Inventory and Evaluate Burrowing Crayfish Monitoring Methods in the MS Sandhill Crane NWR

Final Report on Inventory and evaluate burrowing crayfish monitoring methods in the MS Sandhill Crane NWR

Interagency Agreement Number F16PG00142

Creaserinus oryktes--MSCNWR. Photo by G.A. Schuster. ©US Forest Service

September 11, 2018

Submitted by Susan B. Adams, Ph.D. USDA Forest Service, Southern Research Station, Center for Bottomland Hardwoods Research 1000 Front St., Oxford, MS 38655 E-mail: [email protected]; Phone: 662-234-2744 ext. 267

Submitted to: Mr. Scott Hereford Senior Wildlife Biologist, Mississippi Sandhill Crane NWR 7200 Crane Lane, Gautier, MS 39553

EXECUTIVE SUMMARY

The study was undertaken in part to assess how habitat management for Mississippi sandhill cranes influenced priority at-risk crayfish species on the Mississippi Sandhill Crane National Wildlife Refuge (hereafter, Refuge). Additional goals were to assess methods for sampling and monitoring crayfishes on the refuge and to provide educational materials about the Refuge’s crayfishes. We compared: (1) crayfish burrow densities to vegetation characteristics across Refuge units with differing management histories, (2) various methods for sampling burrowing crayfishes on the Refuge, and (3) various methods for sampling crayfishes and fishes in aquatic habitats. Crayfish burrow densities were negatively associated with woody plant coverage and height and with tree densities, indicating that habitat management to create and maintain prairie and savanna habitat for cranes is also beneficial to burrowing crayfish populations. Burrowing species collected were the speckled burrowing crayfish (Creaserinus oryktes) and the spinytail crayfish (Procambarus fitzpatricki). The latter is a FWS priority, at-risk species. For sampling burrowing crayfishes, burrow excavation and suctioning were more successful than trapping, but neither approach could be readily standardized. The former varied dramatically among collectors, and the latter varied in relation to rainfall. In aquatic habitats, we caught red swamp crayfish (Procambarus clarkii) and least crayfish (Cambarellus diminutus) during systematic sampling and added ditch fencing crayfish (Faxonella clypeata), Gulf crayfish (P. shermani), and spinytail crayfish during non-systematic sampling. In systematic sampling, active methods (dipnetting and seining) produced a higher crayfish catch-per-unit-effort and more fish species than passive methods (baited minnow and habitat traps), but passive methods caught more adult crayfish and all of the amphibians. We caught 10 fish and 2 amphibian species during systematic aquatic sampling. Crayfish photographs and information for educational materials were provided to Refuge staff.

TABLE OF CONTENTS EXECUTIVE SUMMARY ...... 2 INTRODUCTION ...... 4 TERRESTRIAL ...... 5 Methods--Terrestrial ...... 5 Habitat and burrow counts—systematic sampling ...... 5 Crayfish sampling ...... 6 Statistical analyses ...... 7 Results--Terrestrial ...... 8 Habitat and burrow counts—systematic sampling ...... 8 Crayfish capture and sampling efficiency ...... 8 AQUATIC...... 10 Methods--Aquatic ...... 10 Statistical analyses ...... 11 Results—Aquatic ...... 11 Standardized sampling ...... 11 Non-standardized sampling ...... 12 DISCUSSION ...... 12 Sampling recommendations ...... 15 ACKNOWLEDGEMENTS ...... 15 LITERATURE CITED ...... 16 TABLES ...... 17 FIGURES ...... 22 Appendix A. Collectors ...... 32 Appendix B. Terrestrial sampling and results ...... 33 Appendix C. Aquatic sampling and results...... 36

INTRODUCTION Habitat management on the Mississippi Sandhill Crane National Wildlife Refuge (hereafter, Refuge) has focused primarily on creating open-canopy prairie and savanna habitat for Mississippi sandhill cranes (Grus canadensis pulla). Burning is the preferred habitat management method, but in units where frequent burning is not feasible, mechanical treatment (cutting and mulching vegetation) has been used. Such management potentially benefits an entire community adapted to wet prairie and pine savanna habitats. However, responses of some US Fish and Wildlife Service (FWS) priority at-risk species to the habitat management approaches are unknown. The FWS sought more information about the distribution of priority crayfishes on the Refuge and how the land management influenced primary burrowing crayfish species. This study was designed to fill information gaps regarding four crayfish species designated as priority at-risk species by the FWS region and listed as target species for the Refuge: angular dwarf crayfish (Cambarellus lesliei), burrowing bog crayfish (Creaserinus burrisi), speckled burrowing crayfish (Creaserinus danielae), and spinytail crayfish (Procambarus fitzpatricki). Note that a recent taxonomic revision moved all Fallicambarus species in Mississippi to the genus Creaserinus (Crandall and De Grave 2017). Crayfish species are often placed into one or more overlapping categories as primary, secondary, or tertiary burrowers (Hobbs 1942, 1981). Tertiary burrowers live in surface waters, occasionally occupying simple burrows to escape a drying habitat or very cold temperatures, brood eggs, and perhaps avoid predation. Secondary burrowers spend part of most years in surface waters and part in burrows. Primary burrowers rarely or never use surface waters, instead occupying complex burrows that intersect the water table throughout their lives and emerging to forage, find mates, and probably to disperse. Primary burrowers may be further categorized as habitat generalists or specialists (Rhoden et al. 2016). Generalists occupy woodland or open- canopy habitats, whereas many specialists are primarily restricted to open-canopy habitats, often with specific soil types. Primary burrowing crayfishes that are habitat specialists may use mowed road right of ways (Rhoden et al. 2016) or prairie or savanna habitats maintained in an early successional stage by fire or other means (Welch 2004). In the Ouachita Mountains, Arkansas, and Camp Shelby, Mississippi, burrow densities for several burrowing crayfishes were positively related to open canopy and availability of water relatively near the soil surface (Welch 2004, Rhoden et al. 2016). Because the water table is relatively shallow throughout most of the Refuge, canopy cover was a logical candidate as a driver of habitat-specialist burrowing crayfish population densities. The study goals were to assess crayfish population responses to the ongoing habitat management, to begin documenting crayfish distributions on the Refuge, and to test sampling methods on the Refuge. Our objectives were to: 1) document presence of and habitat types used by the four priority at-risk crayfish species thought to occur on the Refuge, 2) test the efficiency of various sampling methods for a potential crayfish monitoring program, 3) compare burrowing crayfish densities among sites with differing vegetation management histories, 4) survey other crayfish species present on the Refuge, and 5) photograph crayfishes for educational materials.

Ancillary to the study, we also collected information about fishes and amphibians incidentally sampled. Because aquatic and terrestrial sampling were conducted independently of each other in most cases, the Methods and Results are separated by habitat type.

TERRESTRIAL METHODS--TERRESTRIAL

Habitat and burrow counts—systematic sampling We sampled during three weeks in late winter/early spring 2017: January 23-27, February 6-10, and February 27-March 3. We used transects and quadrats to systematically sample habitat and crayfish burrow densities in four terrestrial sites per week. We sampled crayfishes quantitatively using passive (trapping) and active methods. Each week we trapped for two nights in two of the sites and one night in the other two. At the end of the study, we actively sampled crayfishes in two additional terrestrial sites where we did not trap or assess habitat. Thirteen people participated in various aspects of the sampling (Appendix Table A1). Site selection criteria included management history, years since previously burned, distribution across the Refuge, and access. We avoided sites where our activity was likely to disturb cranes. We grouped sites into three management history classes: frequently burned, mechanically treated, and infrequently burned (Appendix Table B1). One site was infrequently burned but trees had been removed mechanically. Our infrequently burned sites did not include sites with very dense mid- and understory vegetation, because we could not use our quadrat sampling approach in such sites. The 12 terrestrial sites sampled systematically (Figure 1) were distributed among management classes as follows: frequently burned (N=5), infrequently burned (N=3), mechanically treated (N=3), combination of infrequently burned and mechanical tree removal (N=1). In each systematically sampled site, we established six transects (A-F) approximately 100 to 150 m long and perpendicular to the access road. Transects were spread roughly evenly across the site and in large sites were staggered, with starting points at varying distances from the road. The goal was to cover as much of a site as possible to capture subtle variations in elevation, groundwater depth, and vegetation across the sites. We assessed habitat and burrow numbers in 15, 0.75 x 0.75 m quadrats per transect, so in total we sampled 1,080 quadrats along 72 transects. Quadrats were located by haphazardly tossing PVC squares to alternating sides of a transect, with a minimum of 6.6. m along the transect between quadrats. Quadrats landing on fire ant mounds were re-thrown. We tallied the number of trees <1.2 m tall and >1.2 m tall within 2 m to either side of transects. In each quadrat, we visually estimated the percent cover of each of the following to the nearest 10% (summing to 100%): grasses/forbs, woody plants, leaves/small wood (≤10 cm diameter), large wood (>10 cm diameter), and bare soil. We initially included presence/absence of carnivorous plants, but abandoned that because of their near-ubiquity and the time required to find small carnivorous plants. After the first week we added % of quadrat covered by standing

water in addition to the 100% accounted for by the other categories. We measured the height (cm) of the tallest herbaceous and tallest woody plant in each quadrat. We then counted the number of burrow entrances in the quadrat. If a burrow intersected the edge of the quadrat, it was included. Burrow entrances were classified as Active or Inactive, with three categories of each, as follows: Active—chimney, fresh mud, or open (opening clear of spider webs and detritus); Inactive--volcano-shaped pile of old mud, burrow opening with accumulated debris or spider webs, or plugged (perhaps a flat area of soil with no vegetation). This system was not foolproof; for example, active burrows could have been plugged, and the difference between an inactive crayfish burrow versus soil disturbance by another animal was not always obvious. We flagged up to 2 burrows per quadrat for later crayfish sampling.

