean number of herpetofauna species encountered during the three sampling periods (VES=visual encounter survey, CB=cover board survey, LLB=leaf litter bags, QU=quadrat leaf litter search, DF=drift fence survey, EL=electrofishing).

Table 3. Herpetofauna species minimum, maximum, mean, and standard error values for each technique for each month. Sampling Method

Month

Minimum

Maximum

Mean

SE



0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3 1 2 2 4 5 2 1 1 2 4 4 3 1 2 1 4 4

0.95 0.42 0.16 0.33 1.68 1.68 0.84 0.16 0.11 0.21 1.53 1.58 1.05 0.11 0.21 0.05 1.16 1.89

0.21 0.12 0.11 0.15 0.27 0.31 0.17 0.09 0.07 0.12 0.32 0.26 0.22 0.07 0.12 0.05 0.32 0.26

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3.5

Number of Individuals

3 2.5 VES CB

2

LLB QU

1.5

DF EL

1 0.5 0 1

2

3

Month (2006)

Figure 14. Mean number of salamander individuals encountered during the three sampling periods (VES=visual encounter survey, CB=cover board survey, LLB=leaf litter bags, QU=quadrat leaf litter search, DF=drift fence survey, EL=electrofishing).

Table 4. Salamander individuals minimum, maximum, mean, and standard error values for each technique for each month. Sampling Method

Month

Minimum

Maximum

Mean

SE



0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 4 3 2 27 2 1 1 1 1 14 1 2 2 4 1 9 2

0 0.58 0.16 0.16 3.11 0.37 0.05 0.11 0.05 0.05 1.95 0.05 0.31 0.16 0.31 0.05 0.79 0.31

0.00 0.22 0.16 0.11 1.53 0.14 0.05 0.07 0.05 0.05 0.90 0.05 0.15 0.11 0.22 0.05 0.49 0.15

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Considering all herpetofauna individuals encountered in the analysis, electrofishing produced significantly more individuals than cover boards, drift fence surveys, leaf litter bags, and quadrat searches in June and July (Fig. 15, Table 5). Visual encounter surveys produced significantly more individuals than cover boards, leaf litter bags, and quadrat searches in July and August.

Comparison of Months Counts of species and individuals were pooled across sampling methods to test for overall differences between the three months sampled. In comparing total species richness between months, any anuran species heard calling was included in the analysis. This included Acris crepitans, Hyla versicolor/chrysoscelis, Rana catesbeiana, R. clamitans, and R. sphenocephala. No significant difference existed between the three sampling months of June, July, and August 2006 for total species, salamander species, total individuals, and salamander individuals. Individually by sampling method, however, electrofishing in June and July produced significantly more salamander individuals than in August (Fig. 14).

Effect of Increased Effort The data from June illustrate that an increase in effort may increase the yield for both VES and electrofishing (Fig. 16). Increasing the visual encounter survey effort from 25m to 50m and 75m suggests an increase in number of species (Fig. 16a), number of salamander species (Fig.16b), number of individuals (Fig. 16c), and number of

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7

Number of Indivduals

6 5 VES CB

4

LLB QU

3

DF EL

2 1 0 JUNE

JULY

AUGUST

Month (2006)

Figure 15. Mean number of herpetofauna individuals encountered during the three sampling periods (VES=visual encounter survey, CB=cover board survey, LLB=leaf litter bags, QU=quadrat leaf litter search, DF=drift fence survey, EL=electrofishing).

Table 5. Herpetofauna individuals minimum, maximum, mean, and standard error values for each technique for each month. Sampling Method

Month

Minimum

Maximum

Mean

SE



0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

4 4 4 2 27 9 3 1 1 2 22 16 5 2 4 1 18 24

1.16 0.58 0.26 0.31 6.42 3.79 1.16 0.16 0.11 0.21 5.11 4.26 1.53 0.16 0.31 0.05 3.42 4.53

0.28 0.22 0.21 0.15 1.60 0.71 0.26 0.09 0.07 0.12 1.55 0.96 0.33 0.11 0.22 0.05 1.18 1.23

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2.5 A

VES EL

2

VES EL

B Cumulative Number of Salamander Species

Cumulative Number of Species

2.5

1.5

2

1.5

1 0.5

1

0.5

0

0 25

50

75

25

50

75

Length of Section

6

3.5 C

VES EL

5

Cumulative Number of Salamander Individuals

Cumulative Number of Individuals

Length of Section

4 3 2 1 0

3

D

VES EL

2.5 2 1.5 1 0.5 0

25

50 Length of Section

75

25

50

75

Length of Section

Figure 16. Effect of increasing effort on species (A), salamander species (B), individuals (C), salamander individuals (D), during the month of June (VES=visual encounter survey, EL=electrofishing).

