**1. Background and Objectives**

**[1.a. Introduction]{.underline}**

The National Park Service's (NPS) Great Lakes Inventory and Monitoring
Network (GLKN) has developed a prioritized list of 46 indicators, termed
'Vital Signs,' for monitoring long-term ecosystem health in nine NPS
units in the Great Lakes ecoregion (Route 2004). This amphibian
monitoring protocol is one of several protocols that GLKN intends to
implement under the umbrella of an overall monitoring plan (Route and
Elias 2005). Amphibians were initially considered for monitoring under a
combined Amphibians and Reptiles Vital Sign. However, because of
differences in methods for monitoring both taxa and constrained funding
and logistics, we decided to focus on amphibians initially with the
intention of adding reptiles as the GLKN monitoring program matures.
Hence, this protocol is for amphibians only. However, snapping turtles
will be monitored for toxicants, deformities, and lesions under the
Trophic Bioaccumulation Vital Sign (Bowerman and Route 2005).

**[OTHER EXISTING EFFORTS]{.underline}** -- Implementation of a viable,
long-term monitoring program requires partnering with other agencies and
universities around the Great Lakes region. Implementation of this
amphibian monitoring protocol could be a collaborative effort between
the GLKN, the Upper Mississippi Region (UMR) of the US Geological
Survey's (USGS) national research program for amphibians, the Amphibian
Research and Monitoring Initiative (ARMI), and the Lake Superior Basin
Herpetological Monitoring Program of the U.S. Environmental Protection
Agency's (EPA) Great Lakes National Program Office (GLNPO) (Hecnar and
Casper, personal communication). The design of this protocol has been a
collaborative effort between the GLKN and ARMI and we hope to broaden
this partnership in developing this program further.

In addition to ARMI, State departments of natural resources in Indiana,
Michigan, Wisconsin, and Minnesota use variations of the North American
Amphibian Monitoring Program protocol (NAAMP 2005) and volunteers to
monitor amphibian populations. Similarly, the Marsh Monitoring Program
is a joint Canada/USA effort to monitor amphibian populations around the
Great Lakes. The U.S. Forest Service and the U.S. Fish and Wildlife
Service use variations of the NAAMP protocol to monitor amphibian
populations on national forests and wildlife refuges in the region.

The sampling designs and methods described in this protocol are based
largely upon experiences surveying amphibians in three parks in the GLKN
from 2002 to 2005: the Mississippi National River and Recreation Area
(MISS), the St. Croix National Scenic Riverway (SACN), and Voyageurs
National Park (VOYA) (Sadinski et al. in preparation). Conducting those
surveys enabled investigators to test many of the designs and methods we
recommend in this protocol in a variety of habitats and conditions. We
are confident that they will produce data that are statistically robust
and comparable either directly or indirectly with other programs that
monitor amphibians using similar methods and analytical approaches. This
includes all programs that conduct call surveys regionally and
nationally and especially those such as ARMI and other NPS networks that
analyze data via the same methods we recommend here.

**[1.b. Rationale for monitoring amphibians]{.underline}**

Amphibians were ranked among the top ten vital signs across the parks of
the GLKN. Reasons to monitor amphibians include their:

-   **Sensitivity** - Amphibians generally are sensitive to changes in
    environmental factors, including temperature, precipitation,
    humidity, hydrology, land cover, nutrients, toxicants, and exotic
    species, among others, and are highly-linked in food webs (Jones
    1986; Sadinski and Dunson 1992; Blaustein et al. 1997; Welsh &
    Ollivier 1998; Fauth 1999; Adams 2000; Pollet and Bendell-Young
    2000; Gibbs and Breisch 2001; Marco et al. 2001; Sparling et al.
    2001; Veldhoen and Helbing 2001; Beebee et al. 2000; Rowe et al.
    2003; Johnson and Chase 2004; Lannoo 2005).

-   **Role as ecological integrators** - Many species of amphibians live
    in both terrestrial and aquatic habitats and, thus, their fitness is
    a function of environmental conditions across habitats (Skelly 2001;
    Lannoo 2005).

-   **Ease of study** - Various life stages of amphibians typically are
    available and accessible for study. These traits allow for rigorous
    sampling designs for monitoring (Heyer et al. 1994); Fellers and
    Freel 1995; Corn et al. 2000). They also are invaluable for
    conducting manipulative experiments when further studies are
    necessary to establish the causes and effects of any declines
    (Sadinski and Dunson 1992).

-   **Conservation concern** - Declines of populations of amphibians are
    among the most prominent global issues in conservation biology (for
    example, Young et al. 2001; Blaustein and Kiesecker 2002; Stuart et
    al. 2004).

-   **Regional relevance** - All four states in which the GLKN is
    located, as well as Federal agencies in the region, are currently
    monitoring amphibian populations; results of these efforts will
    provide the NPS with a broader context for data collected in parks
    of the GLKN.

**[1.c. Goals and objectives]{.underline}**

The NPS does not intend to monitor every amphibian species present in
the nine parks of the GLKN (Table 1). This would be impractical and
unnecessary, given the resources available and NPS's objective to
monitor amphibians as useful indicators within the context of vital
signs. Conveniently, populations of the majority of species that live in
these nine parks breed in wetlands during well-known periods from early
spring to mid-summer (Table 1; Conant and Collins 1998; Lannoo 2005).
These life-history traits allow for efficient and effective sampling
designs (Heyer et al. 1994; Fellers and Freel 1995; Crouch and Paton
2000; Crouch and Paton 2002) as well as a sensible approach to selecting
species to monitor.

Our overall goal is to design a monitoring program for populations of
wetland-breeding amphibians that will provide data to test if changes in
their distributions and abundances occur over time. We will analyze and
interpret amphibian results in concert with data from simultaneous
monitoring of environmental conditions, including potential stressors,
to examine whether any trends in populations are associated with trends
in ambient conditions. (We will continue to refine this protocol in the
future to include additional species or environmental stressors as
appropriate.) Data from monitoring amphibians will be integrated with
data from monitoring other vital signs to assess the long-term variance
in ecological conditions in the GLKN.

Based upon our overall goal, and from scoping and focus meetings with
park staff and other scientists, we have formulated the following
specific objectives and monitoring questions. We believe that these
objectives and questions translate into a realistic set of variables
that can be monitored effectively long-term. Further rationales and
details for these objectives can be found in section 2.

OBJECTIVES

1\. **Assessing potential trends in site occupancy**

> a\) Annually measure the presence of several wetland-breeding species
> at predetermined locations throughout the breeding season in each
> park, including species that breed during early, middle, and later
> periods of the summer season.

b)  Based upon measures of occupancy at monitoring locations, use the
    Proportion Area Occupied (PAO; MacKenzie et al. 2002b and MacKenzie
    et al. 2003) as the principal metric to quantify occupancy of
    breeding sites for targeted species across parks each year.

c)  Assess the PAO over time to determine if the distribution or number
    of breeding subpopulations of any monitored species changes
    significantly and compare these results with results from other
    national and state programs.

> 2\. **Correlating trends in distribution to covariates likely to be
> important**
>
> a\) Use portable meters during surveys, nearby remote sensors (such as
> weather stations operated by the National Oceanic and Atmospheric
> Administration), and remotely sensed data from satellites to measure
> and analyze environmental conditions, including covariates and
> potential stressors such as weather, land cover and use, and water
> chemistry.
>
> b\) Use measures of environmental variables as covariates in
> calculating PAO, and in basic statistical tests of association, to
> test whether trends in occupancy are correlated with environmental
> covariates.
>
> 3\. **Use data on the numbers of animals of each species observed at
> each sampling location to complement information on occupancy.**

**[1.d. Specific monitoring questions (as associated with the above
objectives)]{.underline}**

OBJECTIVE 1:

*Question 1*: Do the distributions of monitored species agree with
predictions for each park based upon known historical distributions?

*Question 2*: Are there any trends in the PAO for monitored species
across parks that indicate potential changes in environmental
conditions?

*Question 3*: How do the magnitudes and directions of any changes in the
PAO of targeted species compare with results from other regional and
national surveys?

OBJECTIVE 2:

*Question 4*: Are any trends in PAO associated with any trends in
environmental variables measured during amphibian monitoring or
monitoring other vital signs in the GLKN?

OBJECTIVES 1,3:

*Question 5*: Do many individuals of each species occupy a site?

*Question 6*: Are trends in PAO associated with trends in the numbers of
individuals?

**2. Sampling Design**

**[2.a. Rationale for this sampling design over others]{.underline}**

Because all sampling methods have strengths and weaknesses (for example,
Corn et al. 2000; Buech and Egeland 2002), an ideal monitoring scheme
for amphibians would include a variety of methods (Corn and Bury 1990;
Heyer et al. 1994) executed intensively enough to 1) ensure the highest
likelihood of detecting species when they are present, 2) provide
accurate information on numbers of adults, reproductive success, and
relative abundance of different age classes, 3) produce data on
frequencies and types of malformations, and, among others, 4) provide a
set of measurements of environmental variables for both long-term trend
analysis and (more importantly for this protocol) interpretation of
amphibian results. Such a combination of methods could include intensive
visual-encounter surveys of various types (Corn and Bury 1990; Heyer et
al. 1994), trapping with drift fences and pit-fall traps 2), trapping
with submerged traps (Adams et al. 1997; Wilson and Pearman 2000),
surveys of egg masses (Crouch and Paton 2000; Sadinski 2004), sweeps
with dip nets (Smith et al. 2003), daytime and nighttime call surveys
(Corn et al. 2000; Gibbs and Breisch 2001; Crouch and Paton 2002;
(Stevens and Paszkowski 2004), metamorph surveys (Converse et al. 2000;
Eaton et al. 2004; Pounds and Crump 1994) surveys with cover objects
(Jung et al. 2000), and measurements of water temperature (Moore 1939;
Douglas 1948; Herreid II and Kinney 1967; Freidenburg and Skelly 2004;
Sadinski 2004), water chemistry (Sadinski and Dunson 1992; Rowe et al.
1998; Brodman et al. 2003), water levels and duration of hydroperiod
(Pechmann et al. 1989; Skelly et al. 1999; Ryan and Winne 2001), weather
(Sadinski 2004), and climate (Pounds and Crump 1994; Beebee 1995; Pounds
2001; Beebee et al. 2002), among others.

