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
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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 TE 795930 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. Fifty uL of alkaline lysis buffer (NaOH 25mM, disodium EDTA0.2mM, pH 8.0) was added to each tube. The decapsulated cysts (embryos) were crushed using a
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 6
sterile pipette tip. The tubes, containing crushed embryos, were incubated for 30 minutes at 95o C and then placed on ice for 5 minutes. After chilling, 50 uL of neutralizing solution (Tris HCl 40mM, pH 5.0) was added to the tubes. The tubes were then vortexed and contents spun down. Approximately 2 5 uL of the contents of each tube was extracted and used in subsequent polymerase chain reactions (PCRs).
A 680 base pair fragment of the mitochondrial cytochrome oxidase I (COI) gene using universal primers was amplified (Folmer et al. 1994). Each PCR reaction contained 5 l of DNA, 1x Amplitaq PCR buffer (Applied Biosystem Inc.), 2.5 mM MgCl2, 0.1 mMdNTPS, 0.3 pmoles of each primer and 0.5 units of Amplitaq (ABI) in a 30 l reaction. Thermocycling was performed on an Applied Biosystems 2720 cycler under the following temperature profile: initial denaturation for 3 min at 94o C, 30 cycles at 94o C for 30 sec, 52o C for 45 sec and 72o C for 30 sec, followed by a 5 min extension at 72o C. PCR products were cleaned with the Qiaquick PCR Purification kit (QiagenInc) and sequenced on an ABI3730 automated sequencer.
The PCR approach of Vandergast et al. (2009) was not utilized because this approach can only discriminate three species (B. lindahli, B. lynchi, and B. sandiegonensis). In addition, Vandergast et al. (2009) assay does not account for the amount of sequence divergence that is found in B. lindahli (Aguilar, unpubl. data).
Amplification products that were sequenced in both directions were aligned to an existing database of Branchinecta sequences that have been generated in the laboratory. This includes the following species: B. campestris, B. coloradensis, B. conservatio, B. gigas, B. hiberna, B. lindahli, B. lynchi, B. mackini, B. mesovallensis, and B. sandiegonensis. Linderiella occidentalis was used as an out group. Aligned sequences were used to reconstruct a Maximum Likelihood tree using the Kimura 2 parameter (K2P) model with 1000 bootstrap replicates with the program PhyML (Guindon and Gascuel 2003).
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 7
RESULTS
A total of 57 wetland habitats occurring within the BSA were evaluated for their potential to support federally listed large branchiopods (Figure 2). Twelve of these wetlands were not considered large branchiopod habitat (W9, W10A, W34, W48, W62, W63, W92, W123, W126, W136, W137, and W146). Dry season sampling was conducted in the remaining 45 wetlands.
Soil Analysis
Soils collected from the basins of 45 seasonally inundated wetlands were examined for the presence of federally listed large branchiopods. Cysts belonging to the genus Branchinecta were observed in soils collected from six wetlands (W 32, W 41, W 42, W 65, W 66, and W 128) (Table 1).
Genetic Analysis
Of the original 192 cysts provided by HBC for genetic analysis, DNA was isolated from 83 viable cysts, with 81 successful PCR reactions and 73 usable sequences (Table 2). The versatile fairy shrimp (B. lindahli) was identified from cysts obtained from three wetlands (W 41, W 65, and W 66). The vernal pool fairy shrimp (Branchinecta lynchi) was identified from cysts obtained from three wetlands (W 32, W 42, and W 66).
Representative photographs of the wetlands sampled are in Appendix B.