Crayfish sampling Crayfish sampling prioritized burrows in quadrats, but where not enough burrows were present in quadrats--or in sites where we did not assess habitat--burrows were haphazardly selected for sampling. Passive sampling employed two types of burrowing crayfish traps. The first was a modified Norrocky trap (Norrocky 1984), consisting of a PVC tube with one end capped and the other having a metal, one way flap (Figure 2). We used Norrocky traps of three diameters: small (3.2 cm), medium (3.8 cm), and large (5.1 cm). When possible, we selected a trap closest in size to the burrow opening, although we encountered few burrows openings ≥5 cm. We removed the chimney, if present, from the burrow and placed a trap in the opening at an angle such that the metal flap would close (Figure 2). Traps were inserted far enough into burrow to be secure, but care was taken to ensure that soil did not force the flap open. Where the water table was low, we sometimes poured water into the burrow when installing traps. The second trap type was a mist net trap (Welch and Eversole 2006). This was a square of avian mist net folded and tied with string in the middle (Figure 2). A burrow was opened, the mist net was inserted completely into the opening, and the string was tied to a stake flag. We typically installed 20 Norrocky (10 small, 5 medium, and 5 large) and 20 mist net traps per site, but there were slight variations in numbers. Traps were distributed approximately evenly among and within transects, and if insufficient numbers of burrows were found in quadrats, traps were placed in haphazardly located burrows. In sites where traps were set for two nights, they were checked after the first night, and if a crayfish was captured, the trap was moved to a different burrow. Active sampling was conducted in 45 minute time periods and included excavating, suctioning, baited line sampling, and visual searching. Each person was instructed to record the time spent sampling, the number of burrows sampled, and the crayfishes caught. If time was not recorded or if efforts of two people were combined on the data sheet, those data were not included in sampling efficiency analyses. We excavated burrows by digging with our hands in sandy soils and using Japanese soil knives when necessary to cut roots or dig in soils with higher clay content. Some crayfish were captured immediately near the burrow entrance. Sometimes one could disturb the water in the burrow and wait several minutes for the crayfish to surface. Collectors tended to either excavate one burrow until capturing a crayfish or exhausting all burrow paths or quickly move on to a new 6

burrow if not capturing a crayfish in the first few minutes of digging. Suctioning employed several apparatuses: two sizes of Suction (Slurp) Guns (55 and 69 cm long; Trident, Chatsworth, CA; Figure 2) and one homemade, PVC slurp gun. The suctions sometimes extracted a crayfish from its burrow or merely disturbed the burrow water to the extent that the crayfish surfaced after several minutes. In sites with high water tables, occasionally we suctioned burrows without excavating, but typically the method included a combination of excavating down to water and then suctioning. We briefly tested the final two methods before abandoning them. Visual searching involved using a red or white light to search for crayfish wandering about or stationed at a burrow entrance at night. We tested this on two nights—one dry and one wet--and found no crayfish. In theory, baited line sampling uses nightcrawlers on a hook to lure crayfish from their burrows and is intended for use at night after a crayfish is spotted at its burrow entrance (Loughman et al. 2013). Because we did not locate any crayfishes at burrow entrances, the method was not used in that way. Instead, we used baited lines in several active burrows during the day but without success (although some crayfish were captured when excavating down to the water table to deploy the baited lines). These methods will not be addressed further.

Statistical analyses We summarized quadrat habitat data by site without transforming the data. We compared the mean number of active burrows (all 3 categories combined) per site among the three management history groups (excluding the site that was infrequently burned but had mechanical tree removal) using an independent samples median test. We repeated the test comparing combined frequently burned and mechanically treated sites to infrequently burned sites. We then fit linear and exponential curves to bivariate scatter plots of the habitat variables versus the number of active burrows and selected the curve with the best fit for each variable. The SPSS 25 statistical package (IBM) was used for analyses. We compared sampling efficiencies in several ways. First, we calculated catch-per-unit- effort (CPUE) of each sampling method for each site and date. For terrestrial passive methods, CPUE was the number of crayfish per 15 traps. We compared CPUE among Norrocky traps of different diameters and between mist net traps versus all Norrocky traps and versus small Norrocky traps. We used data from only the first night of trapping and paired samples within sites using the related-samples Wilcoxon signed rank tests. We qualitatively compared CPUE of traps in place for one versus two nights. We also examined total trap CPUE to temperature and rainfall. We obtained hourly weather data from the Refuge during the study period (Station SHCM6, MS Sandhill Crane, 30.452778, -88.661667, elevation 25 feet), calculated mean temperature (oC) and total rainfall during the 24 hours beginning at 0851 hours the day prior to trap checking. We graphed crayfish CPUE in traps versus average temperature and total rainfall over the previous 24-hour period. For terrestrial active sampling methods, CPUE was the number of crayfishes caught per 10 minutes of sampling. For comparisons among individual collectors, we calculated average CPUEs per site and excluded effort by the three collectors who never caught a burrowing crayfish. We first compared CPUE between excavation and excavation/suctioning using the 7

related-samples Wilcoxon signed rank tests. Statistically comparing CPUE among collectors was difficult because often different collectors worked at different sites. Collector 4 was the only person to excavate burrows at all sites. Therefore, we made only descriptive comparisons among collectors. Next, we calculated three versions of average active sampling CPUE, and all three included sampling by excavation and by suctioning but excluded efforts by the three collectors who never caught a burrowing crayfish: (1) active CPUEtotal included effort and results of all remaining collectors at a site; (2) active CPUEcollector4 included only effort and results of Collector 4; and (3) active CPUEothers included effort and results of collectors at a site other than Collector 4. We plotted the average active CPUEcollector4 and CPUEothers at each site to see if they showed similar patterns in CPUE among sites. Finally, we used non-parametric correlation (Kendall’s tau) to compare each of the three active sampling CPUEs to the number of active burrows in quadrats at a site to determine whether crayfish were easier to catch in higher density sites. RESULTS--TERRESTRIAL

Habitat and burrow counts—systematic sampling Vegetation cover varied among sites from nearly 100 % grass/forbs in a crop unit to >25% woody plant cover in an infrequently burned site (Appendix Table B2). Burrow counts did not differ significantly among treatment types (excluding site T11 [O19E]) whether comparing the three treatments (independent samples median test, P=0.176) or combining the frequently burned and mechanically treated categories (P=0.182; Figure 3). However, the low number of sample sites created low statistical power to detect differences. Results of univariate linear and exponential line fitting to compare habitat variables summarized within sites to the total numbers of active burrow per site showed a consistent, exponential relationship between woody plant variables and active burrow numbers (Table 1; Figure 4). Linear and exponential lines for the following variables had significant slopes, indicating a relation to active burrow numbers: average percent of woody plant coverage in quadrats, the average maximum woody plant height in quadrats, and the total number of trees (all heights) within 2 m of transects. Additionally, the exponential, but not the linear, model for trees taller than 1.2 m was significant.

Crayfish capture and sampling efficiency We caught burrowing crayfishes in all 12 of the terrestrial sites sampled systematically. Species included the flatwoods digger (Creaserinus oryktes) and the spinytail crayfish (Procambarus fitzpatricki) which was on the priority at-risk species list. The identification of C. oryktes is not certain. The taxonomy and identification of Creaserinus danielae (on the priority at-risk species list) versus C. oryktes remain unclear. Adams examined the type specimens of both in the Smithsonian National Museum of Natural History. Although the specimens were distinctive from one another, it is unclear whether the distinctive features vary continuously within and among populations or if they truly represent distinct species. Creaserinus specimens from the Refuge fit portions of the descriptions of both species, but I tentatively identified them as C. oryktes. I sent specimens to three other experts who tentatively concurred with this

identification. I retained tissue samples from many specimens in case funds become available for genetic analyses. In systematically sampled sites, we sampled 771 burrows and caught 42 crayfish in 42 quadrats and 102 crayfish outside of quadrats for an overall success rate of 19 %. Active methods produced 83 % of the crayfishes captured (Figure 5). Crayfishes were caught in burrow traps in 9 of 12 sites. The crayfish CPUE (#/15 trap- nights) did not differ significantly among burrow trap types, due in part to low capture rates and high variability. The CPUE did not differ between mist nets and all Norrocky traps or small Norrocky traps (related-samples Wilcoxon signed rank tests, p-values > 0.80), nor among the three sizes of Norrocky traps (related-samples Friedman’s two-way analysis of variance by rank, p-value = 0.247). However, the medium-sized Norrocky traps captured crayfish in only one site and the large traps in two sites. The mist net traps and small Norrocky traps captured crayfishes in 7 and 6 sites, respectively, with mist net traps having the higher CPUE in 5 sites and small Norrocky traps in 4 sites (Figure 6). We caught 21 crayfish in traps that were in place for 1 night versus 4 in traps in place for 2 nights. We concluded that little was gained by trapping for a second night, unless rain is anticipated during the second 24 hours (see below); however even during rains, we caught more crayfish in traps that were out for one compared to two nights. Many traps that did not capture a crayfish showed signs of overnight crayfish activity in or next to the trap (e.g., a trap newly plugged with soil). The plot of crayfish CPUE from traps versus rainfall suggests that even a small amount of rain in the previous 24, or perhaps even 48, hours stimulated higher CPUE in traps (Figure 7). Conversely, the plot of CPUE and average temperature revealed no obvious pattern (Figure 7). We have insufficient data to statistically test the pattern, but each measurable rainfall event (0.01” ending 26 January 2017; 2.55” ending 8 February; and 0.11” ending 2 March) produced a higher trap CPUE than previous night(s) in the same week. Active sampling produced many more crayfishes than did passive sampling (Figure 5; Appendix Table B3). Whereas passive sampling appeared subject to disparities based on rainfall, active sampling varied dramatically among collectors (Figure 8). Collector 4 was the only person to excavate at all sites and also had the highest CPUE overall (Figure 8) and at most sites. We compared the average active CPUEcollector4 at each site to the average CPUEothers at the sites, and the patterns were not concordant (Figure 9). Active sampling CPUE was also not correlated with the number of burrows in quadrats, regardless of whether CPUE was calculated with or without Collector 4 or other collectors (Figure 10). In the 12 systematically sampled terrestrial sites, we caught 110 C. oryktes (excluding hatchlings) and 9 P. fitzpatricki (plus 29 from dipnetting) by excavating and suctioning and caught 24 and 1, respectively, by trapping (Appendix Table B3). So, P. fitzpatricki constituted 7.6 % of actively sampled and 4.0 % of passively sampled crayfishes.