In combination with appropriate sampling designs, such an ideal scheme
would allow for monitoring populations closely in relation to
environmental variables known to affect the fitness of amphibians. Such
an effort would require either more resources than are available for the
GLKN's monitoring program for amphibians or monitoring so few
subpopulations in each park as to render the results useless
statistically and obviate the GLKN's ability to detect any changes at
meaningful spatial and temporal scales. Thus, we have developed an
approach that is less than ideal, but which still allows the NPS to meet
the goals of the GLKN's monitoring program. To derive our approach, we
examined the most critical issues regarding the goals of the program,
the resources we expect to have at our disposal, the biology of
amphibians, and the physical nature of each park.

GOALS OF THE PROGRAM

This amphibian monitoring protocol is one of several protocols that the
NPS intends to implement under the umbrella of an overall plan to
monitor long-term ecosystem health in the nine parks of the GLKN (Figure
1; Route and Elias in preparation). Thus, this protocol ultimately has
to provide data on amphibians that are useful for indicating ecosystem
health given known financial, biological, and physical constraints
(Figure 2).

![](media/image1.png){width="4.059722222222222in" height="3.0375in"}

![](media/image2.png){width="3.85in" height="2.8944444444444444in"}

RESOURCES AVAILABLE

The availability of funding will be the ultimate determinant of how and
where this monitoring protocol is implemented. We considered likely
resource limitations strongly up front so that this protocol is
cost-effective and flexible, yet will allow meaningful data to be
collected with statistical rigor across parks.

BIOLOGY OF RESIDENT AMPHIBIAN SPECIES

In addition to considering the goals of the program and the resources
available, we based this protocol upon our knowledge of the species that
live in each park. This knowledge is from historical information (Vogt
1981; Oldfield and Moriarty 1994; Casper 1998; Conant and Collins 1998;
Hecnar et al. 2002; Casper 2004; Lannoo 2005), surveys conducted during
the inventory phase of the GLKN's Inventory and Monitoring Program
(Hecnar et al. 2002; Newman 2003; Casper 2004; Glowacki and Grundel
2005; Sadinski et al. in preparation), and our work with these species
outside of the parks of the GLKN (Sadinski et al. in preparation). This
knowledge enabled us to evaluate important characteristics of each
species that determined 1) their overall usefulness in providing
information to help meet the NPS-GLKN's overall goal of monitoring
long-term ecosystem health, 2) their detectability, and 3) how they can
be sampled. For our purposes, four important characteristics for species
in the GLKN include 1) when they breed during the year, and whether
they: 2) breed in wetlands; 3) live in terrestrial habitats; and 4) call
during breeding (Table 1).

We considered these characteristics carefully to design a plan for the
GLKN by which the most species could be monitored with reasonable
effort. We evaluated the candidacy of each species for monitoring in
terms of if, where, when, and how they could be sampled effectively
(Figure 3) and identified those with overlapping habitats and similar
life history traits and, thus, with similar potential to be sampled. We
also considered whether or not a species' life history traits qualified
them as good potential indicators of ecosystem health and the statuses
of other amphibian species not monitored (Figure 4).

![](media/image3.png){width="4.459722222222222in"
height="3.3520833333333333in"}

![](media/image4.png){width="4.6in" height="3.442361111111111in"}

*Species that breed in wetlands*

Note: We use common names throughout the text of this document. For a
list of corresponding scientific names, please see Table 1.

We designed this protocol to monitor populations of wetland-breeding
amphibians. Twenty-two of the 24 species that do or could live in the
GLKN breed in wetlands (Table 1), ranging from permanent to temporary
(Conant and Collins 1998; Lannoo 2005). The two amphibian species that
do not breed in wetlands, the redback and slimy salamanders, are
terrestrial salamanders. The slimy salamander lives in only one park at
most, Indiana Dunes National Lakeshore (INDU), whereas the redback might
be found in all nine parks (Vogt 1981; Oldfield and Moriarty 1994;
Conant and Collins 1998; Casper 2004; Lannoo 2005). Of the 22 species
that breed in wetlands, several often breed in portions of the same
wetland in the same park, allowing for sampling several species in one
location. For example, in the St. Croix National Scenic Riverway (SACN),
we have observed spring peepers, blue-spotted salamanders, spotted
salamanders, wood frogs, chorus frogs, northern leopard frogs, eastern
American toads, and gray treefrogs breeding in the same wetland
(Sadinski et al. in preparation). Given that we are trying to maximize
benefits relative to costs, monitoring the distributions and abundances
of breeding subpopulations in such wetlands makes sense.

*Ecological information obtained from species*

The ecological information obtainable from certain species was another
important consideration in selecting species to monitor. We do not
recommend monitoring redback and slimy salamanders, but we are not
disregarding species that can indicate the health of the terrestrial
environment. Post-metamorphs of 20 of the 22 species that breed in
wetlands also live in terrestrial habitats to varying extents (Vogt
1981; Oldfield and Moriarty 1994; Casper 1998; Conant and Collins 1998;
Lannoo 2005; Table 1) and studies have shown that survival as sub-adults
and adults in terrestrial habitats can limit the sizes and distributions
of wetland-breeding amphibian populations (Schmidt 2003; Blackwell et
al. 2004). Thus, monitoring populations of several wetland-breeding
species provides information about the health of terrestrial habitats as
well.

*Wetland-breeding, but not terrestrial species*

Mudpuppies likely are relatively widespread among the parks of the GLKN,
while lesser sirens possibly exist only in Indiana Dunes National
Lakeshore (Conant and Collins 1998; Casper 2004; Lannoo, 2005). Both
species breed in permanent wetlands, but are either completely
(mudpuppies) or almost exclusively (lesser sirens) aquatic (Conant and
Collins (1998). Trapping could be effective for sampling these species
(Johnson and Barichivich 2004), but would require more visits to sites
to survey their habitat than are necessary to survey other
wetland-breeding species. Given the projected financial constraints for
monitoring amphibians in the GLKN, the costs of monitoring mudpuppies
and lesser sirens exceeds the benefits and preclude them from further
consideration at this time. This is especially true because bullfrogs
and green frogs live in some of the same wetlands as mudpuppies and
lesser sirens (Conant and Collins 1998; Lannoo 2005) and can serve to
some extent as indicators of the health of those habitats.

*Calling and non-calling species*

Further identification of the target set of species to monitor was more
challenging because all 20 of these wetland-breeding species can be
surveyed by visual methods or trapping (although using drift fences and
submersible traps is not feasible because of the number of sites
necessary to monitor (section 2.e.), but six of the 20 do not call and
cannot be monitored by call surveys. Furthermore, nighttime call surveys
cannot be conducted in sufficient numbers in VOYA, APIS, and ISRO
because of limited roads. Important differences in the efficiency and
effectiveness of these daytime and nighttime methods required a thorough
comparison of both to justify recommending one over the other and
possibly removing some species from the pool of candidates for
monitoring in some parks (Table 2). We also considered these comparisons
within the context of the primary analytical tool we are recommending,
Percent Area Occupied.

PERCENT AREA OCCUPIED

The data obtained by using this protocol will be analyzed primarily via
comparisons of models that Mackenzie and others (2002a, b, 2003, 2004)
have developed and incorporated into the computer program PRESENCE.
Various models that involve different numbers of variables are compared
in terms of their parsimony and fit of the occupancy data using
information-theoretic criteria (Burnham and Anderson 2002). The percent
area occupied (PAO) is a measure of the percent of the known area of
inference that is occupied by a monitored species based upon survey
data. PRESENCE and relevant supporting information are available as a
free download from the USGS's Patuxent Wildlife Research Center's
software archive (http://www.mbr-pwrc.usgs.gov/software.html). Specific
qualities of using PAO to monitor statuses of populations over time
figured prominently into our comparisons of the costs and benefits of
daytime and nighttime surveys.

Two strengths of using Mackenzie et al.'s (2002, 2003) models are
that 1) they allow for calculating detection probabilities based upon
repeated sampling and therefore provide an unbiased estimate of
occupancy (Schmidt 2003; Royle 2004) and 2) measures of variables
describing ambient environmental conditions can be used as covariates in
the models. This latter feature enables the analyst to investigate the
influence of environmental variables on the occupancy of species
monitored. Another important aspect of this method is that it was
developed in collaboration with scientists from ARMI and is used as the
primary analytical tool for data collected under ARMI throughout the
United States. Consequently, use of the PAO has been adopted by other
NPS Monitoring Networks and is likely to be the analytical method of
choice for the GLNPO's Lake Superior Basin Herpetological Program (S.
Hecnar and G. Casper, personal communications). Thus, the NPS will be
able to compare PAO calculated for populations in the GLKN with PAO
calculated for populations regionally and nationally.

Using Mackenzie et al.'s models requires repeated visits to multiple
sites. The requisite number of sites and repeat surveys is a function of
the desired precision of estimates of PAO and the presence and
detectability of each species being monitored (MacKenzie et al. 2004).
For the purposes of this analytical approach, a species is considered
detected at a site simply if one individual of any life stage is
detected at the site during any of the three visits. Values calculated
for PAO for individual species apply only to the areas of inference,
which are a reflection of how sampling locations are chosen for each
species and the sampling method (specifically, the maximum detection
distance that the method permits)).

DAYTIME VISUAL SURVEYS COMPARED TO NIGHTTIME CALL SURVEYS

As stated earlier, daytime visual surveys of one type or another can be
used to monitor all amphibian species in the GLKN; the same is not true
for call surveys because all species do not call. Data collected through
daytime visual surveys or nighttime call surveys can be analyzed for
PAO. Although estimates of PAO resulting from different methods of
sampling the same species might or might not be similar (Sadinski,
personal observations), different methods require different resources
and expertise and can result in important differences in what is known
about populations (Table 2). We considered these differences carefully
to ensure that use of this protocol ultimately addresses the scientific
questions NPS is asking about the statuses of amphibian populations over
time (Section 1.d; Table 2).

Table 2. Comparisons between daytime and nighttime surveys. Bold and
underlined numbers indicate the more advantageous cell in the comparison
of the two cells across that row.

