Appendix E.10 Dry Season Sampling DRY SEASON SAMPLING, INCLUDING GENETIC ANALYSIS OF CYSTS FOR FEDERALLY LISTED LARGE BRANCHIOPODS AT THE CALIFORNIA FLATS SOLAR PROJECT Prepared for: H.T. HARVEY & ASSOCIATES 983 University Avenue, Building D Los Gatos, CA 95032 Contact: Kelly Hardwicke (408) 458-3236 Prepared by: HELM BIOLOGICAL CONSULTING 4600 Karchner Road Sheridan, CA 95681 Contact: Brent Helm (530) 633-0220 September 2013 DRY SEASON SAMPLING, INCLUDING GENETIC ANALYSIS OF CYSTS FOR FEDERALLY LISTED LARGE BRANCHIOPODS AT THE CALIFORNIA FLATS SOLAR PROJECT INTRODUCTION Helm Biological Consulting (HBC) was contracted by H. T. Harvey & Associates (HTH) to conduct dry-season sampling for the presence of large branchiopods (fairy shrimp, tadpole shrimp, and clam shrimp) that are listed as threatened or endangered under the federal Endangered Species Act (e.g., vernal pool fairy shrimp [Branchinecta lynchi] and longhorn fairy shrimp [Branchinecta longiantenna]) at the California Flats Solar Project (aka “Project”). The contract also included the genetic analysis of any fairy shrimp or tadpole shrimp cysts (embryonic eggs) observed to determine species. The proposed Project consists of approximately 2,615.3 acres (ac) and includes the construction and operation of a 280-megawatt alternating current photovoltaic solar power facility. However, surveys for listed large branchiopods were conducted on a much larger area (i.e., 4,653.45-ac) designated as the “Biological Study Area” (BSA) for the Project (Figure 1), as per direction from U.S. Fish and Wildlife Service (USFWS, Douglas pers. comm. 2011). The BSA is located in southeastern unincorporated Monterey County, California, with an access road to Highway 41 that extends south into northern San Luis Obispo County (Figure 1). More specifically, the BSA is located along the eastern rim of the Cholame Valley and occurs on three U.S. Geological Survey (USGS) 7.5-minute quadrangle maps: The Dark Hole, Cholame Valley, and Cholame (Figure 2). Elevations range from 1,180 feet above mean sea level (msl) at the southern end of the access road where it intersects with Highway 41, to approximately 1,860 feet above msl along the northwest edge of the BSA. Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 1 This report discusses the methods and results of dry-season sampling at the California Flats Solar Project and genetic analysis of large branchiopod cysts obtained from this sampling to determine the presence, or presumed absence, of federally listed large branchiopods. “We certify that the information in this survey report and attached exhibits fully and accurately represent our work.” Brent P. Helm Signature _______________________________ Date 9-6-2013 Kelly Hardwicke Signature _______________________________ Date 9-6-2013 Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 2 MERCED ^_ FresnoDetail ·|}þ99 ·|}þ33 ·|}þ269 California SA N BE N I TO FRESNO ·|þ}269 ·|}þ145 Hanford ·|}þ25 ^_ ·|}þ198 ·|}þ41 ·|}þ198 KINGS MONTEREY 5 ¤£101 !#$" Project Vicinity ·|}þ33 ·|}þ46 33 229 ·|þ} 46 ·|}þ ·|}þ KERN ·|}þ58 San Luis Obispo ^_ SAN LUIS OBISPO ·|}þ1 0 ± 20 166 |}þ135 ·|}þ Miles · SANTA BARBARA Biological Study Area k e e r C C d h o o la o m w n e o t Cre t o C e k 3 1.5 ·|þ}46 0 3 Sources: Esri, DeLorme, NAVTEQ, USGS, Intermap, iPC, NRCAN, Esri ± Miles Japan, METI, Esri China (Hong Kong), Esri (Thailand), TomTom, 2013 J:\Reports\BotanyReports\Branchiopod Surveys\2012 Dry SeasonBranchiopod Survey Report\Fig 1 Vicinity Map.mxd Figure 1: Vicinity Map California Flats Solar Project, 2012 Dry Season Branchiopod Survey Report (3308-02) September 2013 P1 LEGEND Biological Study Area Waterbodies Sampled for Branchiopod Cysts W136 Pools with Cysts W146 Pools Found to not Provide Habitat W137 for Large Branchiopods Special Aquatic Sites/ Wetlands W3 ES9 MAIN RANCH RD Other Waters Ephemeral Stream W29 W20A Intermittent Stream W20 W30 W29A W31 Perennial Stream Culvert Vernal pool Vernal pool fairy shrimp cysts fairy shrimp cysts W48 W32 2,200 1,100 0 2,200 W42 ± Feet Versatile fairy shrimp cysts W33 W34 W62 W63 W36 W65 W64 W9 W38 W66 W67 W39 Vernal pool fairy shrimp and versatile fairy shrimp cysts W40 W68 W1A Versatile fairy shrimp cysts W72 W41 W131 W130 W90 W7A W61 W91 W5A W9A W6A W59 W60 W92 W10A W14A W15A W10A Matchline Sheet 1 W129 k e e r C d o o w n t o C o t W118 W115 W114 W110 PD2 W123 W126 W127 W128 Pool with 1 unidentified cyst collected Copyright:© 2013 National Geographic Society, i-cubed Matchline Sheet 2 Copyright:© 2013 National Geographic Society, i-cubed J:\Reports\Botany Reports\Branchiopod Surveys\2012Dry Season Branchiopod Survey Report\Fig 2 ProposedDry Season SampleLocations.mxd Figure 2: Survey Areas and Potentially Suitable Branchiopod Habitat California Flats Solar Project, 2012 Dry Season Branchiopod Survey Report (3308-02) September 