14 September 2018

Mr. Ricky Ingram Refuge Manager Wheeler National Wildlife Refuge 2700 Refuge HQ Road Decatur, AL 35603

Dear Mr. Ingram:

On 25 August 2018, Tommy Inebnit (USFWS) and I led two teams to conduct biological inventories in two sections of Fern Cave (Alabama Cave Survey no. JK597), as part of a two-year project on the fauna of the cave system. Here, I report on the discovery and genetic assessment of a new population at Fern Cave of Alabama Cave Shrimp ( alabamae), a federally endangered known from just four cave systems in Madison County, Alabama, previously.

My team, consisting of Joe Lamb (graduate student at Alabama A & M University), Bradley Jones (Birmingham Grotto), Nathaniel Mann (Nashville Grotto), and myself, entered the Don Davidson Entrance (E7 or Sump Entrance) around 11:45 am. After wading the large pool and water-filled passage at the entrance for ca. 40m, we climbed onto a mudbank leading to main stream passage that can be followed for several hundred meters to the sump. During much of the year, this section of the cave is inaccessible due to high water. However, water levels were quite low during our visit in the first 200m of the passage in “Bottom Cave,” limited to just three small, isolated pools between the large pool at the entrance and the main stream passage.

The first isolated pool measured approximately 5m long, 2m at its widest point, and water depth up to 25 cm, with mud/silt substrate and a couple larger rocks. We encountered a considerable diversity of aquatic life in this pool, including Southern Cave (Orconectes australis), Southern Cavefish (Typhlichthys subterraneus), Cavespring Crayfish (Cambarus tenebrosus), Banded Sculpin (Cottus carolinae), and a large cave shrimp (Palaemonias sp.). The shrimp was observed at the edge of the pool in ca. 4 cm of water. Although shallow, visibility throughout much of the pool was poor because of silt suspended in the water column. This specimen was captured and retained per instructions from Tommy Inebnit to confirm identification. We then surveyed the remainder of aquatic habitat in this section of the cave until passage was halted by the sump. On our exit of the cave, we stopped and searched again

Department of Biological Sciences, 320 Sparkman Drive, Huntsville, Alabama 35899 Phone: 256-824-3077| Cell: 615-427-3049|E-mail: [email protected] http://www.speleobiology.com/niemiller

at this pool and observed three additional cave shrimp ca. 12–18 mm total length (from the rostrum to end of the uropod). These shrimp were only photographed and not captured.

The retained specimen was photographed then preserved in 100% ethanol for morphological and genetic analyses to determine species identification. Fern Cave occurs in the Paint Rock River watershed ca. 10 km to the NE of the nearest historical Alabama Cave Shrimp locality, Hering Cave (ACS. no. MD6) on the southwestern side of Keel Mountain in the Flint River watershed. Because of the complex hydrogeological history of karst in the southwestern Cumberland Plateau of northern Alabama and high levels of cryptic species diversity in other stygobiotic (aquatic, cave-obligate) fauna, such as cavefishes and cave , the potential exists for the Fern Cave population to represent an undescribed species, such as the populations of Palaemonias in caves to the west along the Tennessee River in Colbert and Lauderdale counties.

My lab extracted DNA from three pleopods using a Qiagen® DNEasy Blood & Tissue Extraction Kit. A 539-bp section the mitochondrial 16S ribosomal DNA locus was amplified using primers 16Sar and 16Sbr. This locus exhibits significant genetic variation and has been population in phylogenetic and population genetic studies of . In addition, sequences of this locus are available on GenBank for P. alabamae and the undescribed species in Colbert and Lauderdale counties for comparison (GenBank accession nos. FN995378–FN995383). Results of PCR were visualized via gel electrophoresis and then sent to Eurofins, Inc. (Louisville, Kentucky) for sequencing. A maximum-likelihood phylogenetic was conducted in RAxML.

Morphological examination firmly places the Fern Cave specimen in the genus Palaemonias, as Palaemonias cf. alabamae. This specimen measured 29.9mm in total length (tip of rostrum to end of uropod) and may be the largest P. alabamae reported. Genetic results confirmed that the Fern Cave sample was closely related to other populations of P. alabamae genetically sampled (Hering and Bobcat caves), with 0.4–0.6% uncorrected sequence divergence between Fern Cave and these other two populations.

Based on these results, it is my determination that cave shrimp at Fern Cave represent a new population of Palaemonias alabamae. This species is known historically from just four cave systems in Madison County: Shelta Cave (type locality), Bobcat Cave, Muddy Cave, and the Hering-Glover-Brazelton cave system. This new record extends the range of the species to the east into a new watershed and into Jackson County. In addition, this new discovery suggests that this may be found in other cave systems in the Paint Rock watershed and perhaps further east along the Tennessee River.

Please do not hesitate to contact me with any additional questions you might have regarding this genetic assessment of the new population of cave shrimp at Fern Cave. The specimen is still

Department of Biological Sciences, 320 Sparkman Drive, Huntsville, Alabama 35899 Phone: 256-824-3077| Cell: 615-427-3049|E-mail: [email protected] http://www.speleobiology.com/niemiller

currently in my possession and will be accessioned into a permanent museum collection per recommendation from USFWS, such as the Auburn Museum of Natural History.

Sincerely,

Dr. Matthew L. Niemiller Assistant Professor The University of Alabama in Huntsville Department of Biological Sciences 301 Sparkman Dr NW Huntsville, AL 35899

CC: Rob Hurt William Gates Emory Hoyle Jennifer Grunewald Jeff Powell Eric Spadgenske Thomas Inebnit Robert Handford Steve Pitts

Department of Biological Sciences, 320 Sparkman Drive, Huntsville, Alabama 35899 Phone: 256-824-3077| Cell: 615-427-3049|E-mail: [email protected] http://www.speleobiology.com/niemiller

Figure 1. Alabama Cave Shrimp (Palaemonias alabamae) from Fern Cave, Jackson Co., Alabama.

Figure 2. Joe Lamb and Bradley Jones searching for cave shrimp in an isolated pool ca. 100m from the “Sump” Entrance to Fern Cave on 25 August 2018.

Department of Biological Sciences, 320 Sparkman Drive, Huntsville, Alabama 35899 Phone: 256-824-3077| Cell: 615-427-3049|E-mail: [email protected] http://www.speleobiology.com/niemiller

Figure 3. Maximum-likelihood phylogram of 539-bp of the mitochondrial 16S locus for Palaemonias cave shrimp in northern Alabama. The Fern Cave sample is highlighted in red.

Department of Biological Sciences, 320 Sparkman Drive, Huntsville, Alabama 35899 Phone: 256-824-3077| Cell: 615-427-3049|E-mail: [email protected] http://www.speleobiology.com/niemiller