+-----------------------------------+-----------------------------------+
| **Comparisons between daytime and |                                   |
| nighttime surveys**               |                                   |
+-----------------------------------+-----------------------------------+
| **Daytime Visual and Call**       | **Nighttime Call**                |
+-----------------------------------+-----------------------------------+
| **[1]{.underline}.** Can observe  | 2\. Can observe only adults of    |
| all life stages (embryos, larvae, | potential target species that     |
| metamorphs, sub-adults, adults)   | call, which would exclude six     |
| of all potential target species,  | species of salamanders that breed |
| including species that do not     | in wetlands and also dwell in     |
| call.                             | terrestrial habitats in parks of  |
|                                   | the GLKN.                         |
+-----------------------------------+-----------------------------------+
| **[2]{.underline}**. Requires     | 2\. Requires repeat surveys       |
| repeat surveys during the time    | during the time when calling      |
| when individuals of any life      | males are present. This greatly   |
| stage (embryos, larvae,           | shortens the window of time in    |
| metamorphs, sub-adults, adults)   | that the presence of each species |
| are present. For the purpose of   | can be detected (Figure 5).       |
| simply detecting the presence of  |                                   |
| a species, this affords surveyors |                                   |
| a longer period of time during    |                                   |
| which repeat surveys can be       |                                   |
| conducted (Figure 5).             |                                   |
+-----------------------------------+-----------------------------------+
| 3\. Each two-person crew can      | **[3]{.underline}**. Each         |
| sample x number of individual     | two-person crew can sample x      |
| potential breeding sites.         | number of individual areas, which |
|                                   | with a parabolic microphone can   |
|                                   | be 2 km or more in diameter       |
|                                   | depending upon conditions. Such   |
|                                   | areas often contain more than one |
|                                   | breeding site.                    |
+-----------------------------------+-----------------------------------+
| 4\. Each person in a two-person   | **[4.]{.underline}** One          |
| crew would have to work at least  | two-person crew can conduct three |
| 80 hours to conduct one of three  | repeat surveys of 30 sites in     |
| repeat surveys of 30 potential    | less than 80 hours per person.    |
| breeding sites.                   |                                   |
+-----------------------------------+-----------------------------------+
| 5\. Surveying all life stages of  | **[5.]{.underline}** To conduct   |
| all target species sufficiently   | repeat surveys at listening       |
| to model population dynamics is   | locations during the night when   |
| not possible. Given this          | adults of the target species are  |
| limitation, surveying for adults  | present and calling would require |
| is arguably the best way to       | one two-person to conduct three   |
| obtain useful information about   | sets of three replicate surveys   |
| the status of a population in     | to monitor species that breed     |
| real time because adults have     | earlier and later, which equates  |
| been recruited into the           | to six weeks of surveying.(# 4    |
| population, are capable of        | above), Such a scheme is          |
| reproducing, and can be           | possible.                         |
| long-lived.                       |                                   |
|                                   |                                   |
| To conduct three repeat surveys   |                                   |
| at sites during the day when      |                                   |
| adults of all target species are  |                                   |
| present would require one         |                                   |
| two-person crew to conduct at     |                                   |
| least two sets of three replicate |                                   |
| surveys to account for earlier    |                                   |
| and later breeding species        |                                   |
| (Figure 5). This equates to 12    |                                   |
| weeks worth of surveys (# 4       |                                   |
| above.). Such a scheme is         |                                   |
| prohibitively expensive.          |                                   |
+-----------------------------------+-----------------------------------+
| 6\. Alternatively, it is possible | **[6.]{.underline}** If a site is |
| to survey potential breeding site | surveyed three times during the   |
| three times during the entire     | reproductive season of species X  |
| reproductive season, which        | and ten individuals are heard     |
| provides ample opportunity to     | calling, then we know that        |
| detect one of the life stages of  | species X occupied that site      |
| a species when that species is    | during that season and we could   |
| present. Thus, if ten larvae of   | estimate the PAO, but we would    |
| species X are observed over the   | not know whether breeding         |
| three surveys, we could estimate  | occurred or was successful at any |
| the PAO and we would know that    | level.                            |
| species X occupied that site      |                                   |
| during that season and that       | However, the information obtained |
| hatching had occurred.            | in this scenario is more          |
|                                   | straightforward for calling       |
| However, such information is      | males. Ten adult males at a site, |
| limited in value for indicating   | despite not knowing if females    |
| the size of the breeding          | were present or if breeding had   |
| subpopulation at that site        | occurred, would give some         |
| because ten larvae can result     | estimate of the potential size of |
| from low hatching success in a    | the breeding subpopulation at     |
| single egg mass from a single     | that site. Monitoring calling     |
| pair of adults or they can be the | males over time could potentially |
| only survivors from 100 egg       | provide important information     |
| masses produced by 50 different   | (but see Buckley and Beebee 2004) |
| pairs of adults. Unless numbers   | regarding the variance in the     |
| of breeding adults or egg masses  | size of that subpopulation,       |
| were obtained, we would have no   | information that we cannot obtain |
| way to know what ten larvae       | reliably from larvae alone or     |
| represent.                        | other age classes if repeat       |
|                                   | surveys were not conducted during |
|                                   | the time when those age classes   |
|                                   | are present (see \# 5 above). The |
|                                   | number of calling males also can  |
|                                   | be indicative of the numbers of   |
|                                   | egg masses deposited at a site    |
|                                   | (Sadinski 2004; Stevens and       |
|                                   | Paszkowski 2004).                 |
+-----------------------------------+-----------------------------------+
| 7\. Depending upon their active   | **[7.]{.underline}** Providing    |
| periods during the day, timing of | surveys are conducted during the  |
| breeding activity at a site, the  | period of time when species X is  |
| amount of vegetative cover, the   | breeding and weather conditions   |
| sensitivity of individuals to     | are not preclusive, calling males |
| disturbance, detectability of     | of species that call in the GLKN  |
| each species in general, and      | can be heard and identified       |
| weather, among other factors,     | relatively easily. Thus, data on  |
| detecting *and identifying*       | the presence of a species and     |
| adults and other life stages at a | estimates of the number of        |
| breeding site during the day can  | calling males can be obtained.    |
| be challenging.                   |                                   |
|                                   | Weather conditions can greatly    |
| Most amphibian species that call  | influence the number of calling   |
| in the GLKN call during the day   | males within a night and among    |
| as well as during the evening and | nights. Sufficient numbers of     |
| night. Call surveys during the    | repeat surveys are necessary to   |
| day can be an effective means to  | reduce the variance associated    |
| detect the presence of calling    | with the numbers of animals       |
| males. However, more individuals  | observed calling.                 |
| of the majority of species in the |                                   |
| GLKN call more frequently and     |                                   |
| intensively during the evening    |                                   |
| and night. Exceptions can be      |                                   |
| species that breed very early in  |                                   |
| the season, such as wood frogs,   |                                   |
| when nighttime air and water      |                                   |
| temperatures can be prohibitively |                                   |
| cold.                             |                                   |
|                                   |                                   |
| Thus, daytime visual and call     |                                   |
| surveys do not necessarily result |                                   |
| in consistent or accurate         |                                   |
| measurements of adults present at |                                   |
| a breeding site for all species   |                                   |
| of amphibians in the GLKN.        |                                   |
+-----------------------------------+-----------------------------------+
| 8\. The likelihood of choosing 30 | **[8]{.underline}**. Also         |
| wetlands to sample beforehand and | requires using GIS tools          |
| finding them suitable varies with | beforehand to select listening    |
| location, but can be low.         | locations. However, the           |
| Selecting sites to sample         | likelihood of surveying 30 sites  |
| requires an investment in GIS     | for each species from 30          |
| analyses beforehand to identify   | pre-selected listening locations  |
| potential wetlands followed by    | is greater with call surveys      |
| groundtruthing preselected sites  | using parabolic microphones at    |
| to determine their suitability as | night, especially in parks like   |
| potential breeding sites for the  | several in the GLKN that contain  |
| targeted species. This can be a   | substantial numbers of wetlands.  |
| laborious process given the poor  | This is true because a parabolic  |
| accuracy of GIS data layers that  | microphone enables the surveyor   |
| describe wetlands..               | to hear, and, thus, survey and    |
|                                   | monitor, species calling from     |
|                                   | more than one breeding site at a  |
|                                   | single listening location (i.e.,  |
|                                   | within the hearing radius of the  |
|                                   | parabolic microphone). The area   |
|                                   | within which the surveyor can     |
|                                   | hear amphibians calling from a    |
|                                   | listening location serves as the  |
|                                   | site being monitored.             |
|                                   |                                   |
|                                   | Note: Unless further              |
|                                   | investigations are warranted,     |
|                                   | surveying calling males at night  |
|                                   | does not involve visiting actual  |
|                                   | breeding sites physically.        |
|                                   | Therefore, surveyors will not     |
|                                   | know how many potential           |
|                                   | individual replicate breeding     |
|                                   | sites actually exist within the   |
|                                   | area surveyed from each listening |
|                                   | location.                         |
+-----------------------------------+-----------------------------------+
| **[9]{.underline}**. Daytime      | 9\. Nighttime call surveys        |
| surveys allow for a larger area   | restrict areas of inference to    |
| of inference because boating and  | smaller areas than for daytime    |
| walking to sites can be done      | surveys because call surveys      |
| safely during the day.            | cannot be done safely and         |
|                                   | efficiently at night in areas     |
|                                   | without roads that can be         |
|                                   | traversed easily.                 |
+-----------------------------------+-----------------------------------+
| PHYSICAL CONSTRAINTS              | 10\. Cannot be conducted safely   |
|                                   | in Voyageurs National Park,       |
| **[10.]{.underline}** Can be      | Apostle Islands National          |
| conducted safely in all parks of  | Lakeshore, and Isle Royale        |
| the GLKN.                         | National Park. We will employ     |
|                                   | these only in limited fashion,    |
|                                   | along trails, in pilot work.      |
+-----------------------------------+-----------------------------------+
| 11\. Requires significant         | **[11.]{.underline}** Requires    |
| training and experience for       | relatively less experience for    |
| surveyors to identify egg masses  | surveyors to learn calls of       |
| and larvae (in particular) and to | species in each park and to       |
| conduct surveys correctly         | identify them successfully. Crews |
| (compare sections 3c and 3d).     | drive to listening locations such |
| Also requires that crews are      | that physical fitness is not      |
| physically fit                    | necessary.                        |
+-----------------------------------+-----------------------------------+
| 12\. Depending upon the park and  | **[12]{.underline}**. Roads and   |
| the sampling design, daytime      | trails permitting, night call     |
| surveys require two people, one   | surveys require two people, two   |
| vehicle, one or two types of      | vehicles, two parabolic           |
| boats, bushwhacking, possibly     | microphones, and two digital      |
| occasional transport by float     | recorders. Requires minimal       |
| plane, boat safety training,      | first-aid training.               |
| aircraft safety training, and     |                                   |
| substantial first-aid training.   |                                   |
+-----------------------------------+-----------------------------------+
| 13\. Results from daytime surveys | **[13]{.underline}**. Results     |
| are relatively less comparable    | from nighttime call surveys are   |
| with results from most other      | more comparable with those from   |
| monitoring programs in the region | other regional monitoring         |
| because most other programs       | programs because the majority of  |
| conduct call surveys.             | them conduct nighttime call       |
|                                   | surveys.                          |
+-----------------------------------+-----------------------------------+

> ![](media/image5.png){width="5.959722222222222in"
> height="4.477083333333334in"}
>
> **Figure 5. Typical reproduction season for several species in SACN.
> Yellow = period when males call during warm springs. Orange = period
> when males call more typically. Blue = total reproduction season,
> including larval period.**

Comparisons 1, 2, 9, and 10 (Table 2) favor daytime visual surveys,
whereas comparisons 3, 4, 5, 6, 7, 8, 11, 12, and 13 favor nighttime
call surveys (Table 2). Based upon this analysis, and without attempting
to weight individual comparisons, we recommend nighttime call surveys to
monitor amphibian populations in parks where they can be conducted
safely and only daytime visual surveys otherwise. This equates to
nighttime surveys being conducted in all parks except Voyageurs National
Park (VOYA), Apostle Islands National Lakeshore (APIS), and Isle Royale
National Park (ISRO), where conditions are not suitable to survey at
night to the extent necessary to provide statistically powerful sampling
within the domain of interest. For network-wide questions, the
difference in methods (specifically, the area that is sampled by
parabolic vs. targeted searches) means that these data from each of
these two sets of parks can be pooled across member parks of each set,
but not across sets.