AQUATIC METHODS--AQUATIC We used standardized sampling to assess crayfishes in 10 aquatic sites on the Refuge and non-standardized sampling in 14 additional sites on or near the Refuge (Figure 1; Appendix Tables C1-C3). Sites ranged from temporarily wet ditches to permanent ponds. Systematic sampling consisted of either active sampling and baited minnow trapping (6 sites) or habitat trapping without active sampling (4 sites; Appendix Table C2). Active sampling included 30 minutes of dipnetting (Cumings dipnets, Flint, MI; 41 cm-wide x 30 cm-tall, 32 mm mesh) by two people followed by 30 minutes of seining (3 m-wide x 2 m-deep, 32 mm mesh). If habitat conditions prevented seining, a second 30-minute round of dipnetting was conducted. The second round typically covered areas not included in the first round of active sampling. Non- standardized active sampling consisted of timed dipnetting, and person-minutes of sampling was recorded (Appendix Table C3). For all sampling, we attempted to sample all types of aquatic habitat present at each site. Minnow traps were standard, cylindrical, galvanized metal traps (419 mm long, 190 mm diameter 6.4 mm mesh) with two conical entrances each with a 25 mm diameter opening. Habitat traps (Miller Net Company, Memphis, Tennessee) were circular (46 cm diameter) nets (3 mm mesh) (Barnett and Adams 2018) with ‘crayfish hotels’ secured to the middle. Hotels consisted of variously sized tubes attached with zip ties to the underside of a 20.3 cm square of 1.9 cm expanded metal (Figure 2). The following six tubes were attached parallel to each other: two 15.2 cm long, 3.2 cm diameter PVC tubes; two 12.7 cm long, 2.5 cm diameter corrugated black plastic tubes; and two 12.7 cm long, 1.9 cm diameter corrugated black plastic tubes. Perpendicular to those on one end was a 17.8 cm long, 3.2 cm diameter PVC tube. Where possible, we raked additional cover (leaves, small woody debris) onto the traps. Habitat traps lay flat on the substrate until the trap was lifted and the collapsible sides raised up, trapping any crayfish (Figure 2). Tall stake flags marked trap locations and also alerted cranes to trap presence so they would not become entangled in the traps. Twenty minnow traps were set for one night per site. Half were placed in shallow water so that they protruded from the water and half were fully submerged in deeper water. Deeper traps were marked with stake flags. Minnow traps were baited with Fancy Feast canned cat food. Fifteen habitat traps were set in each of two sites during the first two sampling trips, and removed during the following visit 2-3 weeks later. Half (7 or 8) of the traps were set in shallow water and the remainder in deeper water. Animals could move freely in and out of a trap until it was lifted. At site A04, 20 habitat traps were inadvertently set, so 5 were checked and moved to a new site after 4 nights. Most crayfishes were retained, but at sites with large catches, some were identified to species and form and released. Amphibians and most fishes were identified to species and released. Some fishes were retained as voucher specimens or to confirm identifications in the laboratory. Retained fishes were deeply anesthetized by submersion in a clove oil solution then preserved in 10% formalin (USFS IACUC permit #2016-016).

At each standardized sampling site, we first used a Hydrolab Quanta (HACH-Hydrolab, Loveland, CO; calibrated before each sample week) to measure water temperature, conductivity, dissolved oxygen, pH, and turbidity. After sampling crayfishes and setting traps, we measured the length, width, and maximum depth of the sampled area and described the vegetation. Coordinates were taken on-site with a Garmin GPSII Plus and later checked and adjusted as necessary using Google Earth.

Statistical analyses Crayfish data were summarized as numbers of crayfish caught and as CPUE by method and site. For sites where standardized sampling was used, we calculated CPUE for passive methods as number of crayfish/10 traps, and for active methods (where relevant) as number of crayfish/30 net-minutes. We compared CPUE between trap depths and between rounds of active sampling using Friedman’s related-samples two-way analysis of variance by ranks. Fish data were summarized as numbers by species per site, numbers by method per site, and CPUE by species, method, and site. Because we were interested in which method(s) best characterized fish diversity, we compared the number of fish species caught by shallow versus deep traps of each type and by round 1 versus 2 of active sampling using Friedman’s related- samples two-way analysis of variance by ranks. RESULTS—AQUATIC

Standardized sampling Of the 10 aquatic sites with standardized sampling, we captured crayfishes in 9, fishes in 7, and amphibians in 4. We did not capture more than one crayfish species at any of these sites. We captured red swamp crayfish (Procambarus clarkii) at 8 of the 10 sites, and least crayfish (Cambarellus diminutus) at one site where we did not capture P. clarkii (Table 2). The channel north of the Blue Hole (A07) was the only site where we caught no crayfish, but we did catch freshwater shrimp (Palaemonetes sp.) there. Although water quality was sampled at 1400 hours at this site versus earlier than 1000 hours at all but one of the other sites, site A07 had the highest temperature, dissolved oxygen, conductivity, and pH and the 2nd lowest turbidity of any site (Appendix Table C1). We also caught 208 fishes of 10 species (Tables 3 and 4) and 9 amphibians of 2 species. At five of the six sites where we used active sampling, we caught more crayfish by active sampling than by minnow trapping, and in four of the six sites, we caught more crayfish in the second than in the first round of active sampling (Table 2; Figure 11). However, size classes of crayfish captured by the various methods varied dramatically, and only minnow trapping caught predominantly adults. The percentage of the P. clarkii catch that was adults declined in the order of baited minnow trapping (82 % adults), habitat trapping (27 %), dipnetting (8 %), and seining (0 %). Sample sizes, and thus statistical power of comparisons, were low, and we found no significant differences between CPUE in shallow versus deep minnow or habitat traps, round 1 versus round 2 dipnetting, or round 1 dipnetting versus round 2 seining (related-samples Friedman’s two-way analysis of variance by ranks, all p-values > 0.1). Because the aquatic

crayfish diversity in the sites sampled by standardized methods appeared to be quite limited, we could not assess the best methods for assessing crayfish species diversity.

Effectiveness of sampling methods for fishes declined in the order of active methods to minnow traps to habitat traps (Table 5; Figure 12). Habitat traps could not be compared directly to the other methods because they were used in different sites; however, habitat traps in three sites caught no fish and caught four warmouth in the fourth site, suggesting the method was ineffective for fishes on the Refuge. Our active sampling produced an average of 4.2 fish species per site (range 1-7). Adding an additional 30 minutes of seining or dipnetting to the first 30 minutes produced an additional 0.5 or 1.0 species per site, respectively, on average (range 0-3). In one site, minnow traps caught 2 species not caught in the active sampling, but in the other five sites produced no additional species. Across all sites, several species were caught only with active sampling methods, but only the sharpfin chubsucker (Erimyzon tenuis) was caught exclusively by a passive method (Appendix Table C4, site A01 shallow minnow trap). Both deep and shallow minnow traps produced dollar sunfish in two sites and no species in two sites. In the remaining two sites, different species were caught in shallow versus deep minnow traps.

During standardized sampling we caught amphibians in four sites and only in minnow traps. We caught two-toed amphiumas (Amphiuma means) in Valentine Pond (A01), Ben Williams 6A Savanna Fireline (A02), and Roadside Ditch O6SE (A10)(5, 2, and 1 individuals, respectively). Six of the eight were caught in deep traps. One lesser siren (Siren intermedia) was caught in a shallow minnow trap in the West Fireline site (A06).

Non-standardized sampling

During non-standardize aquatic sampling, we captured two crayfish species not captured during any other sampling: ditch fencing crayfish (Faxonella clypeata) and Gulf crayfish (P. shermani; Table 6). All of the sites where we caught F. clypeata and all but one site with P. shermani were along or south of Highway 90. In addition, we caught P. fitzpatricki (also caught during terrestrial sampling) and P. clarkii and C. diminutus (also caught during standardized aquatic sampling; Table 6).

DISCUSSION Crayfish burrow densities were higher where woody plants were shorter and sparser. This was consistent with predictions based on studies of other habitat specialist primary burrowing crayfishes (Welch 2004, Rhoden et al. 2016) and suggested that the FWS habitat management for cranes was also beneficial to primary burrowing crayfishes. Larger samples sizes would be needed to detect any differences between the effects of frequent burning versus mechanical treatments on burrow densities by site. The Utah crop unit site was one outlier in the plot of percent woody plant cover versus the number of active burrows (Fig. 4), with far fewer burrows than expected. Reasons for this are unknown, but lower than expected burrow numbers could be due to soil compaction from machinery or to reduced diversity of native plants or animal prey. Further investigation of the causes would be interesting as it may provide insight into how