2013 METHODS Dr. Brent Helm of HBC and Dr. Kelly Hardwicke of HTH conducted dry-season sampling on November 15, 2013 as authorized by the USFWS (Appendix A). Sampling was conducted under permits TE795930-8 and TE-797267-16 of Section 10(a)(1)(A) of the federal Endangered Species Act, 16 U.S.C. 1531 et seq., and its implementing regulations. Methods generally followed USFWS’s (1996) Interim Survey Guidelines to Permittees for Recovery Permits under Section 10(a)(1)(A) of the Endangered Species Act for the Listed Vernal Pool Branchiopods and are described below. All areas that potentially could support federally-listed large branchiopods were sampled. Potential habitat for federally-listed large branchiopods is defined as any seasonal inundated depression that on average ponds water 2.0 inches or greater in depth for 14 or more consecutive days for fairy shrimp and 30 or more consecutive days for tadpole shrimp. Potential habitat characteristics of large branchiopods are based on the life history of Central Valley endemics (Eriksen and Belk 1999; Helm 1998, 1999; Helm and Vollmar 2002). The presence of water marks, algae mats, driftlines, hydrophytic vegetation (“water- loving plants”), slope, contributing watershed, maximum potential ponding depth, and aquatic arthropods (i.e., crustaceans and insects) exoskeletons were helpful indicators for evidence of ponding depth and duration. Habitats that swiftly flow water (e.g., creeks, streams, and ephemeral drainages) or semi-to-permanently inundated areas that support population of predators (e.g. bullfrogs, fish, and crayfish) were not generally considered suitable habitat for federally-listed large branchiopods. Dry-season sampling involved the collection of a minimum of ten-soil sub-samples mainly from the lowest topographic areas within each wetland considered potential habitat onsite. Soil samples were placed in liter-size plastic sealable bags and marked with the project name, basin number, and date. The soil was then transported to HBC’s laboratory for processing and analysis. Soil Analysis In the laboratory, a brine solution was prepared by mixing table salt (NaCl) with lukewarm tap water in a large container. The collected soil material was placed in the brine solution. The soil material was then gently worked by hand to breakdown any persistent soil structure. The organic material rising to the top of the brine solution was skimmed off and placed in a 600-micron diameter pore-size sieve stacked atop a 75-micron diameter pore- Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 5 size sieve. The soil material was processed through the top sieve by flushing it with lukewarm tap water while gently rubbing it with a soft-bristle brush. The soil retained from the 75-micron diameter pore size sieve was then removed and thinly (≈1.0 mm) spread into plastic petri dishes. The contents of each petri dish were examined under a 10 to 252-power zoom binocular microscope. A minimum of 0.5-hour was spent searching the contents of each petri dish for large branchiopod cysts (embryonic eggs). Dr. Helm’s large branchiopod cyst reference collection and scanning electron micrographs of cysts (Rabet 2010, Brendock et al. 2008, Hill and Shepard 1998, Mura 1991, Belk 1989, and Gilchrist 1978) were used to identify and compare any cysts observed within the soil samples. There are several species of fairy shrimp within the genus Branchinecta that could occur or are known to occur within the vicinity of the BSA. Hence, any cysts belonging to the genus Branchinecta that was observed within the soils collected onsite were carefully removed from the petri dishes and placed in microcentrifuge tubes. Each tube containing cysts was marked with the wetland number and the number of cysts it contained. Additional soils were collected from W128, W41, W66, W65, W42, and W32 by Kelly Hardwicke on 18 December 2012. Genetic Analysis Vials containing cysts were transported to Dr. Andres Aguilar of the California State University of Los Angeles. In Dr. Aguilar’s laboratory, individual cysts were inspected under a stereo microscope for capsule integrity. Those cysts that were intact were used in DNA isolation steps as follows. Cysts were placed in 1 mL of household bleach (in groups of 10) and incubated at room temperature on a horizontal shaker for 15 minutes. This initial ‘washing’ in bleach decontaminates each cyst from foreign DNA and helps to degrade the tertiary capsule. Following this step, cysts were isolated on filter paper and washed twice in Wash Buffer. Individual cleaned decapsulated cysts were placed in sterile individual 1.5 mL microcentrifuge tubes for DNA extraction. DNA extraction followed the HotShot method (Montero-Jau 2008). To reduce the risk of contamination all extractions were preformed in a physically isolated room under a positive flow hood.