This approach will result in data for fewer species in parks where call
surveys are conducted (Table 1; Figure 5), although the data for these
species potentially will be higher in ultimate value than those
collected for species surveyed during the day (Table 2). Furthermore, we
argue that the statuses of calling species may reflect the statuses of
sympatric, but non-calling species. Using salamanders present in the
GLKN as an example, tiger, spotted, and blue-spotted salamanders and
central newts can breed in wetlands where wood frogs and spring peepers
(and possibly chorus frogs, gray treefrogs, American toads, and others)
breed as well. If evidence for declines in populations of these anurans
is obtained by monitoring them through nighttime call surveys, this
could trigger further studies of whether the quality of their breeding
or terrestrial habitats, and in turn those of the salamanders, has
changed. Thus, monitoring anurans via nighttime call surveys also can
provide the means to monitor habitat conditions for species not being
monitored directly.

**[2.b. Recommended species to monitor]{.underline}**

Based upon all of the above considerations, we recommend that the
targeted group of species to monitor include American toads, spring
peepers, chorus frogs, northern leopard frogs, gray treefrogs, and green
frogs (Table 1; Figure 5). It is quite possible that breeding habitat
potentially suitable for the above species also will be potentially
suitable for blue-spotted, spotted, and tiger salamanders, wood frogs,
and mink frogs.

**[2.c. Recommended survey methods]{.underline}**

As stated above, lack of road access in VOYA, APIS, and ISRO does not
allow for sufficient numbers of locations to be monitored by nighttime
call surveys in these three parks. Road and water access in Grand
Portage National Monument (GRPO) are even more restrictive and do not
allow but for a very small number of sites to monitor either by day or
night. Thus, we recommend that daytime call and visual surveys be used
to monitor amphibians in VOYA, APIS, and ISRO and that nighttime call
surveys be conducted in the remaining six parks. For details on these
surveys, please see section 3 or SOPs 2 and 4.

**[2.d. Detecting meaningful changes in PAO]{.underline}**

The meaningfulness of changes in PAO to amphibian populations varies
with the initial statuses of those populations. If the PAO and the
number of animals per population are low when monitoring begins,
especially if they already have been declining, further declines in PAO
can indicate more dramatic threats to the persistence of those
populations than for populations with higher initial PAO and abundances.
Furthermore, distributions and abundances of amphibian populations, as
well as how frequently individuals breed, are known to fluctuate over
time (Pechmann and Wilbur 1994). Thus, attempting to describe meaningful
biological changes in PAO before monitoring begins and baseline PAO have
been estimated is not straightforward.

Monitoring amphibian populations because of concern over their statuses
and monitoring them to indicate changes in environmental conditions are
not necessarily the same. The fundamental question from the perspective
of the GLKN's Monitoring Program is that if the PAO of amphibian
populations are declining over time, which point(s) in the declines
should trigger managers to conclude that environmental conditions have
changed enough to be concerned? We do not know enough about the dynamics
of amphibian populations, how ecological systems in the parks work, and
the levels of change that concern park managers to answer this question
with confidence. Given the above limitations, predicting a level of
meaningful change over a given period of time is highly speculative and
is perhaps best left to be answered by resource managers in the GLKN. We
will suggest that a 50% decline in the PAO for a species over ten years
of monitoring definitely would be cause for concern about potential
environmental changes in the GLKN.

**[2.e. Sampling replication]{.underline}**

Theoretically, the optimal number of repeat surveys depends upon the
proportion of survey sites species X occupies (ψ~x~, psi) and the
probability of detecting that species when it is present (*p*) (Table
3), not the number of sites surveyed or the total number of surveys
(MacKenzie et al. 2004). Practically speaking, these numbers (Table 3)
should be considered the minimum numbers of repeat surveys required
(MacKenzie et al. 2004).

We currently do not have values for ψ and ρ available for our proposed
targeted species in the GLKN. Therefore, we must rely upon estimates of
ψ and ρ initially for this protocol. Based upon our personal
observations surveying for these species in VOYA, SACN, and other areas
outside of the GLKN, we estimated values of 0.5 for ψ and and 0.7 for ρ.
These values appear reasonably conservative as average occupancies and
detection probabilities. Theoretically, at least two repeat surveys of
sites are required if ψ = 0.5 and ρ = 0.7 (Table 3), but statisticians
often recommend at least three total visits per season (MacKenzie et al.
2004).

**Table 3. The minimum number of repeated surveys required for
combinations of ψ and ρ (MacKenzie et al. 2004). ψ = the proportion of
sites occupied by species X; ρ = the probability of detecting species X
when it is present**

  --------- --------- --------- --------- --------- --------- --------- --------- --------- ---------
            **ψ**                                                                           

  ***p***   **0.1**   **0.2**   **0.3**   **0.4**   **0.5**   **0.6**   **0.7**   **0.8**   **0.9**

  **0.1**   14        15        16        17        18        20        23        26        34

  **0.2**   7         7         8         8         9         10        11        13        16

  **0.3**   5         5         5         5         6         6         7         8         10

  **0.4**   3         4         4         4         4         5         5         6         7

  **0.5**   3         3         3         3         3         3         4         4         5

  **0.6**   2         2         2         2         3         3         3         3         4

  **0.7**   2         2         2         2         2         2         2         3         3

  **0.8**   2         2         2         2         2         2         2         2         2

  **0.9**   2         2         2         2         2         2         2         2         2
  --------- --------- --------- --------- --------- --------- --------- --------- --------- ---------

Given this information, three repeat surveys are recommended for the
following reasons.

1.  We are estimating average ψ and *p* for several target species
    across nine parks and we currently do not know how good these
    estimates are. Given that two is the minimum number of visits per
    season required to model detectability, we recommend three visits to
    help ensure that the number of repeat surveys is adequate for a
    larger proportion of the monitored species (Johnson 2002; MacKenzie
    et al. 2002 a, b; 2003; Buckley and Beebee 2004; MacKenzie et al.
    2004).

2.  In addition to PAO considerations regarding the number of repeat
    surveys, the nighttime call surveys should enable monitoring the
    numbers of males calling at breeding sites, an important capability
    for monitoring the statuses of populations in real time (Table 2) as
    a complement to PAO. In our experience, and that of others (for
    example, (Jones 1986; Pearman et al. 1995; Bridges and Dorcas 2000;
    Brooke et al. 2000; Buech and Egeland 2002), peaks in the numbers of
    calling males vary considerably with species, time of day, and
    weather conditions within and among days. The best way to minimize
    such variance is to increase the number of samples (*N*), as
    variance is a function of *n* (Neter et al., 1985). Three repeat
    surveys per season (Figure 6) would consistently provide a
    substantially improved estimate of the number of males calling than
    would two such surveys.

> ![](media/image6.png){width="5.979861111111111in"
> height="4.4847222222222225in"}
>
> **Figure 6. Typical reproduction season for several species in SACN.
> Yellow = period when males call during warm springs. Orange = period
> when males call more typically. Blue = total reproduction season,
> including larval period. Columns 1, 2, and 3 = two-week time periods
> during which nighttime surveys should be conducted.**

3.  Similarly, a third repeat survey for daytime surveys in VOYA, APIS,
    and ISRO will increase the chances of finding all life stages of
    each species (and, thus, help reduce the variance in surveyors'
    observations), including the numbers of egg masses and metamorphs
    (Figure 7). A third survey also increases the chances of detecting
    species when they are present and maximizing the information
    obtained about breeding subpopulations at a site.

> ![](media/image7.png){width="5.9in" height="4.425in"}
>
> **Figure 7. Typical reproduction season for several species in SACN.
> Yellow = period when males call during warm springs. Orange = period
> when males call more typically. Blue = total reproduction season,
> including larval period. Columns 1, 2, and 3 = two-week time periods
> during which daytime surveys should be conducted.**

4.  Three repeat surveys per season seemed realistic logistically and
    financially at the start of this program, but are especially
    challenging at APIS. As the NPS learns about detection probabilities
    for each species in the initial years of this program, opportunities
    to be more efficient by reducing the number of repeat surveys to two
    for some species might exist. However, if resources are not limited,
    we recommend an even larger number of repeat surveys to reduce the
    variance further and maximize the information obtained about each
    species. Thus, although detectability will be able to be only weakly
    modeled across the year (and for only a few species), we will use
    only one repeat survey per period (repeated by two observers in
    later sampling periods).

**[2.f. Number of sampling locations]{.underline}**

The GLKN has elected to monitor amphibian populations because they can
be good indicators of the quality of the habitats in which they live
(section 1.b.). If monitored amphibian populations decline over time,
especially in concert with changes in other vital signs being monitored
in the GLKN, the NPS might need to investigate potential causal factors
to understand which environmental conditions have changed and the
broader ramifications of those changes. We are recommending that NPS
managers primarily use the PAO of each monitored species over time to
evaluate changes in amphibian populations across the GLKN. Thus, precise
estimates of PAO are essential to detect trends over time and to serve
to trigger appropriate concern about environmental conditions in the
parks.

The prudent approach to designing the amphibian monitoring program is to
maximize the precision of estimates of PAO to enhance the ability of the
NPS to detect trends. This requires reducing the variances associated
with estimates of PAO, which decrease with increases in the number of
units sampled (MacKenzie et al., 2004). Thus, monitoring a sufficient
number of sites is critical within the context of what is possible based
upon logistics and resources.