12 burrower assemblages along mowed roadsides compare to those in more natural prairie or savanna habitats. If future terrestrial crayfish research or monitoring is undertaken, for habitat assessment, we recommend focusing on characterizing woody plant prevalence and size. Including variables indicative of water table depth and of duration of surface or near-surface water may prove useful. Potential variables include percent sphagnum moss or abundance of certain carnivorous plants, elevation, and soil type. We initially included presence/absence of carnivorous plants, but abandoned that because of the time required to find very small carnivorous plants and the near- ubiquity of them; however, including a metric for only larger carnivorous plants would be feasible. None of the methods we tested were consistently efficient for sampling the burrowing crayfishes themselves, and thus none provided a means to make valid comparisons among sites based on actual crayfish captures. Two arguments that are sometimes made for trapping rather than excavating burrowing crayfishes are that trapping is easier to standardize and to conduct. Certainly trapping removed much or all of the variability among people; however, because its effectiveness appeared to be highly subject to rainfall during our study, it did not actually standardize sampling among sites, at least as implemented during our study. If many sites were trapped simultaneously, then trapping may provide more standardized results. Also, it appeared that P. fitzpatricki were less susceptible than C. oryktes to trapping. Installing and checking traps was physically easier than active sampling, but in terms of total time spent per crayfish caught, active methods were probably equally efficient because they did not require multiple trips to a site. However, extremely high variability among collectors in CPUE of active sampling made it difficult to standardize effort. Given this, we recommend (1) having the same individuals sample crayfishes at all sites or preferably (2) restricting quantitative assessments of crayfish activity to counts of burrow entrances, and using actual crayfish sampling only to assess species presence/absence, relative density, and habitat use. Modified Norrocky traps and mist net traps produced similar CPUE overall. Mist net traps are cheaper and easier to make and are easier to store and transport; however, removing crayfish from mist net traps is sometimes difficult, and crayfish are much more likely to be damaged when caught in a mist net compared to a Norrocky trap. Note that these results cannot necessarily be translated to other soil types or other crayfish species, as both are likely to influence trap effectiveness (personal observation). Strategies for burrow excavation by various collectors fell along a continuum. At one extreme, collectors would excavate a burrow until all possible tunnels were excavated to the extent possible or until a crayfish was caught, sometimes excavating only two or three burrows in 45 minutes. At the other extreme, a collector may spend only a few minutes on any one burrow, moving on to the next if a crayfish was not caught quickly. The most successful collector followed the latter approach. To maximize captures on the Refuge, we recommend having a soil knife available and not devoting more than roughly 5-7 minutes to most burrows. Use of a slurp gun in combination with excavating seemed particularly beneficial in sites where

the water table was high enough that burrow water could be reached just by removing the chimneys. The FWS at-risk species list includes C. danielae, but not C. oryktes. Identification and taxonomy of the two continue to baffle astacologists. The limited genetic data available are consistent with the uncertainty about morphology of the two. Ainscough et al. (2013) noted that “Fallicambarus (Creaserinus) danielae and F. (C.) oryktes are difficult to distinguish from each other, and further work is needed to determine whether they truly are separate species.” That said, we believe that only one Creaserinus species was collected throughout the Refuge. Any morphological differences observed among specimens appeared to represent continuous variation rather than distinct forms. In particular, color was highly variable among the Creaserinus specimens, with brown, blue, and nearly white specimens all collected from one site. Resolving this taxonomic uncertainty is critical to effectively managing these taxa. Procambarus fitzpatricki is a priority at-risk species and a target species on the refuge. Although relatively much less abundant than C. oryktes in our burrow sampling, we found P. fitzpatricki in 7 of the 14 terrestrial sites and in one additional aquatic site. It may have occurred at more of our terrestrial sites but was not collected due to its low abundance. It seemed to be slightly more aquatic compared to C. oryktes and was caught during dipnetting of small temporary pools in two sites. In sampling elsewhere in southern Mississippi, we have collected P. fitzpatricki much more often than C. oryktes while dipnetting in temporary waters such as roadside ditches. Two other at-risk crayfish species are considered by the FWS region to be both priority species and target species for the Refuge: the burrowing bog crayfish (Creaserinus burrisi) and the angular dwarf crayfish (Cambarellus lesliei). We did not collect either species during the study. Creaserinus burrisi has not been collected as far south as the Refuge and probably does not occur there. Cambarellus lesliei, on the other hand, has been collected close to the refuge (personal communication with Bob Jones, MS Museum of Natural Science). We sampled on and near the Refuge specifically for this species during the final sampling week but did not find it. However, we did collect Cambarellus diminutus from two aquatic habitats, both small, probably perennial pools. Although not on the target species list for the Refuge, C. diminutus may have a more restricted distribution than C. lesliei. It is ranked as “G3” by NatureServe, “threatened” by the American Fisheries Society (Taylor et al. 2007), and Data Deficient by the IUCN Red List (Adams and Jones 2010), whereas C. lesliei is ranked Least Concern by IUCN (Adams et al. 2010). The other three species that we collected in aquatic habitats, P. shermani, P. clarkii, and F. clypeata, are common, secure species. None of the 10 fish species we caught on the Refuge are of conservation concern. For aquatic sampling, we found that active sampling (dipnetting and seining) produced the most crayfish and the highest fish species diversity. On the other hand, baited minnow traps were more effective for catching adult crayfish and amphibians but added only one fish species to those caught by active sampling. Habitat traps caught few fishes but appeared to be fairly effective for crayfishes, although the results cannot be directly compare to other methods due to

our sampling design. In general, deep habitat traps caught more crayfish than shallow ones, and the opposite was true for minnow traps, except in one site. Fully submerged minnow traps can be problematic because they can drown herpetofauna. Based on our results, one solution that may be effective for crayfish sampling would be to use baited minnow traps in shallow water and habitat traps in deeper water. The advantage of habitat traps is that they can be left in place indefinitely and checked periodically. The disadvantage is that they need to be in place for some time to be effective. The optimal soak time for habitat traps is unknown, but interestingly, in site A04 where we accidentally set too many habitat traps and removed five after 4 nights, we caught slightly more crayfish per trap after 4 nights (2.6) than after 13 nights (2.5).

Sampling recommendations

For terrestrial crayfishes, assessing burrow densities currently appears to be the best method for comparing crayfish densities among sites. Habitat assessment for burrowing crayfishes should focus on woody plant metrics and perhaps on variables related to water table depth, soil type, and elevation. For actually collecting burrowing crayfishes, burrow excavation, possibly combined with suctioning, is the most promising method. Burrow traps should only be used if trapping can be implemented to coincide with rain events, and trapping for one night was the most efficient. Further assessment of night sampling on warm, wet nights is may also be worthwhile. Dipnetting small, isolated pools in savannas is a promising method for collecting P. fitzpatricki.

For aquatic sampling, active sampling is recommended for obtaining large numbers of crayfishes and for maximizing fish species caught. A second round of active sampling slightly increased the number of fish species caught, although effort may be better applied to sampling additional sites. Crayfish diversity was low in habitats large enough for our systematic sampling. That said, baited minnow traps captured the most adult crayfishes. We caught more crayfish species by dipnetting in smaller water bodies than by the combined efforts of our systematic sampling in larger aquatic habitats. Thorough sampling of an aquatic habitat for crayfish could be accomplished using deep habitat traps, shallow baited minnow traps, and at least one round of active sampling; however, the best application of effort for assessing crayfish distributions across the refuge would be to use active sampling in as many diverse aquatic habitats as possible. Minnow traps were the only method that captured sirens and amphiumas. Compared to shallow traps, deep minnow traps maximized amphibian captures but contributed additional fish species in only two sites.

ACKNOWLEDGEMENTS We thank the employees, interns, and volunteers who helped on this research. We are particularly grateful to those who moved beyond their typical areas of interest and expertise and willingly embraced the challenges of burrowing crayfishes sampling! G. McWhirter led the aquatic sampling and identified all fishes in his typical capable way. Thanks to the incomparable sampling machine, M. Bland, for his work as “Collector #4”! Z. Barnett organized sampling for several days during round 2. We thank G. Schuster for taking and editing the photographs of live

crayfishes. S. Hereford initiated the study and assisted throughout. A. Dedrickson assisted with sampling and logistics. G. Henderson created Figure 1. Finally, thanks to the numerous FS and FWS administrative staff who facilitated the funding agreement. Research funding was provided by the US Fish and Wildlife Service via an Interagency Agreement (F16PG00142) to the FS. The FS Southern Research Station provided salary and in-kind support for the project.

LITERATURE CITED Adams, S. and R.L. Jones. 2010. Cambarellus diminutus. The IUCN Red List of Threatened Species 2010: e.T3670A10010991. http://dx.doi.org/10.2305/IUCN.UK.2010- 3.RLTS.T3670A10010991.en. Downloaded on 13 December 2017. Adams, S., R.L. Jones, and G.A. Schuster. 2010. Cambarellus lesliei. The IUCN Red List of Threatened Species 2010: e.T3671A10012114. http://dx.doi.org/10.2305/IUCN.UK.2010- 3.RLTS.T3671A10012114.en. Downloaded on 11 September 2018. Ainscough, B. J., J. W. Breinholt, H. W. Robison, and K. A. Crandall. 2013. Molecular phylogenetics of the burrowing crayfish genus Fallicambarus (Decapoda: Cambaridae). Zoologica Scripta:1-11. Barnett, Z. C., and S. B. Adams. 2018. Comparison of two crayfish trapping methods in Coastal Plain seasonal wetlands. North American Journal of Fisheries Management 38:911-921. Crandall, K. A., and S. De Grave. 2017. An updated classification of the freshwater crayfishes (Decapoda: Astacidea) of the world, with a complete species list. Journal of Crustacean Biology 37:615-653. Hobbs, H. H., Jr. 1981. The crayfishes of Georgia. Smithsonian Contributions to Zoology 318:1- 549. Hobbs, H. H., Jr. . 1942. The crayfishes of Florida Biological Science Series, University of Florida Publications 3:1-79. Loughman, Z. J., D. A. Foltz, II, and S. A. Welsh. 2013. Baited lines: an active nondestructive collection method for burrowing crayfish. Southeastern Naturalist 12:809-815. Norrocky, M. J. 1984. Burrowing crayfish trap. Ohio Journal of Science 84:65-66. Rhoden, C. M., C. A. Taylor, and W. E. Peterman. 2016. Highway to heaven? Roadsides as preferred habitat for two narrowly endemic crayfish. Freshwater Science 35:974-983. Taylor, C. A., G. A. Schuster, J. E. Cooper, R. J. DiStefano, A. G. Eversole, P. Hamr, H. H. Hobbs, III, H. W. Robison, C. E. Skelton, and R. F. Thoma. 2007. A reassessment of the conservation status of crayfishes of the United States and Canada after 10+ years of increased awareness. Fisheries 32:372-389. Welch, S. M. 2004. Relationships between burrow densities of the Camp Shelby burrowing crayfish (Fallicambarus gordoni) and vegetation structures in the Cypress Creek watershed of the Camp Shelby training site, MS. Technical report of The Nature Conservancy, Camp Shelby Field Office, Camp Shelby, Mississippi, USA. Welch, S. M., and A. G. Eversole. 2006. Comparison of two burrowing crayfish trapping methods. Southeastern Naturalist 5:27-30.