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salamander individuals (Fig. 16d). Increasing the electrofishing effort from 25m to 50m and 75m suggests an increase in number of species (Fig. 16a) and number of individuals (Fig.16c). However, these patterns may be misleading because comparisons were based on data from only four sites for VES and five sites for electrofishing, only one of which had a 75m observation for each method. The standard error for all samples was relatively high. July and August were similarly inconclusive. Only five species and 11 total individuals were captured using quadrat leaf litter searches which did not constitute a large enough sample to examine the effect of sampling additional quadrats (Table 6).

Monitoring Microhabitat Features Salamanders were encountered in a variety of microhabitats. The total number of salamanders encountered included 22% found on the streambank, 10.7% found in riffles, 38.7% found in runs, and 27.4% found in pools (Table 7). Streambank encounters consisted of 24.3% in leaf litter, 56.8% in mud, and 18.9% in seeps. In riffles, 50% of encounters occurred in a water depth of 1-3cm, and 50% in a water depth of 4-6cm. In runs, 21.5% of encounters occurred in a water depth of 1-3cm, 64.6% in a water depth of 4-6cm, 4.6% in a water depth of 7-9cm, 6.1% in a water depth of 10-12cm, and 3.1% in a water depth of greater than 12cm. Pool encounters consisted of 4.2% in a water depth of 1-3cm, 10.4% in a water depth of 4-6cm, 22.9% in a water depth of 7-9cm, 60% in a water depth of 10-12cm, and 2.1% in a water depth of greater than 12cm. Seven species were detected on the streambank, three in riffles, two in runs, and four in pools.

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Table 6. Number of individuals of each taxon encountered with each technique. Taxa Acris crepitans Ambystoma maculatum Ambystoma opacum Bufo americanus Bufo fowleri Chelydra serpentina Chrysemys picta Coluber constrictor Desmognathus fuscus Elaphe obsoleta Eumeces fasciatus Eurycea bislineata Hyla cinerea Nerodia sipedon Notophthalmus viridescens Plethodon cinereus Pseudacris crucifer Pseudotriton montanus Psuedotriton ruber Psuedotriton spp. larvae Rana catesbeiana Rana clamitans Rana palustris Rana sphenocephala Rana sylvatica Scincella lateralis Terrapene carolina Thamnophis sirtalis Total

VES

CB

LLB

QU

DF

EL

11 --5 3 1 1 -1 1 1 10 -1 --2 1 4 -2 155 17 23 1 -3 -243

--1 --------7 ---1 -----1 -3 1 ---14

----1 ------10 -------6 --------17

-----------4 1 -----1 --4 --1 ---11

2 -1 8 6 --1 -1 -3 1 -1 --1 1 -4 37 2 3 1 1 -1 75

2 27 -1 -1 -----47 ------2 39 9 150 4 9 ----291

29

Table 7. Numbers of salamanders encountered in each microhabitat type (AMMA=Ambystoma maculatum, AMOP=Ambystoma opacum, DEFU=Desmognathus fuscus, EUBI=Eurycea bislineata, NOVI=Notopthalmus viridescens, PLCI=Plethodon cinereus, PSMO=Pseudotriton montanus, PSRU=Pseudotriton ruber, PSLV=Pseudotriton spp. larvae). HABITAT TYPE STREAMBANK LEAF LITTER IN TRAP UNDER LOG UNDER SAMPLING UNIT MUD IN TRAP LEAF DEBRIS ROOT MASS UNDER LOG UNDER SAMPLING UNIT SEEP IN TRAP UNDER LOG UNDER SAMPLING UNIT RIFFLE 1-3 CM WATER DEPTH GRAVEL GRAVEL/SAND GRAVEL/WOODY MUD/LEAF DEBRIS 4-6 CM WATER DEPTH GRAVEL/LEAF DEBRIS GRAVEL/SAND LEAF/WOODY DEBRIS RUN 1-3 CM WATER DEPTH GRAVEL/SAND MUD/LEAF DEBRIS MUD/SILT ROOT MASS SAND SAND/LEAF DEBRIS