Although pushing the limits of projected resources available, this
survey scheme appears plausible on a per park basis. Thus, three repeat
surveys of 30 listening locations (nights) or potential breeding sites
(days) are recommended per park per year (sections 3c and 3e). For some
of the target species, such as spring peepers, this could equate to 244
sites (eight parks with 30 sites each plus GRPO with four) sampled
across nine parks in one season (Table 1). For other species, such as
salamanders in VOYA, APIS, and ISRO, the number of sites sampled could
be 90 per season across the network (Table 1). In the pilot work,
however, we will attempt to sample only the eight sites with known
permanent water bodies.

**[2.g. Level of change detectable with this design]{.underline}**

The levels of change detectable with this design will vary with ψ, ρ,
the number of sites surveyed per season, and the number of seasons
sampled for each species. We do not have results from computer
simulations to describe changes detectable for each combination of these
parameters. However, we offer the following estimates based upon the
calculations of MacKenzie (personal communication).

-   If ψ = 0.5 initially, ρ = 0.7, 200 sites are sampled three times per
    season for ten seasons, and occupancy declines at a rate of 0.1, a
    change in ψ to 0.27 would be detectable as significant statistically
    based upon 95% confidence intervals. This would equate to a decline
    of 46% over ten years.

-   If ψ = 0.8 and all other values stayed the same as above, a change
    in ψ to 0.6 would be significant. This would equate to a decline of
    25% over ten years.

-   In contrast, if ψ = 0.5 for 50 sites surveyed over ten seasons with
    a decline trend of 0.2 and all other values the same as above,
    statistical significance would occur at ψ = 0.12, a decline of 76%
    over ten years.

-   If ψ = 0.8 for 50 sites surveyed over ten years with a decline trend
    of 0.2 and the same values otherwise, then significance would occur
    at ψ = 0.35, a decline of 56% over ten years.

Monitoring 200 sites affords substantially greater opportunity to detect
significant changes in occupancy than does monitoring 50 sites. Thus,
the NPS will be able to detect smaller changes in occupancy faster in
species that are monitored over a large number of sites, all else being
equal (this also is true for species that occupy a higher proportion of
sites). We anticipate that most of the target species monitored via
nighttime call surveys will be monitored over a large number of sites
(Table 1), so this design will be relatively sensitive to detecting
changes in occupancy across parks for those species.

The relationship between detecting levels of change and the number of
sites sampled per season also demonstrates the limits of this design to
detect changes in amphibian populations in each park. The above
detection levels for 50 sites clearly show that very meaningful declines
of populations will occur before they are detected as statistically
significant based upon monitoring 30 sites. Because surveying more sites
per park is not realistic, we recommend that a single PAO be calculated
for each target species based upon sampling across all parks in which it
is present per season. This will enable monitoring the occupancy of each
species over time across all such parks.

We also recommend that PAO be calculated for each species for the sites
at which they are monitored in each individual park, except Grand
Portage National Monument, which has too few sites (SOP 1, 3). While the
accuracy of estimates of PAO will be relatively poor in some parks,
plotting these PAO over time will enable at least a qualitative
assessment of how PAO for individual parks compare with PAO for all the
parks. These data also can be complemented by data describing the
numbers of animals observed during surveys to observe potential trends
that might not be significant statistically, but might provide important
biological information. Managers from the GLKN can then decide to invest
more resources into sampling a larger number of sites in that park if
necessary.

**[2.h. Timing of sampling]{.underline}**

NIGHTIME SURVEYS

*Frequency* -- It is recommended that three repeat surveys be conducted
at each of 30 listening locations per park per season during the time
when each targeted species (section 3.f.5) is calling (Figure 5). This
equates to three surveys being conducted at each of 30 listening
locations over the course of nine nights (section 3.c.4 and 3.c.18)
during each of three portions of the breeding season: early, middle, and
late (Table 4; Figure 6). At APIS, VOYA, and ISRO, however, nighttime
surveys have limited utility, due to the logistical considerations. We
will sample at night in APIS during pilot sampling only on islands with
hydric-soil components near the permanent wetlands to be sampled. This
will be done on established trails, using headlamps. S

*Timing* -- Ideally, nighttime surveys would be timed to occur when the
maximum number of males of each species calls. Realistically, timing
surveys to maximize the chances of hearing calling males is challenging,
especially for species that call over protracted periods (Figure 5). In
general, we recommend that the first round of surveys begin within three
days after the first choruses of males of the target species for that
round (wood frogs, for example) are heard and that the remaining two
sampling periods are scheduled to maximize surveying during periods when
males are calling (Figure 6).

DAYTIME SURVEYS -- ***to be used at SLBE in only a (limited) pilot
manner***

*Frequency* -- We recommend that three repeat surveys be conducted at
each of 30 potential breeding sites per park per season during the time
when at least one age class could be present at a breeding site (Figure
5). This equates to three surveys being conducted over longer time
intervals than for the nighttime surveys (Figures 6, 7), although still
during early, middle, and late portions of the breeding season (Table 4;
Figure 7).

*Timing* -- Ideally, daytime surveys would be timed to maximize the
chances of observing adults, egg masses, and metamorphs of each target
species. Realistically, more than three repeat surveys are necessary to
make such observations for all of the target species. In general, we
recommend that the first round of surveys begin within five days after
the first choruses of males of the target species for that round (wood
frogs, for example) are heard and that the remaining two sampling
periods are scheduled to maximize surveying when as many life stages of
different target species are present at breeding sites (Figure 7).

Table 4. Recommended frequency and timing of sampling. E = early in the
breeding season, M = during the middle of the breeding season; L = later
in the breeding season (Figure 5)

  ---------------------------- --------------- ------------- ---------- --------- --------------
                               **Surveys**                                        

                               **Nighttime**   **Daytime**                        

  **Frequency and Timing**     Call surveys    Call surveys  Physical   Dip-net   Perimeter,
                                                             and        surveys   metamorph,
                                                             chemical             malformation
                                                             measures             surveys

  Frequency (# of replicates   3               1             1          1         1
  per sampling period)                                                            

  Timing                       E, M, L         E, M, L       E, M, L    E, M, L   E, M, L
  ---------------------------- --------------- ------------- ---------- --------- --------------

**[2.i. Parameters to be measured during surveys]{.underline}**

The parameters measured will vary with each type of survey. Data
collected for each type of survey will be used to analyze for PAO for
each species for each year.

Table 5. Measurements that will be taken during each type of survey in
parks of the GLKN. Please see the details of each method in Section 3.

+---------------------------+-------+------+--------+------+---------+
|                           | *     |      |        |      |         |
|                           | *Surv |      |        |      |         |
|                           | eys** |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
|                           | **N   | **D  |        |      |         |
|                           | ightt | ayti |        |      |         |
|                           | ime** | me** |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| **Parameters**            | Call  | Call | Ph     | Dip  | Per     |
|                           | su    | sur  | ysical | -net | imeter, |
|                           | rveys | veys | and    | sur  | met     |
|                           |       |      | ch     | veys | amorph, |
|                           |       |      | emical |      | malfo   |
|                           |       |      | me     |      | rmation |
|                           |       |      | asures |      | surveys |
+---------------------------+-------+------+--------+------+---------+
| 1.  Air temperature       | X     |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 2.  Relative humidity     | X     |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 3.  Barometric pressure   | X     |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 4.  Wind speed            | X     |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 5.  Water temperature     | X     |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 6.  Water pH              |       |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 7.  Water conductivity    |       |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 8.  Type of wetland       |       |      | X      |      |         |
+---------------------------+-------+------+--------+------+---------+
| 9.  Reasons why the       |       |      | X      |      |         |
|     wetland is considered |       |      |        |      |         |
|     potential breeding    |       |      |        |      |         |
|     habitat (1^st^ visit  |       |      |        |      |         |
|     to the site only)     |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 10. GPS coordinates of    |       |      | X      |      |         |
|     breeding site (1^st^  |       |      |        |      |         |
|     visit to the site     |       |      |        |      |         |
|     only)                 |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 11. Length and width of   |       |      | X      |      |         |
|     wetland basin         |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 12. \% of basin with      |       |      | X      |      |         |
|     standing water        |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 13. \% of basin with      |       |      | X      |      |         |
|     canopy cover          |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 14. \% of standing water  |       |      | X      |      |         |
|     that contains         |       |      |        |      |         |
|     emergent vegetation   |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 15. Type of emergent      |       |      | X      |      |         |
|     vegetation            |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 16. Primary and secondary |       |      | X      |      |         |
|     types of surrounding  |       |      |        |      |         |
|     land cover            |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 17. GPS coordinates of    | X     |      |        |      |         |
|     listening location    |       |      |        |      |         |
|     (1^ST^ time only)     |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 18. Estimate of the       | X     | X    |        |      |         |
|     number of each        |       |      |        |      |         |
|     amphibian species     |       |      |        |      |         |
|     calling               |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 19. Directions in which   | X     |      |        |      |         |
|     calling individuals   |       |      |        |      |         |
|     are located           |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 20. Estimate of the       | X     |      |        |      |         |
|     distance calling      |       |      |        |      |         |
|     individuals are from  |       |      |        |      |         |
|     the listening         |       |      |        |      |         |
|     location              |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 21. Number and age class  |       |      |        | X    | X       |
|     of each amphibian     |       |      |        |      |         |
|     species observed      |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 22. Number and Order of   |       |      |        | X    | X       |
|     each amphibian        |       |      |        |      |         |
|     predator observed     |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 23. Number and of egg     |       |      |        | X    | X       |
|     masses found of each  |       |      |        |      |         |
|     species               |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 24. Proportion of embryos |       |      |        | X    |         |
|     alive per egg mass    |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 25. Number of malformed   |       |      |        | X    | X       |
|     and deformed          |       |      |        |      |         |
|     metamorphs            |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 26. Type of each          |       |      |        | X    | X       |
|     malformation and      |       |      |        |      |         |
|     deformity             |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+
| 27. Any relevant          | X     | X    | X      | X    | X       |
|     observations not      |       |      |        |      |         |
|     called for            |       |      |        |      |         |
|     specifically above    |       |      |        |      |         |
+---------------------------+-------+------+--------+------+---------+

**2.j. [Relationships to other Vital Signs]{.underline}**

The GLKN has elected to monitor amphibian populations because they can
be good indicators of the quality of wetland habitats in which they live
(section 1.b.). To the extent possible the GLKN vital signs program will
provide 'weight of evidence' data from a suite of different protocols to
indicate environmental change. For example, these data on amphibian
monitoring could potentially show population declines in concert with
changes in land use and degraded water quality -- both being monitored
under other GLKN vital signs protocols. These vital signs have been
shown to be linked ecologically in three different conceptual models
developed by GLKN (Gucciardo et al. 2004). However, if causal
relationships are needed, the NPS may need to conduct more intensive
research to understand which environmental conditions have changed and
the broader ramifications of those changes. Initially, the NPS will
analyze data collected through this protocol with data from other vital
signs such as climate/weather (Hart in preparation), water quality in
lakes (Axler and Elias 2005), water quality in streams (Magdalene and
Elias 2005), trophic bioaccumulation (Bowerman and Route 2005), and land
use/cover (Bolstad and Gafvert in preparation) to look for statistical
associations in any trends.