TABLES

Table 1. Curve-fitting results for univariate models of quadrat or transect habitat features versus number of burrows. Independent variables were habitat parameters averaged over each site, except for numbers of trees, which were summed over each site. The dependent variable was the total number of active burrows in quadrats for each site. Models with P<0.05 in bold.

Model Summary Parameter Estimates Equation R-Square F df1 df2 Sig. Constant b1 PERCENT COVER % woody plants Linear 0.42 7.12 1 10 0.024 92.60 -2.90 Exponential 0.56 12.67 1 10 0.005 116.39 -0.08 % grass/forbs Linear 0.24 3.22 1 10 0.103 1.93 0.80 Exponential 0.27 3.69 1 10 0.084 10.23 0.02 % bare Linear 0.08 0.92 1 10 0.361 84.29 -3.18 Exponential 0.01 0.10 1 10 0.755 57.30 -0.03 % leaf or small wood Linear 0.08 0.91 1 10 0.364 65.48 -0.58 Exponential 0.09 0.94 1 10 0.356 52.78 -0.01 % large wood Linear 0.19 2.40 1 10 0.153 73.51 -66.03 Exponential 0.08 0.84 1 10 0.380 57.18 -1.03 PLANT HEIGHTS Woody plant height Linear 0.40 6.52 1 10 0.029 92.79 -0.88 Exponential 0.54 11.78 1 10 0.006 118.10 -0.03 Herbaceous plant height Linear 0.00 0.01 1 10 0.927 63.53 -0.04 Exponential 0.01 0.05 1 10 0.831 55.10 0.00 TREES Total trees Linear 0.30 4.36 1 10 0.063 78.46 -0.55 Exponential 0.44 7.73 1 10 0.019 79.10 -0.02 Trees > 1.2 m Linear 0.24 3.17 1 10 0.105 71.36 -0.55 Exponential 0.38 6.03 1 10 0.034 65.04 -0.02 Trees < 1.2 m Linear 0.07 0.77 1 10 0.400 68.70 -0.61 Exponential 0.07 0.78 1 10 0.399 57.07 -0.02

Table 2. Numbers and CPUEs of crayfishes from aquatic sites with standardized sampling. Number of nights that habitat traps were in place are given in parentheses after site names. CPUE for active sampling is number of crayfish per 30 minutes of sampling (per person for dipnetting) and number per 10 traps for passive sampling. Procambarus clarkii were caught with all methods, but Cambarellus diminutus were caught only by habitat trapping and only in one site. Abbreviations: D1 = dipnetting, round 1 active sampling; D2 = dipnetting, round 2 active sampling; S = seine, round 2 active sampling; MTS = minnow trap shallow; MTD = minnow trap deep; HTS = habitat trap shallow; HTD = habitat trap deep.

D1 D2 S MTS MTD HTS HTS HTD HTD Crayfish CPUE (#/30 min. or #/10 traps) # C. # C. Total Total Site # P. # P. # P. # P. # P. # P. dim- # P. dim- P. C. dim- code Site name (habitat trap nights) clarkii clarkii clarkii clarkii clarkii clarkii inutus clarkii inutus clarkii inutus D1 D2 S MTS MTD HTS HTD A01 Valentine Pond 21 0 0 0 21 0 10.5 0.0 0.0 0.0 A02 Ben Williams 6A Fireline 33 84 10 8 135 0 16.5 84.0 10.0 8.0 A03 Duck Pond 1 13 4 0 0 17 0 6.5 4.0 0.0 0.0 A06 West Fireline 88 158 23 46 315 0 44.0 158.0 23.0 46.0 A08 Beasley Road Drain 30 41 92 49 212 0 15.0 41.0 92.0 49.0 A10 Roadside Ditch 43 53 9 9 114 0 21.5 26.5 9.0 9.0 A04 Elbert Shorebird Pond (13) 12 0 26 0 38 0 15.0 37.1 A04 Elbert Shorebird Pond (4) 7 0 6 0 13 0 23.3 30.0 A05 Firetower Pond (12) 0 1 0 2 0 3 1.3 2.9 A07 Channel N of Blue Hole (23) 0 0 0 0 0 0 0.0 0.0 A09 Maverick Pond (24) 9 0 16 0 25 0 11.3 22.9 Grand Total 228 94 246 134 112 28 1 48 2 890 3

Table 3. Fishes caught with standardized sampling in aquatic sites in MS Sandhill Crane NWR, 2017. All but 4 warmouth were caught by active sampling or in minnow traps. Abbreviation Latin name Common name Number ASAY Aphredoderus sayanus pirate perch 12 EZON Ellasoma zonatum banded pygmy sunfish 68 ETEN Erimyzon tenuis sharpfin chubsucker 1 EAME Esox americanus grass pickerel 33 EFUS Etheostoma fusiforme swamp darter 4 FNOT Fundulus notti southern starhead topminnow 11 GAFF Gambusia affinis mosquitofish 32 LGUL Lepomis gulosus warmouth 7 LMAC Lepomis macrochirus bluegill 4 LMAR Lepomis marginatus dollar sunfish 36

Table 4. Number of fish of each species by site. Bold indicates sites sampled with active sampling and baited minnow traps. Sites not in bold were sampled with habitat traps only. Fish species abbreviations as in Table 3.

Site Total Total code Site ASAY EAME EFUS ETEN EZON FNOT GAFF LGUL LMAC LMAR fishes species A01 Valentine Pond 2 1 2 5 1 2 2 15 7 Ben Williams 6A A02 Fireline 7 1 10 11 29 4 A03 Duck Pond 1 3 4 10 6 13 1 22 59 7 A04 Elbert Shorebird 0 0 A05 Firetower Pond 0 0 A06 West Fireline 55 55 1 Channel N of Blue A07 Hole 4 4 1 A08 Beasley Road Drain 8 2 5 15 3 A09 Maverick Pond 0 0 A10 Roadside Ditch 4 19 3 4 1 31 5

Table 5. Number of fishes captured by site and method. Abbreviations: D1 = dipnet 1; S = Seine; D2 = dipnet 2; MTD = minnow trap deep; MTS = minnow trap shallow; HTD = habitat trap deep; HTS = habitat trap shallow.

Site code Site Date D1 S D2 MTD MTS HTD HTS Total

A01 Valentine Pond 1/24/2017 6 1 -- 1 7 -- -- 15

A02 Ben Williams 6A Fireline 1/25/2017 1 20 -- 6 2 -- -- 29

A03 Duck Pond 1 2/7/2017 16 36 -- 1 6 -- -- 59 1/27/17; A04 Elbert Shorebird 2/8/17 ------0 0 0

A05 Firetower Pond 2/8/2017 ------0 0 0

A06 West Fireline 2/9/2017 35 20 -- 0 0 -- -- 55

A07 Channel N of Blue Hole 2/28/2017 ------4 0 4

A08 Beasley Road Drain 2/28/2017 10 5 0 0 -- -- 15 A09 Maverick Pond 3/1/2017 ------0 0 0

A10 Roadside Ditch, O6SE 3/1/2017 8 19 1 3 -- -- 31 Total 76 77 24 9 18 4 0 208

Table 6. Number and species of crayfishes caught at each aquatic site sampled with timed, haphazard dipnetting. See Figure 1 and Appendix Table C3 for site details.

Site P. P. P. C. F. code Site name clarkii shermani fitzpatricki diminutus clypeata Total 10 isolated pools in NW A11 Fontainebleau (F1C) 0 0 29 0 0 29 A12 Ditch, ephemeral pools along Crane Ln. 0 0 4 0 0 4 A13 Susie's Pool 0 18 0 0 0 18 A14 Angie's Pool 9 0 0 46 0 55 A15 Isolated pools (2) in Semmes 9 O19E 0 0 0 0 0 0

A16 Ditches, roadside; west of O3W 97 0 0 0 0 97 A17 11 Isolated pools in O5S 0 0 0 0 0 0 A18 Ditches, along Old Spanish Trail Rd. 115 0 0 0 332 447 A19 Isolated pool 0 5 0 0 46 51 A20 Isolated pools 14 0 0 0 31 45 A22 Ditches, roadside 7 6 0 0 8 21 A23 Unnamed stream 15 13 0 0 8 36 A24 Unnamed stream 6 4 0 0 15 25 Total 263 46 33 46 440 828

FIGURES

Figure 1. Map of sites sampled on and near the Mississippi Sandhill Crane National Wildlife Refuge, Jackson County, MS. Labels coincide with site codes in Appendix Tables B1, C1, and C3. A = aquatic sites and T = terrestrial sites.

Figure 2. A selection of sampling tools used. Modified Norrocky trap, size small (top row), mist net trap being extracted from burrow with Creaserinus oryktes (middle left), and a Trident slurp gun used for suctioning burrows (middle right). Habitat trap collapsed (bottom left) and lifted (bottom right).

Figure 3. Total number of active burrow entrances in quadrats per site and habitat management history. Habitat management codes represent: F—frequently burned; M—mechanically treated; IM—infrequently burned but trees removed mechanically in 2011; I—infrequently burned.

Figure 4. Linear and exponential curves of total burrows per site versus the three most informative habitat variables summarized within sites: average of % of each quadrat covered by woody vegetation, average maximum woody plant height, and total trees within 2 m of transects. See Table 1 for model results.