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 8
Table 1. Results of Soil Examinations Micro- Ostracods Insect Exo- Turbellaria Cladocera Live/Cysts/C Branchinecta Hydracarina Basin No. Skeletons Cysts Ephippia arapaces sp. Cysts Live Nematoda Collembola ES-9 X X X P-1 X Pd-2 X X W-1A X X X W-3 X X W-5A X X W-6A X X X W-7A X X W-9A X X W-14A X X W-15A X X X W-20 X W-20A X X X X W-29 X X X W-29A W-30 X X W-31 X W-32 X 3 (2) X W-33 X X W-36 X W-38 X X X W-39 X X X X W-40 X X X W-41 12 (29) W-42 X 34 (>100) X W-59 X X W-60 X W-61 X X W-64 X W-65 X 6 (18) W-66 X 26 (36) W-67 X X W-68 X X X W-72 X X W-90 X X W-91 X X W-110 X W-114 X X W-115 X X W-118 X X W-127 X X X X W-128 X 1 broken (0) X W-129 X X W-130 X X X W-131 X X X ( ) = the number of cysts obtained from additional soil collections.
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 9
Table 2. Results of Genetic Analysis Number of Cysts Viable/ Basin Provided Extracted Successful Successful Branchinecta Branchinecta Number By HBC Isolated DNA PCR Sequence lindahli lynchi W-32 5 5 5 5 4 4 W-41 41 41 10 10 9 9 W-42 60 60 26 26 24 24 W-65 24 24 16 16 16 16 W-66 62 61 26 24 20 17 3 Total 192 191 83 81 73 42 31
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 10
REFERENCES
Literature Cited
Belk, D. 1989. Identification of species in the Conchostraca genus Eulimnadia by egg shell morphology. Journal of Crustacean Biology. 9(1): 115 125.
Brendock, L., D. C. Rogers, J. Olesen, S. Weeks, and W. R. Hoch. 2008. Global diversity of large branchiopods (Crustacea: Branchiopoda) in freshwater. Hydrobiologia. 595: 167 176.
Eriksen, C. H., and D. Belk. 1999. Fairy shrimps of California’s puddles, pools, and playas. Mad River Press, Inc. Eureka, CA. 196 pp.
Folmer, O., M. Black, W. Hoeh, R. Lutz, R. Vrijenhoek.1994 DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology & Biotechnology 3: 294 299.
Gilchrist, B. M. 1978. Scanning electron microscope studies of the egg shell in some Anostraca (Crustacea: Branchiopoda). Cell Tiss. Res. 193: 337 351.
Guindon, S., and O. Gascuel. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: 696 704.
Helm, B. P. 1998. Biogeography of eight large branchiopods endemic to California. Pages 124 139 in Witham, C. W., E. T. Bauder, D. Belk, W.R. Ferren Jr., and R. Ornduff. (eds.). Ecology, conservation, and management of vernal pool ecosystems – proceeding from a 1996 conference. California Native Plant Society, Sacramento, CA. 285 pp.
Helm, B. P. 1999. Feeding ecology of Linderiella occidentalis (Dodds) (Crustacea: Anostraca). Doctoral thesis. University of California, Davis. 158 pp.
Helm, B. P., and J. E. Vollmar. 2002. Vernal pool large brachiopods. Pages 151 190 in John E. Vollmar (ed.). Wildlife and rare plant ecology of eastern Merced County’s vernal pool grasslands. Sentinel Printers, Inc. CA. 446 pp.
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 11
Hill, R. E., and W. D. Shepard. 1998. Observation on the identification of California anostracan cysts. Hydrobiologia 359: 113 123.
Montero Pau, J., A. Gomez, and J. Munoz. 2008. Application of an inexpensive and high throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs. Limnol.Oceanogr.: Methods 6: 218–222
Mura, G. 1991. SEM morphology of resting eggs in the species of the genus Branchinecta from North America. J. Crust. Biol. 11: 432 436.
Rabet, N. 2010. Revision of the egg morphology of Eulimnadia (Crustacea, Branchiopoda, Spinicaudata). Zoosystema 32 (3): 373 391.