AMMA

AMOP

DEFU

EUBI

NOVI

PLCI

PSMO

PSRU

PSLV

----

----

----

-2 4

1 ---

--1

--1

----

----

------

1 ---1

------

-3 1 7 3

------

------

-1 ----

1 1 -2 --

------

----

----

-1 --

2 2 1

----

----

----

-1 --

----

-----

-----

-----

-3 ---

-----

-----

-----

1 ----

-1 3 1

----

----

----

1 4 --

----

----

----

----

1 -3

-------

-------

-------

1 ---7 --

-------

-------

-------

-------

-3 1 1 -1

30

Table 7. Continued. HABITAT TYPE 4-6 CM WATER DEPTH GRAVEL/LEAF DEBRIS GRAVEL/MUD GRAVEL/SAND LEAF DEBRIS MUD/LEAF DEBRIS MUD/WOODY DEBRIS SAND/LEAF DEBRIS SAND/MUD SAND/WOODY DEBRIS 7-9 CM WATER DEPTH LEAF/WOODY DEBRIS SAND/LEAF DEBRIS 10-12 CM WATER DEPTH GRAVEL/LEAF DEBRIS MUD/LEAF DEBRIS >12 CM WATER DEPTH MUD POOL 1-3 CM WATER DEPTH MUD/LEAF DEBRIS 4-6 CM WATER DEPTH GRAVEL/LEAF DEBRIS MUD/LEAF DEBRIS MUD/SAND 7-9 CM WATER DEPTH LEAF/WOODY DEBRIS MUD/LEAF DEBRIS MUD/LEAF/UNDERCUT 10-12 CM WATER DEPTH CLAY/GRAVEL LEAF DEBRIS/MUD LEAF DEBRIS/SILT >12 CM WATER DEPTH MUD/CLAY

AMMA

AMOP

DEFU

EUBI

NOVI

PLCI

PSMO

PSRU

PSLV

----------

----------

----------

5 1 7 3 1 -6 1 4

----------

----------

----------

----------

3 1 2 1 2 1 1 -3

---

---

---

-1

---

---

---

---

2 --

---

---

---

1 --

---

---

---

---

-3

--

--

--

--

--

--

--

--

2

--

--

--

--

--

--

--

--

2

----

----

----

1 2 1

----

----

----

----

-1 --

----

----

----

3 1 --

----

----

----

-1 --

2 3 1

-27 --

----

----

--1

----

----

----

1 ---

----

--

--

--

1

--

--

--

--

--

31

Mud Salamander Surveys Two adult mud salamanders were captured in August; one in Cedarville State Forest (Prince George’s County) with a visual encounter survey, and one in the Doncaster Demonstration Forest (Charles County) with a drift fence survey. The mud salamander encountered with the drift fence was found underneath a funnel trap. Both were captured on the streambank (Table 7). No mud salamanders were sampled in an area where mud salamanders are known to occur, the Jug Bay Wildlife Sanctuary in Anne Arundel County (Chris Swarth, personal communication), although three sites were established there.

32

DISCUSSION

The results of this study suggest that the most effective sampling methods in the Coastal Plain region of Maryland are VES and electrofishing. Other studies have found VES to perform better than other methods such as cover board surveys and drift fence surveys (Foley and Smith 1999, Hyde and Simons 2001, Paszowski et al 2002, Fogarty and Jones 2003). Foley and Smith (1999) found that most often VES produced more species and more individuals than drift fence surveys, although the rate of accumulation of species did not differ significantly between the two methods. In a comparison of VES and pitfall traps, Fogarty and Jones (2003) captured 27 salamanders with pitfall traps and 328 salamanders with VES, which accounted for 9% and 50% of amphibians captured, respectively. In this study there often were no significant differences between VES, electrofishing, and drift fence surveys; however the cost in labor, maintenance, and time (drift fence surveys require at least two visits for results) may cause the latter method to be less attractive than VES and electrofishing (Corn 1994, Enge 1997). Also, Fogarty and Jones (2003) reported depredation of amphibians in pitfall traps by raccoons, which may present a source of bias. In this study, a dead snake (T. sirtalis) which appeared to have been bitten to death through the trap, possibly by a raccoon, was found in a funnel trap. Drift fence surveys may be useful for monitoring mud salamanders, as 25% of records at the Jug Bay Wildlife Sanctuary were caught with drift fence surveys; however the majority were captured by hand (Chris Swarth, unpublished data).

33

Cover board surveys and leaf litter bags also require at least two visits to establish and sample sites, and the low yield may not be worth the effort. Also, it has been suggested that snakes may become entangled in leaf litter bags and die (Stuart and Watson 2001). Previous studies have shown leaf litter bags to perform better than turning over rocks in the stream channel (Pauley and Little 1998); however, electrofishing unquestionably produced the most larval salamanders in this study, and a combination of electrofishing and VES may be ideal. These data also suggest that the sampling of amphibians and reptiles in Maryland’s Coastal Plain does not vary significantly between the months sampled. This is consistent with data from a watershed in the highlands of Maryland (Strain, G.F., in preparation). However peak activity periods for the majority of temperate amphibians may occur in earlier months (Duellman and Trueb 1994, Stebbins and Cohen 1995) and an amphibian monitoring program may benefit from sampling earlier. Also, sampling only during the summer months may not completely characterize the amphibian assemblage at a site, given different life histories and emergence times of some species (Stebbins and Cohen 1995). Smith and Grossman (2003) suggested that differences in larval southern two-lined salamander abundance from season to season were due to differences in seasonal microhabitat availability. Temperature and rainfall may also influence the number of species encountered at a given site as well (Duellman and Trueb 1994), and differences between sites may be an artifact of weather or temperature. The microhabitat data suggest that salamanders are most often encountered in runs and pools. However, the observations in pools included one event in which 27 individuals of A. maculatum were found in an isolated pool of a dry streambed, which