120 C. oryktes 100 P. fitzpatricki 80

20 Number of crayfish caught Number of crayfish 0 Excavate/suction Mist net traps Norrocky traps Method Figure 5. Numbers of crayfishes captured in the 12 terrestrial sites with systematic sampling by active (excavate and excavate/suction) and passive (mist net traps and modified Norrocky traps) methods during all three sample periods. Hatchlings from the same burrow as an adult are excluded.

5 Mist net Norrocky small 4

3 nights) - 2 Crayfish CPUE Crayfish

(#/15 trap (#/15 1

Site

Figure 6. Burrowing crayfish CPUE in mist net traps and small modified Norrocky traps from the 12 sites with standardized sampling. Crayfish were all C. oryktes except for one P. fitzpatricki in a small Norrocky trap.

2 CPUE 25 Temperature 20 C) o 15 1 10 Crayfish CPUE Crayfish Temperature ( Temperature 5

0 0

Date

2 7 CPUE 6 Rainfall 5 4 1 3

2 (cm) Rainfall Crayfish CPUE Crayfish 1 0 0

Date Figure 7. Terrestrial crayfish CPUE (#/15 traps) and mean temperature (top) and rainfall (bottom) during the previous 24 hours by sample date. CPUE is for burrowing crayfishes caught in modified Norrocky and mist net traps. Note that each date along the x-axis represents a date when traps were checked, and dates are discontinuous along the axis.

Figure 8. Average CPUE (number/10 minutes) for crayfish, excluding hatchlings, collected by excavating burrows. Collectors were assigned numbers (x-axis). Error bars represent standard deviation, and numbers in italics represent number of sampling periods (typically 45 minutes each). Some collectors excavated for two sampling periods at some sites.

Figure 9. Average crayfish CPUE (number/10 minutes, excluding hatchlings) collected by excavating and excavating/suctioning burrows by all collectors except #4 (black bars) and average CPUE from excavations by collector 4 (grey bars).

Avg. total active CPUE Avg. Collector 4 excavation CPUE 1.8 Avg. active CPUE without collector 4 1.6

1.4

1.2

1.0

0.8

0.6 Crayfish CPUE (#/10 min.)Crayfish 0.4

0.2

0.0 0 20 40 60 80 100 120 140

Number of active burrows in quadrats

Figure 10. Numbers of active burrows in quadrats compared to three measures of crayfish CPUE (# crayfish/10 minutes of sampling) from active sampling: CPUEtotal (green), CPUEcollector4 (orange); and CPUEothers (blue). Within each color, circles represent sample sites. Kendall’s tau ≥0.26, P-value >0.1 for all three relationships.

180 D1 D2 160 S 140 MTS MTD 120 HTS HTD 100

CPUE 80

0 A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 Site code

Figure 11. Catch-per-unit-effort (CPUE) of crayfishes in aquatic standardized sampling sites. Habitat traps were used in different sites than other methods, and the second round of active sampling employed either dipnetting or seining, but never both. The CPUE for active sampling is number of crayfish per 30 net-minutes of sampling and number per 10 traps for passive sampling. All crayfish captured were Procambarus clarkii except in site A05, where all were Cambarellus diminutus. Legend indicates sampling methods with abbreviations as follows: D1 = dipnetting, round 1 active sampling; D2 = dipnetting, round 2 active sampling; S = seine, round 2 active sampling; MTS = minnow trap shallow; MTD = minnow trap deep; HTS = habitat trap shallow; HTD = habitat trap deep.

1 Average number of of number fish species Average 0

Method

Figure 12. Average number of fish species caught using each method in standardized sampling sites. Totals for combined methods shown in darker shades. For habitat traps, the combined number of species was the same as the number from deep habitat traps because no fish were caught in shallow habitat traps.

APPENDIX A. COLLECTORS

Appendix Table A1. Names, initials, and affiliations of people assisting on project. US Forest Service = USFS; US Fish and Wildlife Service = FWS. Name Initials Affiliation Position Susan B Adams SBA USFS employee Zanethia C Barnett ZCB USFS employee Mickey R Bland MRB USFS employee J Gordon McWhirter JGM USFS employee Carl G Smith CGS USFS employee Angela J Dedrickson AJD FWS employee Scott G Hereford SGH FWS employee Valerie D Schneider VDS FWS intern James W Stockdale JWS FWS intern Victoria E Thorpe VET FWS intern Virginia M VanVianen VMV FWS intern Stewart C Ray SCR FWS volunteer Marlene Schmitt MS --- volunteer

APPENDIX B. TERRESTRIAL SAMPLING AND RESULTS Appendix Table B1. Terrestrial site locations, sampling types and dates, and management histories. Years since burn are years as of 2017 sampling date. For standardized sampling sites, latitude and longitude are from the end of transect C farthest from the road, and for the non-standardized sites are near the starting point of sampling. Years Site FWS Systematically Sample Sample Nights Habitat Vegetation since code Unit Site name sampled? Y/N round date(s) trapped type treatment burn Latitude Longitude West Valentine 1/24/2017; T01 G5N Savanna Y 1 1/25/2017 1 savanna Frequently burned 2 30.45482 -88.68491 North Valentine 1/24/2017; T02 G6N Savanna Y 1 1/25/2017 2 savanna Frequently burned 1 30.46354 -88.68929 1/25/2017; T03 G15S Church Savanna Y 1 1/26/2017 2 savanna Frequently burned 1 30.41642 -88.66350 NW 1/26/2017; T04 F1C Fontainebleau Y 1 1/27/2017 1 savanna Mechanical 18 30.39786 -88.74356 S Duck Pond 2/7/2017; T05 O10SW Savanna Y 2 2/8/2017 2 savanna Frequently burned 1 30.45395 -88.75887 T06 O9W Savanna Y 2 2/8/2017 1 savanna Infrequently burned 7 30.45770 -88.76931 2/8/2017; T07 O16E Savanna Y 2 2/9/2017 2 savanna Frequently burned 1 30.46423 -88.78165 2/9/2017; T08 O20 Utah Crop Unit Y 2 2/10/2017 1 crop unit Mechanical_mowed 5 30.46815 -88.81761 T09 G11NW Martin Pasture Y 3 2/28/2017 1 pasture Mechanical_mowed 10 30.43331 -88.64503 T10 G11SE Martin NW Y 3 3/1/2017 1 Infrequently burned 2 30.43135 -88.64306 Infrequently savanna/ burned/ mechanical T11 O19E Semmes 9 Y 3 3/1/2017 1 prairie in 2011 3 30.47113 -88.75497 savanna/ T12 O5S Utah North Y 3 3/2/2017 1 prairie Infrequently burned 3 30.46989 -88.81612 T13 O3W Savanna O3W N 3 3/2/2017 0 savanna Frequently burned 2 30.48464 -88.81197 Fontainebleau T14 F2N SW N 3 3/2/2017 0 savanna Mechanical >18 30.39454 -88.74862

Appendix Table B2. Averages and standard deviations of habitat data from quadrats for each terrestrial site with systematic sampling. Habitat variables are as described in Methods. In each quadrat, percentages of organic cover categories and bare soil added to 100. Percent standing water was in addition to those and was not included during the first week of sampling.

Avg. Avg. Avg. Avg. Avg. Avg. stand- SD Avg. woody SD grass/ SD woody SD leaf/ SD Avg. bare SD ing stand- herb. SD plant woody Site FWS forbs grass/ plants woody SWD leaf/ LWD SD soil bare water ing height herb. height plant code Unit (%) forbs (%) plants (%) SWD (%) LWD (%) soil (%) water (cm) height (cm) height T01 G5N 93.0 12.85 5.89 10.69 0.22 2.11 0.00 0.00 0.89 3.56 -- -- 90.41 17.05 18.29 24.15 T02 G6N 86.0 10.79 7.56 9.75 0.11 1.05 0.00 0.00 6.56 6.21 -- -- 95.76 13.81 23.59 21.72 T03 G15S 77.8 13.88 13.56 12.48 0.22 1.48 0.11 1.05 8.56 7.43 -- -- 101.00 13.51 30.39 17.12 T04 F1C 84.1 15.35 7.44 12.41 0.44 2.07 0.00 0.00 8.22 8.94 -- -- 83.13 21.84 25.05 23.53 T05 O10SW 74.1 20.93 12.78 12.00 0.56 3.13 0.44 2.56 12.11 11.85 0.00 0.00 80.88 19.61 33.97 17.54 T06 O9W 65.2 17.30 23.11 12.95 5.22 7.68 0.00 0.00 6.44 8.91 0.00 0.00 81.66 21.44 56.76 23.47 T07 O16E 91.1 77.94 11.78 10.56 0.22 1.48 0.67 6.32 4.33 7.94 19.89 31.85 84.94 19.82 36.87 21.39 T08 O20 95.2 8.24 0.00 0.00 0.00 0.00 0.00 0.00 4.78 8.24 1.33 10.62 33.13 14.26 0.00 0.00 T09 G11NW 90.3 11.75 0.22 1.48 2.11 4.86 0.00 0.00 7.22 10.60 0.00 0.00 31.41 9.79 4.63 7.81 T10 G11SE 42.7 31.15 27.56 20.19 22.56 20.48 0.22 1.48 6.67 7.34 0.22 2.11 79.66 39.32 91.40 32.19 T11 O19E 66.6 23.33 14.89 13.43 4.67 12.65 0.33 2.35 13.00 12.93 2.56 8.42 76.62 21.95 46.19 25.36 T12 O5S 18.7 13.92 7.33 9.81 63.22 20.27 0.56 4.33 10.44 11.01 2.22 9.57 18.22 8.53 70.26 52.41

Appendix Table B3. Crayfish data from active sampling of terrestrial sites by excavating and suctioning. Excludes hatchlings and baited line and dipnetting sampling data. For standardized sites T01-T12, active CPUE are averages among Collectors who caught crayfish from burrows at any site (excludes effort for the three collectors who never extracted a crayfish from a burrow). ‘Number of crayfish, active,’ is the total number of crayfish from burrows. Trap CPUE is number per trap-night. ‘Number of crayfish, traps,’ is for C. oryktes (CORY) unless P. fitzpatricki (PFIT) is indicated. ‘Grand total from burrows’ is the total number of non-hatchling crayfish collected from burrows. Standardized sampling was not conducted on sites below the dashed line.