**3. Field Methods**

**[3.a. Preparations for the field season]{.underline}**

1.  Applications for any required permits should be submitted far enough
    in advance to ensure compliance by the beginning of the field
    season. \[Should be clear that both NPS permits, by park, and state
    DNR permits will be needed.\]

2.  Training and updates for surveyors should begin at least one month
    prior to the beginning of the field season to allow for scheduling
    safety training in addition to training in field methods.

3.  Staff should begin to assemble, test, and organize all requisite
    equipment beginning approximately one month prior to the likely
    beginning of the field season (Figures 6, 7).

4.  Based upon local weather conditions, staff should arrange to monitor
    indicator breeding habitats in each park to determine when breeding
    begins and be prepared to begin surveys in a timely fashion. This
    will include identifying random sequences of sites to survey and
    making certain that updated forms and GPS coordinates and maps are
    loaded into PDAs and GPS units, respectively.

**[3.b. Sequence of events during field season]{.underline}**

See sections 2.h., 3.c., and 3.e.

**[3.c. Site selection -- Nighttime surveys]{.underline}**

(INDU, MISS, PIRO, SACN, SLBE, GRPO)

Note: The text in this section has been copied into a separate SOP (#
1).

We have already used the following algorithm to select sites for INDU,
MISS, PIRO, SACN, SLBE, GRPO. We provide these details so that future
investigators can replicate our site selection. Coordinates and shape
files for sites at each park can be found in the GLKN master amphibian
geodatabase (see also section 4).

1.  Use GIS tools to demarcate the legal boundaries of each park.

2.  Use GIS data layers to identify all roads within each park.

3.  Divide the area of each park that contains roads into three
    proportional segments, based roughly upon the quantity and density
    of roads.

4.  Use GIS tools to locate 150 points randomly along roads in each
    segment of each park.

5.  Use GIS tools to create 1000-m buffers around each point (Figure 8).

    i.  The distance over which calling males can be heard varies with
        species, land cover, topography, weather conditions, and
        background noise. Our goal is to select listening locations that
        are far enough apart to minimize likelihood of surveying the
        same breeding site from two different listening locations. We
        have not tested distances at which different species can be
        heard with a parabolic microphone and the naked ear under
        different conditions. Based upon our experiences using both over
        the course of four seasons of surveys, we suggest that 1000 and
        300 m for hearing calling amphibians with the parabolic
        microphone and naked ear, respectively, should be conservative
        for most species under most conditions.

> ![](media/image8.png){width="4.009722222222222in"
> height="4.252083333333333in"}
>
> **Figure 8. Random points and 1000-m buffers selected for Sleeping
> Bear Dunes National Lakeshore**

6.  Select all non-overlapping buffers in each segment randomly, to
    establish a random sequence of points and buffers within each
    segment.

7.  Select the first 15 non-overlapping buffers in each segment
    according to the sequence identified in \# 6. If 15 are not
    available, select the first (at least) 10 buffers that do not
    overlap (Figure 9).

> ![](media/image9.png){width="3.6in" height="3.75in"}
>
> **Figure 9. The selection of the pool of listening locations with
> 1000-m buffers at SLBE.**

8.  These 15 points will serve as the pool of potential listening
    locations per block.

    i.  Because of its long, linear structure, we first blocked SACN
        into 12 blocks based upon driving distance, then selected six of
        those blocks randomly in which to survey points chosen according
        to the procedures above (Figures 10, 11).

> ![](media/image10.png){width="4.889583333333333in"
> height="3.667361111111111in"}
>
> **Figure 10. The six blocks selected randomly for SACN**
>
> ![](media/image11.png){width="5.009722222222222in"
> height="3.7645833333333334in"}
>
> **Figure 11. The sites for monitoring amphibian populations in section
> 8 of SACN**

9.  Select the first ten listening locations from the pool of 15 in each
    block.

10. Sample these locations according to SOP 2.

**[3.d. Nighttime call surveys]{.underline}**

We will be using a combination of parabolic microphones and the naked
ear to conduct call surveys at night, which will be the main focus of
our pilot work. Parabolic microphones can at least double the area being
surveyed from a listening node and substantially improve the ability of
surveyors to estimate the numbers of animals calling at a site (W.
Sadinski, personal observations). However, GLKN staff have experienced
resistance among some in the herpetological community regarding their
utility. Sites will be selected for nighttime surveys randomly from road
segments that lie within soil polygons that are poorly drained. All
these sites will be on the mainland, though we will discuss with SLBE
staff the possibility of nighttime surveys along trails on the islands
in later activity periods.

**Requisite Equipment and Supplies (per team per park)**

1.  GPS units with the capacity to have topographic maps installed for
    each park (2)

2.  DC converter for the GPS units (2)

3.  pocket weather instruments that can measure temperature, relative
    humidity, barometric pressure, and wind speed (2)

4.  personal digital assistants (PDA) with at least 8K of memory and
    that can be loaded with Pendragon Forms software (2)

5.  digital stopwatches with timer lights (2)

6.  compasses (2)

7.  vehicles (2)

8.  parabolic microphones (for example, a Telinga stereo.dat with
    phantom-powered handle) (2)

9.  digital audio recorders (for example, a HHb MDP 500) (2)

10. DC converters for the digital recorder (2)

11. headphones (for example, AKG K240M) (2)

12. tripods (2)

13. tripod mounts for microphones (2)

14. small to medium-sized flashlights (2)

15. rechargeable batteries (if available) and chargers for GPS and PDA

STANDARD OPERATING PROCEDURES -- NIGHTTIME CALL SURVEYS

The range of sampling dates in which sampling should occur in each park
unit (during each activity period) is prescribed by the Frog and Toad
Survey programs (often associated with state Departments of Natural
Resources) in each state. If such data are available from local sources
(e.g., personal observations, anuran listserves), we recommend that
surveys within each activity period begin within 3 days of the
initiation of calling activity within the region.

1.  **Sampling should not occur** on evenings with lightning, heavy rain
    (though mist and light rain that does not affect hearing are OK), or
    when wind speeds are consistently \>12 mph. When parabolic
    microphones are used, sampling must be suspended or ended to prevent
    them from getting wet. Sampling should not occur when air
    temperatures are less than 5.6º, 10º, or 13 º C (42 º, 50 º, or 55
    º F) for repeat survey periods 1 (i.e., early Spring), 2, and 3,
    respectively.

2.  Of the randomly selected listening stations within each park, select
    a subset to sample each evening. On the first survey of the group of
    sites, survey them in order from the lowest-numbered station to the
    highest-numbered station, keeping the order. On the second survey,
    reverse the order. On the third survey, survey only every other
    station (starting at the second) on the way from lowest number to
    highest, then perform the alternate, remaining (odd) points on the
    return trip. At the start of implementing these protocols in each
    park, be prepared to have to delete preselected listening locations
    in cases where preselections by GIS methods prove inaccurate in the
    field. Reasons to discard pre-selected listening locations include
    lack of a safe location to park a vehicle and too much noise to
    allow for listening for calling amphibians effectively.

3.  Monitor weather conditions and breeding activity throughout the
    reproductive season so that surveys 1, 2, and 3 are initiated at
    appropriate times after targeted species have begun to breed.

4.  Begin each set of call surveys each evening 30 minutes after sunset.

5.  Name and record the geospatial coordinates (one-minute average) for
    each listening location in the GPS unit and the PDA the first time
    each location is visited.

6.  Measure and record windspeed (in mi/hr; averaged over one minute),
    air temperature and relative humidity (in ˚C and %, as soon as
    readings are stable for 20 sec), barometric pressure (in hPa), and
    water temperature (in ˚C) if there is a water body within 15 m of
    the road **at each listening location**. Also note at each stop the
    number of cars passing by during the 5 min period, and, if ambient
    noise seriously or profoundly affects sampling (e.g., continuous
    traffic nearby, 6-10 cars passing, or construction noise), the
    number of minutes for which time was interrupted and the phase of
    the moon at the first and last listening location each evening.
    Measure the type and intensity of precipitation, and whether or not
    the moon is visible **at first and last stops**.

7.  Each surveyor conducts one five-minute calling survey (as measured
    with a stopwatch) at each of the preselected listening locations per
    evening. All naked-ear surveys are conducted outside of and away
    from the vehicle.

8.  Surveyor records all species heard, and the
    **[maximum]{.underline}** calling index (found at the bottom of the
    data sheet or on the PDA) for each species detected

9.  **[At every third station]{.underline}**, extend the survey period
    for an extra 5 min, and write in the lower half of each cell any new
    species that are detected. **[If not doing a 10-min
    survey]{.underline}**, note time to first detection for each
    species.

10. We are also field-testing the utility of parabolic microphones to
    extend the listening area, and thus sample a greater proportion of
    the landscape -- likely allowing for higher detection rates if
    domain is not limited to wetlands alone. Follow this preliminary
    survey with a ten-minute survey conducted by the surveyor sitting
    inside the vehicle with the parabolic microphone attached to a
    tripod and extending out of the window and above the roof of the
    vehicle and the digital recorder inside the vehicle. **Perform
    side-by-side comparison of naked ear vs. parabolic one night for
    each of the three portions of the sampling domain in each of the
    three seasons**, meaning an extra 9 sampling events per year.

11. Surveyor listens in all directions for approximately the same amount
    of time. An easy method to follow is to divide the rotation of the
    microphone into four arcs of 90º each and spend 2.5 minutes over the
    course of each survey listening within each quadrant.

12. Anytime noise from passing vehicles, storms, etc. makes hearing
    impossible, elapsed time should be stopped on the stopwatch at the
    point of interruption and continued afterwards until the survey has
    been conducted for the full ten minutes.

13. Parabolic microphones should not be permitted to get wet.

14. Data recorded in the PDA at the end of each ten-minute survey are
    backed up on the memory module attached to the PDA upon completion
    of entering the data. Data on the PDA are uploaded to computers as
    soon as possible after the completion of each evening's surveys.

NOTES ON THE USE OF PARABOLIC MICROPHONES

1.  We recommend using parabolic microphones during nighttime call
    surveys because they can greatly increase a surveyor's ability to
    detect calling amphibians and to decipher the number of amphibians
    calling compared with the naked ear.