U. S. Fish and Wildlife Service. 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. 11 pp.Vandergast, A. G., D. A. Wood, M. Simovich, and A. J. Bohonak. 2009. Molecular identification of co occurring Branchinecta fairy shrimp species from encysted embryos using multiplex polymerase chain reaction. Mol. Ecol. Res. 9: 767 770.
Personnel Communications
Cooper, Douglas . 2011. Email from Mr. Douglass Cooper of the USFWS on 17 November 2011, authorizing HTH to conduct wet season surveys within the BSA.
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 12
APPENDIX A. USFWS AUTHORIZATION LETTER
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 13
81440-2008-B-0025 (Helm) 08EVEN00-2012-B-0260 (California Flats Solar Project)
Dr. Hardwicke. This electronic correspondence serves as the Ventura Fish and Wildlife Office's authorization for Brent Helm to conduct dry season sampling for listed vernal pool branchiopod cysts at the California Flats Solar Project site located in southeastern Monterey County, California. Sampling will be conducted in those features identified on Figures 1 and 2 (attached) received in this office on October 22, 2012, as well as any other feature that are deemed to be potential suitable habitat for listed vernal pool branchiopod species. Dr. Helm will subsequently identify the cysts to the genus level in accordance with our 1996 interim survey guidelines and the conditions of his recovery permit (TE-795930).
During the course of the wet season surveys conducted at this project site, no large vernal pool branchiopod species were identified. You did note, in an electronic correspondence to Douglass Cooper dated August 20, 2012, that rainfall totals this year were estimated to be only 54 percent of average rainfall for the area. For this reason, as we are doing with other project surveys conducted in areas where less than 70 percent of average rainfall was observed, the Service will not be accepting negative results of wet season surveys for this season.
Soil samples from the identified features may only be collected when the soil is dry to the touch both at the surface and one inch below. Extra care must be taken to ensure that soil samples are collected and stored in such a way that the ability to detect and identify any cysts that may be present is not compromised.
If you or Dr. Helm have any questions, feel free to contact me. We look forward to the results of this survey effort.
Regards,
Julie M. Vanderwier, Senior Fish & Wildlife Biologist U.S. Fish & Wildlife Service, Ventura Fish & Wildlife Office 2493 Portola Road, Suite B Ventura, California 93003 desk: 805.644.1766, ext. 222 facsimile: 805.644.3958 everything is connected to everything else. everything must go somewhere. nature knows best. there is no such thing as a free lunch. -- barry commoner
(See attached file: Fig 1 Proposed Dry Season Sample Locations.pdf) (See attached file: Fig 2 Proposed Dry Season Sample Locations.pdf)
APPENDIX B. REPRESENTATIVE PHOTOGRAPHS
Dry-season Sampling and Genetic Analysis Ph: (530) 633-0220 California Flats Solar Project Fax: (530) 633-0230 14
California Flats Solar Project – Basin W31.
California Flats Solar Project – W48. Not large branchiopod habitat. No basin. Occurs on side of gentle slope.
California Flats Solar Project – Basin W131.
California Flats Solar Project – W92. Not large branchiopod habitat. No depression. Occurs on side slope of swale.
California Flats Solar Project – Basin W64. Large wetland within bottom of drainage.
California Flats Solar Project – W41. Small stock pond created by the placement of earthen fill for road crossing within a natural drainage. Branchinecta sp. cysts were observed within soils collected from this basin and genetically analyzed as belonging to Branchinecta lindahli.
California Flats Solar Project – P1. Pond created by the placement of earthen fill within a natural swale system.
California Flats Solar Project – W30.
California Flats Solar Project – W39. Plunge pool within drainage.
California Flats Solar Project – W123. Not large branchiopod habitat. No depression, perennial seep on steep slope of hill.
California Flats Solar Project – W126. Not large branchiopod habitat. No depression - steep drainage.
California Flats Solar Project – W128. A single broken Branchinecta sp. cyst was observed within soils collected from this basin.
California Flats Solar Project – Basin W3. Drainage outfall from culvert.