a Avg. CPUE Number of crayfish, active Trap CPUE (Number per 15 trap-nights) Number of crayfish, traps Grand Norrocky Norrocky Total Norrocky Norrocky Total total Site FWS CORY PFIT Mist Norrocky Norrocky small small Norrocky Mist Norrocky Norrocky small small Norrocky from code unit CORY PFIT CORY PFIT dipnet dipnet net large medium CORY PFIT both spp. net large medium CORY PFIT both spp. burrows T04 F1C 0.61 0.22 8 3 0 29 0.00 0.00 0.00 0.00 1.50 0.75 0 0 0 0 1 1 12 T09 G11NW 0.56 0.00 5 0 -- -- 0.00 0.00 0.00 0.00 0.00 0.00 0 0 0 0 0 0 5 T10 G11SE 0.19 0.04 5 1 -- -- 0.00 0.00 0.00 0.00 0.00 0.00 0 0 0 0 0 0 6 T03 G15S 0.32 0.00 14 0 -- -- 0.38 0.00 0.00 0.00 0.00 0.00 1 0 0 0 0 0 15 T01 G5N 0.31 0.06 16 3 -- -- 0.00 0.00 0.00 0.00 0.00 0.00 0 0 0 0 0 0 19 T02 G6N 0.22 0.00 10 0 -- -- 0.38 0.00 0.00 0.00 0.00 0.00 1 0 0 0 0 0 11 T05 O10SW 0.19 0.00 6 0 -- -- 0.38 1.50 0.00 0.75 0.00 0.75 1 1 0 1 0 2 9 T07 O16E 0.26 0.00 8 0 -- -- 0.75 0.00 0.00 0.83 0.00 0.39 2 0 0 1 0 1 11 T11 O19E 0.55 0.05 10 1 0 0 1.50 0.00 0.00 1.36 0.00 0.79 2 0 0 1 0 1 14 T08 O20 0.64 0.00 13 0 -- -- 2.25 0.00 0.00 0.00 0.00 0.00 3 0 0 0 0 0 16 T12 O5S 0.56 0.06 10 1 0 0 0.00 0.00 0.00 5.45 0.00 3.16 0 0 0 4 0 4 15 T06 O9W 0.28 0.00 5 0 -- -- 2.25 3.00 3.75 1.36 0.00 2.25 3 1 1 1 0 3 11 T13 O3W 0.78 0.06 14 1 0 0 ------15 T14 F2N 0.67 0.04 15 1 0 0 ------16 Total number 139 11 0 29 13 2 1 8 1 12 a Average CPUE (# crayfish/30 min) for active sampling across collectors and sampling rounds (when applicable) for each site.

APPENDIX C. AQUATIC SAMPLING AND RESULTS

Appendix Table C1. Water quality and water permanence for aquatic sites with standardized sampling. Date and time indicate when water quality was measured. Initials of collectors as in Appendix Table A1. Habitat Perennial date (P) or (month/ Max. H2O DO Site intermittent day, depth temp. DO (% Cond. Turbidity Latitude Longitude code Site name (I) 2017) Time (cm) (oC) (mg/L) sat.) (µS/cm) pH (NTU) Collectors (DD) (DD) A01 Valentine Pond P 1/24 818 43 13.03 5.03 47.80 0.037 5.98 7.60 JGM, AJD 30.45980 -88.69248 Ben Williams 6A A02 Savanna Fireline I 1/25 859 23 14.43 5.55 54.10 0.023 5.24 36.60 JGM, AJD 30.41827 -88.69823

A04 Elbert Shorebird Pond P 1/23 JGM, AJD 30.45950 -88.69001 A03 Duck Pond 1 P 2/7 930 83 18.44 7.35 78.30 0.022 4.65 14.4 JGM, CGS 30.46369 -88.75122 JGM, AJD, A05 Firetower Pond P 2/8 950 74 17.82 4.79 50.70 0.020 5.31 8.9 CGS 30.43197 -88.65987 JGM, CGS, A06 West Fireline, O16E I 2/8 825 20 14.30 4.54 44.00 0.040 5.32 19.6 AJD 30.46033 -88.79600 JGM, AJD, A04 Elbert Shorebird Pond p 2/8 843 60 17.35 4.80 50.50 0.029 5.61 10.20 CGS 30.45954 -88.68999

Channel N of Blue JGM, AJD, A07 Hole P 2/28 1400 37 24.10 8.34 98.90 0.055 6.86 4.60 CGS, JWS 30.46115 -88.79118 A08 Beasley Road Drain P 2/27 833 67 19.62 2.35 25.30 0.032 4.45 5.80 JGM, JWS 30.42870 -88.66496 JGM, AJD, A09 Maverick Pond p 3/1 1244 37 22.98 5.16 59.90 0.022 4.61 4.40 CGS, VDS 30.45667 -88.75733 A10 Roadside Ditch, 06SE P 2/28 752 51 19.63 5.21 57.10 0.030 5.33 15.00 JGM, JWS 30.47164 -88.78308

Appendix Table C1 continued. Site code Site description On Valentine Road about 150m from intersection with Entry Road (Brown's Trail). Sampled area 64m x 24m. Sampled area included dense stand of young bald cypress (20% of area), grass (20%), and a zone of sparse baldcypress and grass (60%). Gives way to open water. 80% of area had little or A01 no canopy cover. 104m x 8m. Sparse tree canopy - slash pine, baldcypress, longleaf pine. Dense shrub/scrub riparian vegetation - gallberry, sweetbay, smilax. Grass A02 intermittent in water, dense on bank. Substrate - thick grass detritus. on Valentine Road, about 380m from intersection with Entry Road (Brown's Trail). Notes say "at Valentine Road". Sampled area 37m x 7m + 15m x 62m. Riparian vegetation along road bank primarily grasses with 4 small slash pines. Deep traps set along road bank. Pond vegetation in sampled area A04 (shallow)--grasses and a few very small trees. Pond Dimensions 43.7m x 27.8m; open site, no tree canopy. Vegetation: grasses, herbaceous veg., and small woody shrubs. Large quantity of A03 filamentous algae. 35m x 13m. Tree canopy sparse slash pine. North bank--shrubs, grass, vines. South (pond area--dense grass, a very few young trees, shrubs. Ditch A05 along road was >1m deep. Flooded fire line. Wetted dimensions 4.5m x 77.0m. Lined with overstory of young pines (about 8-10" dbh). Understory on both sides continuous A06 grasses, herbaceous veg., and shrubs. on Valentine Road, about 380m from intersection with Entry Road (Brown's Trail). Notes say "at Valentine Road". Sampled area 37m x 7m + 15m x 62m. Riparian vegetation along road bank primarily grasses with 4 small slash pines. Deep traps set along road bank. Pond vegetation in sampled area A04 (shallow)--grasses and a few very small trees. Channel on north side of Blue Hole Pond. 70m x 8m. Little canopy cover. Banks mostly grass, herbaceous, and shrubs. Very few short pines ~2m tall. A07 Sparse aquatic vegetation. Sampled area 28 x 38m. Forested swamp--cypress, slash pine, assorted water tolerant hardwoods. Numerous woody shrubs in sample area. Margins of A08 sample area recently burned. Abundant small and large woody debris in water. 19 x 39m. 3 to 4, 10" cypress trees, heavy emergent aquatic veg (grasses), cypress saplings in and around pond. Small areas of perennial water in A09 deepest part of pond (no veg). Sample area 2 x 76m. Open canopy. Grasses, herbaceous and shrubs on banks. No visible flow. Aquatic vegetation and algae present. Saw a water A10 snake.

Appendix Table C2. Number and CPUE (# per 30 net-minutes or # per 10 traps) of crayfish from aquatic sites with standardized sampling. Includes depth category for traps, effort, number of traps, nights traps were in place, and start and end times for both active and passive sampling. Procambarus clarkii (PCLA) was the only crayfish species caught except at site A05 where Cambarellus diminutus (CDIM) was caught.

Nights Start/ End/ Site Sample Effort # Total Total in set retrieve code Site name Method Depth date (min) traps PCLA CDIM Crays CPUE place time time Comments A01 Valentine Pond Dipnet 1 1/24/2017 60 21 0 21 10.50 1035 1105 All juveniles. A01 Valentine Pond Minnow Trap deep 1/24/2017 10 0 0 0 0.00 1420 800 No crayfish collected A01 Valentine Pond Minnow Trap shallow 1/24/2017 10 0 0 0 0.00 1420 800 No crayfish collected A01 Valentine Pond Seine 1/24/2017 30 0 0 0 0.00 1303 1333 No crayfish collected Ben Williams 6A A02 Savanna Fireline Dipnet 1 1/25/2017 60 33 0 33 16.50 1043 1113 All juveniles. Ben Williams 6A A02 Savanna Fireline Minnow Trap deep 1/25/2017 10 8 0 8 8.00 1500 900 All adults. At Powerline. Ben Williams 6A A02 Savanna Fireline Minnow Trap shallow 1/25/2017 10 10 0 10 10.00 1500 900 All adults. At Powerline. Ben Williams 6A A02 Savanna Fireline Seine 1/25/2017 30 84 0 84 84.00 1144 1214 All juveniles. A03 Duck Pond 1 Dipnet 1 2/7/2017 60 13 0 13 6.50 920 950 All juveniles. A03 Duck Pond 1 Minnow Trap deep 2/7/2017 10 0 0 0 0.00 1454 830 A03 Duck Pond 1 Minnow Trap shallow 2/7/2017 10 0 0 0 0.00 1454 830 A03 Duck Pond 1 Seine 2/7/2017 30 4 4 4.00 1017 1047 All juveniles. Elbert Shorebird 5 juveniles Set 20 habitat traps by mistake. A04 Pond Habitat Traps deep 1/27/2017 2 6 0 6 30.00 4 1530 720 Checked/ pulled 2 deep traps on 1/27/2017 Elbert Shorebird All juveniles. Set 20 traps initially by mistake. A04 Pond Habitat Traps shallow 1/27/2017 3 7 0 7 23.33 4 1530 720 Checked and pulled 3 shallow traps on 1/27/2017. Elbert Shorebird A04 Pond Habitat Traps deep 2/8/2017 7 26 0 26 37.14 13 1530 837 23 juveniles. Elbert Shorebird A04 Pond Habitat Traps shallow 2/8/2017 8 12 0 12 15.00 13 1530 837 11 juveniles.