2.  However, the power of parabolic microphones can also greatly hinder
    a surveyor's ability to detect calling anurans and the direction in
    which they are calling depending upon the species and environmental
    conditions.

    a.  When one or many individuals are calling loudly from a site near
        the listening node, the surveyor likely will hear those animals
        regardless of the direction they are pointing the microphone and
        dish. This can result in incorrect observations due to the
        surveyor hearing calling animals where such animals do not exist
        or missing observations because the surveyor cannot
        differentiate calling individuals from other species
        sufficiently to record them as observations.

    b.  This requires that the surveyor gains some measure of experience
        with these situations and uses a variety of techniques to avoid
        incorrect or incomplete data.

        i.  These techniques include adjusting the volume and gain on
            the digital recorder to try to reduce ambient calls, being
            aware of how the microphone picks up sounds from directions
            other than the one in which it is pointed, and above all
            else, recognizing when using the parabolic microphone is so
            counterproductive that the survey has to be conducted wholly
            or partially with the naked ear to differentiate calls. In
            such cases, the surveyor should record how much of the
            survey time was conducted with the parabolic microphone and
            how much was conducted with the naked ear in the notes
            section for that listening node.

        ii. Occasionally, the noise of wind or moving leaves also can
            hinder the ability to hear calling amphibians with the
            parabolic microphone. In those cases, the surveyor should
            revert to the naked ear and record the specific time spent
            using both techniques as in section b.i above.

    c.  For the sake of ease and efficiency, we recommend that surveyors
        use the parabolic microphone extended over the top of the roof
        of the vehicle while sitting inside and rotating it in all
        directions. All surveys with the naked ear should be conducted
        outside of vehicles to ensure that calls can be heard in all
        directions.

**[3.e. Site selection -- Daytime surveys]{.underline}**

(being considered for eventual application at Apostle Islands National
Lakeshore, Isle Royale National Park, Voyageurs National Park).

To directly compare the utility of daytime versus nighttime surveys, we
will perform daytime surveys at SLBE at 3-5 sites in the latter two
seasons, but will do "dry runs" of the technique in the first sampling
period.

Islands and limited roads require transport by boat or floatplane
through most of VOYA, APIS, and ISRO. Bushwhacking through the interior
of VOYA, in particular, can be arduous and time-consuming due to the
lack of trails, dense vegetation, and extensive complexes of wetlands.
While floatplanes are a solution in some locations, their availability
and cost are restrictive. These logistical constraints diminish the
benefits of surveying breeding sites far from roads, boat-access points,
and trails, because the additional costs in resources to get to such
sites (subsumed under time) are considerable. For this reason we
recommend limiting the areas of inference in these three parks to areas
within 1000 meters of roads, trails, or points of boat access, and not
selecting any sampling locations beyond such areas. If it becomes
important to extrapolate results of our monitoring to unsampled areas,
the tenability of this assumption can be tested with replicated pairs or
gradients of site accessibility.

We recommend that the NPS locate breeding sites to monitor in these
three parks according to the following algorithm.

1.  Use digital data and GIS tools to define the legal boundaries of
    VOYA, APIS, and ISRO and to lay a grid of 25-ha square cells within
    1000 m of roads, the borders of lakes that provide access by boat,
    or trails (Figure 11).

2.  Use GIS tools and the wetland data layers from the USGS-NPS land
    cover dataset or the National Hydrological Database to identify all
    wetlands in a perennial class that are within the size range of 0.2
    -- 1.0 ha in each square cell.

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a.  The USGS-NPS land cover dataset is recent and highly detailed, but
    available only for VOYA. The only data set that describes wetlands
    sufficiently for APIS and ISRO is the National Hydrological
    Database. The accuracy and resolution of data in this latter set are
    limited.

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3.  Using the numbers assigned to cells automatically when the GIS grid
    is created, select up to 60 non-contiguous cells that contain
    targeted wetlands (as in #2 above) randomly from the pool of all
    cells that contain such wetlands (see Fig. 12). Assign numbers one
    to 60 to these cells based upon the order in which they were chosen.

![](media/image12.png){width="4.411111111111111in"
height="3.7583333333333333in"}

**Figure 12. Pool of twenty-five hectare cells for Voyageurs National
Park. Yellow cells = those that contain targeted wetlands.**

4.  Select one of these wetlands randomly from the pool of such wetlands
    that lie wholly or partly in each cell and create a centroid of the
    wetland. Record the centroids of other wetlands in the same cell and
    keep them in reserve in case the selected one proves inappropriate
    or the total number of cells with appropriate wetlands is
    inadequate.

5.  Ground-truth the targeted wetlands in cells 1 -- 30 during the first
    replicate survey (Figure 5) of year 1. If the target wetland does
    not appear to provide suitable breeding habitat for the target
    (section 2.b.) species, then select another wetland (randomly) from
    the pool of reserve wetlands (see 4 above) in the same cell. If no
    such reserve sites exist, then select an additional cell (greater
    than \# 30) from the original pool of 60 cells. Repeat this process
    until 30 wetlands are located that provide potential breeding
    habitat for the same group of species across all sites (replicate
    sites for each species).

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i.  No more than one wetland per cell should be in the final set of
    wetlands that is monitored.

ii. The primary reason we are recommending the wetland criteria for
    picking potential breeding sites is to try to ensure that a site is
    large and diverse enough to provide microhabitats for most, if not
    all, of the above species.

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6.  Once preselected wetlands are confirmed to be appropriate, survey
    them according to the methods described in section 3.e..

**[3.f. Daytime surveys]{.underline}** -- ***to be used at SLBE in only
a (limited) pilot manner***

Two persons working as a team should use the following methods to survey
breeding sites during the day during each replicate survey in VOYA,
APIS, and ISRO.

1.  Call

2.  Physical and chemical

3.  Dip-net

4.  Perimeter/metamorph

Note: Concerns over spreading pathogens among breeding sites requires
doing as much as possible under field conditions to limit the chances of
this happening (DAPTF, 2005). In many parks of the GLKN, several
wetlands often must be traversed on the way to and from the breeding
site to be surveyed. Unfortunately, it is not practical to carry
sufficient quantities of concentrated or dilute solutions of bleach or
alcohol to remote breeding sites so that hip boots can be sprayed after
walking through each wetland. Therefore, we recommend treating hip boots
and nets with a bleach solution prior to leaving the vehicle to boat or
walk to a sampling site. Each person should spray the surface area of
dip nets and hip boots thoroughly with a solution of 6% bleach (DAPTF,
2005; Appendix B). Surveyors should be careful not to touch the boots or
nets with exposed skin or clothing while the toxic bleach solution is
concentrated on the boots.

  ------------------------------------ ----------------------------------
  **Requisite Equipment and Supplies   
  (per team per park)**                

  1\. GPS units with the capacity to   12\. digital counters or tally
  have topographic maps installed for  devices (2)
  each park (2)                        

  2\. DC converters for the GPS units  13\. digital stopwatches (2)
  (2)                                  

  3\. pocket weather instruments that  14\. meter stick (1)
  can measure temperature, relative    
  humidity, barometric pressure, and   
  wind speed (2)                       

  4\. laser rangefinder (1)            15\. five-gallon buckets with lids
                                       (2)

  5\. personal digital assistants      16\. pH and conductivity standards
  (PDA) with at least 8K of memory and 
  that can be loaded with Pendragon    
  Forms software (2)                   

  6\. field T/pH/conductivity meters   17\. deionized water
  (2, 1 backup)                        

  7\. field guides for amphibians (2)  18\. hip boots (2 -- 4 pairs) and
  and keys for embryos (2) and         a portable spray bottle with a 6%
  malformations (2)                    bleach solution

  8\. dip nets (3, 1 backup)           19\. batteries (rechargeable when
                                       available and battery chargers)

  9\. backpacks (intermediate in size  20\. boat (type appropriate to
  between daypacks and full backpacks  park)
  (2)                                  

  10\. compasses (2)                   21\. boating safety gear

  11\. scuba white boards (2)          22\. vehicle (1)
  ------------------------------------ ----------------------------------

STANDARD OPERATING PROCEDURES -- DAYTIME SURVEYS

1.  Call Surveys (naked ear)

Because bushwhacking to sites through thick vegetation and across
wetlands while carrying expensive, relatively fragile, parabolic
microphones and digital recorders can be cumbersome and costly, we
recommend that daytime call surveys be conducted using the naked ear.

a.  Conduct call surveys from a location approximately ten to 30 meters
    away from the site, hidden from the view of amphibians when possible
    to reduce the likelihood of interrupting calling or breeding.

b.  Remaining still and quiet, each observer listens for any and all
    amphibian species calling at the site during a period of ten minutes
    (stopwatch). Observers do not share information about their
    observations during the survey.

c.  Record the call data in a single PDA after the call survey has been
    completed (for examples of the structure of forms in the PDA, please
    see Appendix A). (To make uploading data simpler at the end of the
    day, this PDA will be the repository for all data collected during
    sampling for a given day.) Enter call data separately for each
    observer (species and number heard) into the PDA. If a species is
    heard outside of the ten-minute survey or at a different site,
    record the name of the species, number heard, direction heard,
    estimated distance, and the observer in the notes section of the
    chorus form.

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2.  PHYSICAL AND CHEMICAL MEASUREMENTS

Measuring physical and chemical variables provides data to use as
covariates in analyzing PAO or for analyzing statistical associations
between the presence of a species, number of individuals, and variables
that can affect presence.

a.  After completing the call survey, continue to the wetland, suspend a
    pocket weather meter from a nearby branch, and activate it. Record
    the air temperature, barometric pressure, wind speed (averaged over
    one minute), and relative humidity after completing the following
    physical and chemical measurements.

b.  Measure water temperature, pH, and conductivity at one location near
    shore.

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i.  Standardize the meter according to the manufacturer's
    recommendations and at least prior to taking measurements at the
    first site of the day. Further standardizations should be conducted
    at intervals consistent with the manufacturer's recommendations.

ii. Rinse the electrode thoroughly with deionized water before and after
    any use.

iii. Haphazardly select a location in water from 20 to 35 cm deep
     (shallower or deeper if this range is not available). Record the
     depth of the water at this location in the PDA.

iv. Immerse electrode and measure water temperature, pH, and
    conductivity. Record the value after each has remained stable for
    ten seconds.