34

may not be representative of general stream sampling. When these observations are removed, the proportions encountered change to 26.2% in streambank habitat, 12.8% in riffles, 46.1% in runs, and 13.5% in pools. This suggests that salamanders may be encountered with greater frequency in streambank and run habitats. The low frequency of salamanders in pools may be due to a general higher number of fish in this habitat, and because salamander larvae can detect chemical cues from fish (Petranka et al. 1987) they may avoid this habitat (Smith and Grossman 2003). The low frequency of salamanders in riffles and pools may also be explained by a lower percentage of these habitats encountered, but this was not quantified. Some sampling methods detected species that others did not (Table 6), and this is consistent with previous studies (Foley and Smith 1999, Fogarty and Jones 2003). Drift fence surveys detected 18 species total and four species that others did not, including one salamander species, N. viridescens. Cover boards detected six species total and one species that others did not, the salamander P. cinereus. Electrofishing detected 11 species total and one species that others did not, the salamander A. maculatum. VES detected 19 species total and six species that others did not, including one salamander species, D. fuscus. Leaf litter bags and quadrat leaf litter searches did not detect any species not detected by other methods. Although only two adult mud salamanders (P. montanus) were identified, that does not mean that only two mud salamanders were captured. An unknown proportion of the 45 larvae of the genus Pseudotriton captured through the use of electrofishing and leaf litter bags may have been mud salamander larvae but were unable to be identified as

35

such, due to the strong resemblance of this species to P. ruber larvae (Scott Stranko, Rebecca Chalmers, personal communication).

36

RECOMMENDATIONS

As VES and electrofishing were the best overall methods for maximizing species and individuals encountered, the sampling protocol that the MBSS currently has in place should be adequate, with a few suggestions. The MBSS Sampling Manual (Kazyak 2001) instructs crews to “collect/positively identify herpetofauna observed during electrofishing or other activities.” Dedicating time and a section of streambank exclusively to VES would most likely maximize encounters by minimizing disturbance to herptiles before they are found. Also, great care should be taken to search all available microhabitats at a site (Foley and Smith 1999). Salamander larvae do not react to electrofishing in the same manner as most fishes. They tend to remain on the bottom, often blending in with the substrate unless they happen to roll over and expose their light bellies. Also, amphibians do not remain shocked as long as most fishes, usually recovering within seconds of being shocked. Therefore extreme care should be taken when in smaller streams to capture salamander larvae. Another difficulty is that smaller larvae easily escape through the 1/8th inch holes of standard dipnets. In this study we switched to a dipnet with 1/16th inch mesh and had no further difficulty. The decrease in mesh size did not appear to interfere with sampling in any way. Salamanders are difficult to detect, and even the most common of salamanders are frequently not found at a particular collecting site. Due to small sample sizes it may be difficult to quantitatively assess population trends and distributions. Determining quantitatively whether or not an increase in the length of stream sampled increases the

37

yield may be beneficial. Along these lines, estimates may be biased if they are calculated with the often incorrect assumption that species are equally detectable (ARMI 2006, Salvidio 2001). In order to be able to quantitatively assess salamander or other herpetofauna populations, it may be important to modify sampling protocols to estimate detection probabilities (MacKenzie et al. 2003, 2002, ARMI 2006). The results of this study suggest that salamanders may be encountered with the greatest frequency on the streambank and in run habitats. However, available habitat was not evaluated and this result may be caused by lower representation of other habitats at study sites. Randomly sampling habitat and utilizing contingency analysis to determine if certain species prefer specific microhabitat features may be beneficial. If one of the specific goals of a salamander monitoring program is to determine the status of the mud salamander, P. montanus, and the majority of Pseudotriton species are captured in their larval form through electrofishing, then utilization of molecular markers to determine P. montanus larvae from P. ruber larvae may be necessary. Many studies have employed molecular techniques to determine morphologically similar species (Mack et al. 1986, Rawson et al. 1996, Brown et al. 1999). The larval form of P. montanus may be more abundant or at least more readily catchable than the adult, and a molecular marker that would allow easy identification of P. montanus individuals would greatly aid a monitoring program.

38

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