Appendix Table C2 continued.

Nights Start/ End/ Site Sample Effort # Total Total in set retrieve code Site name Method Depth date (min) traps PCLA CDIM Crays CPUE place time time Comments A05 Firetower Pond Habitat Traps deep 2/8/2017 7 0 2 2 2.86 12 755 948 Both adults. A05 Firetower Pond Habitat Traps shallow 2/8/2017 8 0 1 1 1.25 12 755 948 Female with juveniles attached. West Fireline, A06 O16E Dipnet 1 2/9/2017 60 88 0 88 44.00 1000 1030 All juveniles. West Fireline, A06 O16E Minnow Trap deep 2/9/2017 10 46 0 46 46.00 1432 820 30 juveniles. West Fireline, A06 O16E Minnow Trap shallow 2/9/2017 10 23 0 23 23.00 1432 820 14 juveniles. West Fireline, A06 O16E Seine 2/9/2017 30 158 0 158 158.00 1052 1122 All juveniles. Channel N of Blue A07 Hole Habitat Traps deep 2/28/2017 7 0 0 0 0.00 23 1320 1350 No crayfish. Many freshwater shrimp. Channel N of Blue A07 Hole Habitat Traps shallow 2/28/2017 8 0 0 0 0.00 23 1320 1350 No crayfish. Many freshwater shrimp. Beasley Road A08 Drain Dipnet 1 2/28/2017 60 30 0 30 15.00 1009 1039 18 juveniles. Beasley Road A08 Drain Dipnet 2 2/28/2017 30 41 0 41 41.00 1100 1130 30 juveniles. Beasley Road A08 Drain Minnow Trap deep 2/28/2017 10 49 0 49 49.00 1435 835 All adults! All released. Beasley Road A08 Drain Minnow Trap shallow 2/28/2017 10 92 0 92 92.00 1435 835 All adults! 11 preserved. A09 Maverick Pond Habitat Traps deep 3/1/2017 7 16 0 16 22.86 24 1223 1300 8 juveniles. A09 Maverick Pond Habitat Traps shallow 3/1/2017 8 9 0 9 11.25 24 1223 1300 3 juveniles. Roadside Ditch, A10 O6SE Dipnet 1 3/1/2017 60 43 0 43 21.50 919 949 42 juveniles. Roadside Ditch, A10 O6SE Dipnet 2 3/1/2017 60 53 0 53 26.50 1038 1108 All juveniles Roadside Ditch, A10 O6SE Minnow Trap deep 3/1/2017 10 9 0 9 9.00 1310 800 1 juvenile. Roadside Ditch, A10 O6SE Minnow Trap shallow 3/1/2017 10 9 0 9 9.00 1310 800 All adults. All released.

Appendix Table C3. Site location, sample date, collectors, and effort for aquatic sites sampled with haphazard dipnetting. Date is month/year in 2017. Effort is # of collectors x sampling period in minutes.

Dipnet Site Date effort code Site name LOCATION Latitude Longitude 2017 Collectors (min.) Comments A11 Isolated pools (10) In NW Fontainebleau (F1C) 30.39736 -88.74311 1/26 ZCB 45 Small, scattered, pools in savanna. Roadside ditch and South side of Crane Lane near road to Sampled at night after big A12 ephemeral pools bunkhouse, and small pools in savanna 30.44973 -88.65648 2/7 SBA rainstorm in pines across dirt road from Martin Shallow pool - probably A13 Susie's pool Pasture, in FWS unit G11SE 30.43217 -88.64385 2/28 SBA 7 intermittent in corner of Martin Pasture G11NM A14 Angie's pool (savanna) 30.43212 -88.64423 2/28 AJD 30 Deep pool - probably perennial

A15 Isolated pools (2) in Semmes 9 (O19E) 30.47113 -88.75497 3/1 SBA 15 along West Perigal Road, west of FWS A16 Ditches, roadside unit O3W 30.48461 -88.81250 3/2 AJD 45 Open savanna Many trees, some elevational gradient. Little understory because A17 Isolated pools (11) Utah North O5S 30.46989 -88.81612 3/2 JGM 43 just burned. Probably intermittent. on both sides of Old Spanish Trail Rd. A18 Ditches, roadside near access to NW Fontainebleau 30.39563 -88.74471 3/2 SBA, MRB 20 Probably intermittent. in dirt access road to NW Fontainebleau, north of Old Spanish A19 Isolated pool Trail Rd. 30.39612 -88.74471 3/2 JGM 10 Large pool in dirt road in fire trail along SW Fontainebleau A20 Isolated pools F2N, south of Old Spanish Trail Rd 30.39410 -88.74884 3/2 VMV 45 Wide, dirt fire lane, flooded SBA, JGM, A21 Ditch, roadside along S side of US Hwy 90 30.39535 -88.67551 3/3 MRB ? All crayfish released. along north side of US Hwy 90 at SBA, JGM, A22 Ditches, roadside Audubon Lane 30.39860 -88.69395 3/3 MRB 30 Most crayfish released. on south (downstream) side of US SBA, JGM, A23 Unnamed stream Hwy 90 crossing. 30.39748 -88.68945 3/3 MRB 30? A24 Unnamed stream on south side of US Hwy 90 crossing 30.39508 -88.67395 3/3 SBA. JGM ?

Appendix Table C4. Fish CPUE (#/30 net-minutes or #/10 traps) by site, method and species. Fish species abbreviations as in Table 3.

Site Sample All code date Site name Method Depth ASAY EAME EFUS ETEN EZON FNOT GAFF LGUL LMAC LMAR fish

A01 1/24/2017 Valentine Pond dipnet 1 0.0 1.0 0.0 0.0 1.0 0.5 0.5 0.0 0.0 0.0 3.0 A01 1/24/2017 Valentine Pond minnow trap deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 1.0 A01 1/24/2017 Valentine Pond Minnow Trap shallow 0.0 0.0 0.0 1.0 0.0 4.0 0.0 0.0 0.0 2.0 7.0 A01 1/24/2017 Valentine Pond Seine 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 1.0 Ben Williams 6A Savanna A02 1/25/2017 Fireline dipnet 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.5 Ben Williams 6A Savanna A02 1/25/2017 Fireline Minnow Trap deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.0 6.0 Ben Williams 6A Savanna A02 1/25/2017 Fireline Minnow Trap shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 2.0 Ben Williams 6A Savanna A02 1/25/2017 Fireline Seine 0.0 7.0 0.0 0.0 1.0 0.0 10.0 0.0 0.0 2.0 20.0 A03 2/7/2017 Duck Pond 1 dipnet 1 0.0 1.5 0.0 0.0 2.0 1.5 3.0 0.0 0.0 0.0 8.0 A03 2/7/2017 Duck Pond 1 Minnow Trap deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 A03 2/7/2017 Duck Pond 1 Minnow Trap shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.0 6.0 A03 2/7/2017 Duck Pond 1 Seine 0.0 0.0 4.0 0.0 6.0 3.0 7.0 1.0 0.0 15.0 36.0 A04 1/27/2017 Elbert Shorebird Pond Habitat Traps deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A04 1/27/2017 Elbert Shorebird Pond Habitat Traps shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A04 2/8/2017 Elbert Shorebird Pond Habitat Traps deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A04 2/8/2017 Elbert Shorebird Pond Habitat Traps shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A05 2/8/2017 Firetower Pond Habitat Traps deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A05 2/8/2017 Firetower Pond Habitat Traps shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A06 2/9/2017 West Fireline, O16E dipnet 1 0.0 0.0 0.0 0.0 17.5 0.0 0.0 0.0 0.0 0.0 17.5 A06 2/9/2017 West Fireline, O16E Minnow Trap deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A06 2/9/2017 West Fireline, O16E Minnow Trap shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A06 2/9/2017 West Fireline, O16E Seine 0.0 0.0 0.0 0.0 20.0 0.0 0.0 0.0 0.0 0.0 20.0

Appendix Table C4 continued. Site Sample All code date Site name Method Depth ASAY EAME EFUS ETEN EZON FNOT GAFF LGUL LMAC LMAR fish A07 2/28/2017 Channel N of Blue Hole Habitat Traps deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.7 0.0 0.0 5.7 A07 2/28/2017 Channel N of Blue Hole Habitat Traps shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A08 2/28/2017 Beasley Road Drain dipnet 1 2.5 0.5 0.0 0.0 0.0 0.0 2.0 0.0 0.0 0.0 5.0 A08 2/28/2017 Beasley Road Drain dipnet 2 3.0 1.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 5.0 A08 2/28/2017 Beasley Road Drain Minnow Trap deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A08 2/28/2017 Beasley Road Drain Minnow Trap shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A09 3/1/2017 Maverick Pond Habitat Traps deep 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A09 3/1/2017 Maverick Pond Habitat Traps shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 A10 3/1/2017 Roadside Ditch, O6SE dipnet 1 0.0 3.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 4.0 A10 3/1/2017 Roadside Ditch, O6SE dipnet 2 2.0 5.5 0.0 0.0 0.0 0.0 1.5 0.0 0.5 0.0 9.5 A10 3/1/2017 Roadside Ditch, O6SE Minnow Trap deep 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 A10 3/1/2017 Roadside Ditch, O6SE Minnow Trap shallow 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 0.0 3.0