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c.  Install a piece of pvc pipe (approximately 3.7 cm in diameter) in
    sediments in the deepest section of each wetland that can be reached
    without going over the top of the boots. Record the GPS coordinates
    of this pipe's location. Use a meter stick to measure water depth
    (to the nearest centimeter) along the south side of this pipe.

d.  Measure the length and width of the basin of the wetland with the
    rangefinder (according to the manufacturer's recommendations). If
    the rangefinder will not work because of obstructed views or the
    lack of reflective objects on the edge of the basin, estimate the
    length and width.

e.  Estimate the percentage of the wetland basin that contains surface
    water.

f.  Estimate the percentage of the surface water in the wetland that
    contains emergent vegetation and by difference, the percentage that
    is open water.

g.  Record the type of emergent vegetation based upon the
    pre-established standard categories in the data forms on the PDA.

h.  Estimate the percentage of canopy cover over the top of the wetland
    basin.

i.  Estimate the primary (most abundant) and secondary (second most
    abundant) types of land cover surrounding the wetland based upon
    observations made walking into the wetland and views from the
    wetland.

j.  Record all of the above data in appropriate data fields in the PDA.

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3.  DIP-NET SURVEYS

> Dip-net surveys enable surveyors to sample a potential breeding site
> for all life stages of all amphibian species present in the submerged
> portions of the site, from embryos to adults. They also enable
> observers to survey for aquatic predators. Dip-net surveys are
> conducted along transects through the relatively shallow portions of
> the wetland that provide habitat for amphibians. They begin after
> measuring the above physical and chemical variables is complete.

a.  Each person evaluates the amphibian habitat contained in the
    submerged portion of the wetland and haphazardly selects a point in
    that habitat to serve as the first node in a visual transect that
    extends outward from that point through the habitat.

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i.  Each person's transect consists of ten nodes, each two minutes of
    slow, careful walking time apart. Therefore, the second node is
    located at the spot where the surveyor is standing after walking
    through the habitat for two minutes. This walk might or might not be
    a straight line, depending upon the distribution of the habitat.

ii. Each person's starting node and projected path of travel along the
    transect should be sufficiently far enough away from the other
    person's to eliminate or reduce the chances of one person's
    activities disturbing animals in the other's sampling area.

iii. Time counts down on the stopwatch only during transit between nodes
     and when sweeping with the net.

iv. Note: Surveyors must not share information regarding their
    observations until the survey is complete.

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b.  Each person conducts one full sweep (approximately 1 m in length)
    with a dip net at each of the ten nodes by submersing the dip net
    and quickly sweeping through the water column as far away from the
    body while facing the direction not yet traveled.

    i.  Sweeps should be indiscriminate relative to amphibians visible
        at the node.

c.  After a sweep at a node, each person suspends time on their
    stopwatch and identifies the number of each species of amphibian
    caught, the life stage of each individual amphibian caught, and the
    numbers and identities (to Order at least) of any potential
    predators of amphibians.

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i.  Record the identities and numbers of animals caught in a sweep at
    each node on the scuba white board suspended around the observer's
    neck, so that the results of each sweep can be transcribed
    straightforwardly to the PDA at the end of the survey. Use the
    digital counter to tally individuals of each species as necessary.

    d.  Amphibian egg masses: Count and identify (field guides)
        individual amphibian egg masses observed in sweeps at nodes or
        during surveys conducted between nodes.

        i.  Estimate and record the typical stage of development (Gosner
            1960; Harrison 1969) among embryos in each mass and the
            proportion alive per mass (based upon gross deformities,
            arrested development, and the presence of water mold).

        ii. Handle egg masses with extreme care so as not to damage
            embryos, especially young ones, or cause them to dislodge
            from egg jellies prematurely. Return them to the water
            gently and try to place them in locations (in or on
            vegetation, for example) and at depths typical for the
            species.

        iii. If communal rafts of egg masses, such as those of wood
             frogs, are observed during the dip-net survey, plan to
             return to them after the completion of the dip-net survey
             to count egg masses and estimate the proportion alive (see
             the procedures described for this in section 3.3.k.).

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e.  Restart the stopwatch after all animals from each sweep have been
    identified, recorded, and released and the observer has begun moving
    to the next node.

f.  Release animals at the same location where they were caught.

g.  Identify and quantify the numbers and identities of amphibians
    observed while moving from one node to another using the dip net (to
    capture observed individuals not identifiable in situ), the digital
    counter, and the scuba white board.

h.  Suspend time on the stopwatch whenever identifying animals caught
    with the dip net or writing on the white board for more than ten
    seconds.

i.  Attempt to identify and quantify all amphibians and predators
    observed or that can be caught without moving off the predestined
    transect or disturbing areas further ahead on the transect.

j.  Record these observations on the scuba white board such that these
    data are identified as being from internode xy.

> k\. Make every attempt not to double sample any animals throughout
> this survey or any others.
>
> l\. Place any captured amphibians that metamorphosed recently into the
> five-gallon bucket (attached to the observer's belt), with lid
> attached, for examination at the end of the survey. Note: Typically no
> recent metamorphs should exist during the first survey conducted early
> in the season.
>
> m\. If the wetland does not contain sufficient water and habitat for
> each observer to complete sweeps at ten nodes, each observer completes
> as many nodes as possible and records that the survey was shorter and
> why in the notes section for this data field in the PDA.

d.  Small quantities of submerged habitat at a breeding site might
    require that surveyors begin their transects near each other prior
    to heading away from each other as they move along their respective
    transects.

e.  After completion of the survey, transcribe each observer's data from
    the scuba white board into the appropriate data fields for each
    individual observer in the PDA.

    i.  Transcribe the data collected at nodes into the nodes data
        field.

    ii. Transcribe the data collected between nodes into the internodes
        data field.

f.  After completion of the survey, examine all recently metamorphosed
    amphibians and record the number of each species and the frequency
    and types of malformations and deformities (Meteyer et al. 2000) per
    individual in the data fields in the malformations section of this
    survey. These observations should be recorded as animals collected
    during the dip-net survey.

g.  Surveying communal egg masses: After completion of the survey,
    evaluation of any metamorphs, and entering all data collected to
    that point into the PDA, return to survey any communal egg masses
    found along the transect and measure them according to the following
    procedures.

    i.  The original observer counts or estimates the number of
        individual egg masses.

    ii. Estimate the number when they are too numerous or advanced
        developmentally to count. To estimate, count (or estimate if
        necessary) the number of egg masses within a column of water (a
        subsample of a typical section of the communal mass) extending
        down from a square approximately 0.3 m on a side at the surface.

    iii. Estimate how many such squares cover the surface of the
         communal mass and multiply this number by the number of egg
         masses that were counted in the subsample.

    iv. If the number of egg masses simply cannot be counted or
        estimated, state why in the notes.

    v.  The observer estimates the range of development among embryos in
        the egg masses (from early cleavage to hatched -- Gosner 1960;
        Harrison 1969) based upon gross examination of embryos in ten
        egg masses selected haphazardly.

    vi. The observer estimates the proportion of embryos alive per egg
        mass out of the total number of embryos per egg mass.

    vii. Haphazardly select ten egg masses, if available; otherwise
         sample all egg masses. Estimate the proportion alive in each
         egg mass based upon gross malformations, lack of development,
         deteriorating tissue, and the presence of water mold.

    viii. Each surveyor records the data on egg masses she/he observed,
          including the identity of the species that deposited the egg
          masses, the number of egg masses, the developmental stage of
          the embryos, and the proportion alive in each of ten egg
          masses in appropriate fields in the PDA.

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4.  PERIMETER/METAMORPH SURVEYS

Conduct this survey after the dip-net survey at each of the daytime
sites. The objectives of this survey are to capture and identify to
species as many recently metamorphosed and postmetamorphic individuals
as possible in non-submerged habitat during 20 minutess.

We are assuming that animals observed during dip-net surveys are not
observed during perimeter/metamorph surveys. If an individual animal is
observed or suspected of moving between the aquatic and terrestrial
habitats, that animal must not be counted twice. If too many
uncertainties exist about whether the movements of animals across these
habitats increases the chances of double sampling, write clear notes in
the PDA regarding specific uncertainties and the species and numbers of
individuals involved.

Note: This survey should be conducted regardless of whether the site is
dry or not.

a.  Identify likely non-submerged habitat for amphibians throughout the
    basin of the wetland, including vegetated areas further away from
    the water.

b.  Each person haphazardly selects a point to begin surveying such
    habitat.

c.  The starting point should allow for maximizing the area surveyed
    between the two surveyors.

d.  Note: Surveyors must not share information about their observations
    until after the survey is complete.

e.  If the quantity of non-aquatic habitat is small, such that two
    surveyors likely will cover the entire wetland prior to surveying
    for the full 20 minutes, then the two persons should start from the
    same haphazardly selected point and head away from each other during
    the survey to maximize the area sampled.

f.  Each person activates their stopwatch and meanders slowly through
    the previously identified habitat in the direction opposite from the
    one in which the other person is traveling.

g.  If the survey is suspended for any reason, stop the stopwatch until
    the survey is resumed.

h.  Each person identifies all life stages of all amphibian species
    observed during the 20-minute survey using the scuba white board as
    necessary. Note: Suspend time on the stopwatch during any writing
    longer than ten seconds.

i.  If necessary for identification and sexing, capture animals with the
    dip net (the need to do this will vary with the experience of
    individual surveyors).

j.  Avoid handling captured animals any more than is absolutely
    necessary.

k.  Release them in the same location they were captured.

l.  Surveyors identify all amphibians observed, captured or not, to the
    extent possible (for example, one unidentified adult anuran).

m.  Each person tries to catch (dip nets and by hand) as many recent
    metamorphs as possible during the 20 minutes.

n.  Continually place captured recent metamorphs in the bucket, with the
    lid attached, for examination at the end of the survey.

o.  If the quantity of habitat available in the basin is insufficient to
    complete a 20-minute survey, then each surveyor should stop the
    stopwatch when finished surveying and record the length of time
    spent conducting the survey.

p.  At the end of the survey, enter the identity, and age class of each
    species in the PDA (Please see Appendix A for examples of the forms
    in the PDA).

q.  Enter observations of each person separately into appropriate fields
    on the data forms that are reserved for each person in the section
    for this survey.

r.  After entering the above data, examine the captured metamorphs and
    record the quantity of each species and the frequency and types of
    malformations/deformities per individual according to Meteyer et al.
    (2000).

s.  Enter each surveyor's data for metamorphs and
    malformations/deformities separately according to specific surveyor
    in the data fields for this survey in the PDA.

t.  Note: Occasionally habitat for metamorphs at a breeding site is so
    thick that one individual working alone is highly inefficient at
    counting, identifying, and capturing animals. We recommend that the
    two surveyors work together as a team under such conditions.

u.  Record data in the PDA as being from one surveyor in such cases and
    record that this survey was a team effort in the notes for this data
    field.