HISTORICAL AFFINITIES AND POPULATION BIOLOGY OF THE BLACK WARRIOR WATERDOG (NECTURUS ALABAMENSIS) FINAL REPORT FY 1998 FEB - 4 1999 Craig Guyer Department of Zoology and Wildlife Science Auburn University Auburn, AL 36849 (334)-844-9232 [email protected] SUMMARY 1) The Black Warrior waterdog is morphologically and genetically distinctive from other waterdogs in the state of Alabama and should be recognized as Necturus alabamensis. 2) The Black Warrior waterdog is most closely related to the mudpuppy, Necturus maculosus. 3) Four waterdogs are present in the state of Alabama, the two listed above, plus two forms from the Coastal Plains; the latter include Necturus beyeri (all rivers draining into Mobile Bay) and Necturus iodingi (Appalachicola to Perdido drainages, inclusive). 4) Populations of Black Warrior waterdogs in Sipsey Fork and Brushy Creek appear to be patchily distributed, creating challenges for determining key features of demography. 5) State and Federal protection of the Black Warrior waterdog as a threatened species is warranted. INTRODUCTION Waterdogs (Necturus: Proteidae) are paedomorphic, stream-dwelling salamanders of the Atlantic and Gulf Coastal Plains. The systematics of these creatures has challenged herpetologists for the past 60 years. The Black Warrior Waterdog, a species restricted to the upper Black Warrior drainages of Alabama, has been particularly problematic. Viosca (1937) originally described this taxon as being similar toN maculosus, but subsequent taxonomic treattnents considered specimens from this drainage to be conspecific with waterdogs from the lower portions of the Mobile drainages (N maculosus: Bishop 1943, Schmidt 1953; N beyeri alabamensis: Hecht 1958, Conant 1958; N puncta/us: Brode 1969; N beyeri: Mount 1975; N alabamensis: Conant 1975, Conant and Collins 1998). Attempts to reconstruct the phylogenetic history of Necturus have failed to provide resolution to this controversy; data from albumin immunology do not resolve relationships offorms from the Gulf Coast (Maxson et al. 1988) and protein electorphoresis suggests that the Black Warrior waterdog are genetically distinctive and more similar to waterdogs from the Chattahoochee (GA) and Econfina (FL) rivers than to other members of Necturus (Gnttman et al. 1990). Further confounding this picture is an assnmption, perpetuated by treattnents of waterdogs in field guides (e.g. Conant !975), that the specimen described by Viosca (1937) was from the lower Coastal Plains and that the specific epithet alabamensis is associated with animals distributed in such streams 1 from the Mobile to the Appalachicola Rivers. For this reason, Ashton and Peavy (1986) considered the Black Warrior waterdog to be an undescribed taxon. However, Bart et al. (1997), building on arguments first presented by Neill (1963), demonstrated that the Viosca specimen (1937) was a Black Warrior waterdog and that the specific epithet alabamensis must be restricted to this taxon. This taxonomic decision left the remaining waterdogs from the lower Coastal Plains without a valid scientific name and Bart eta!. (1997) recommended these specimens be referred to as N sp. cf. beyeri until sufficient data were available to determine the number of distinct taxonomic units represented by these animals. The Black Warrior waterdog is extremely rare, being known from 10 localities, only six of which were known to contain specimens during intensive survey work in 1992, 1994, and 1996-97 (Guyer 1997). Sipsey Fork appears to be the only site that has a population of sufficient size to serve as a reliable source of much-needed demographic data, but this site has a declining population of another taxon restricted to the upper Black Warrior River (Sternotherus depressus; Bailey and Guyer 1998), perhaps indicating that habitat quality is deteriorating at the best remaining locality for the Black Warrior waterdog. Clearly, this taxon deserves consideration for state or federal listing for conservation protection. However, the systematic quagmire characterizing the genus Necturus creates serious problems for such listing efforts. Because the currently recognized scientific name for the Black Warrior waterdog is N. a/abamensis and because most field guides and state lists consider this scientific name to be associated with a much more widespread form in tl1e lower Coastal Plains, clarification of the systematics of Necturus is needed. Two specific decisions are required: I) a valid name (or names) for forms from the lower Gulf Coastal Plains of Alabama, Florida, and Georgia and 2) documentation of systematic affinities oftl1e Black Warrior waterdog within Necturus. The former will allow formation of a stable taxonomy for all currently recognized species, thereby simplifying the listing process; the latter will inclicate the species of Neclurus from which the most appropriate comparative data can be generated for conservation efforts associated with N alabamensis, the Black Warrior waterdog. In addition to these systematic problems, the popnlation biology of the Black Warrior waterdog requires examination to determine key environmental variables associated with critical waterdog habitat and long-term population trends. OBJECTIVES 1) To determine the systematic relationships of all putative species of the genus Necturus within Alabama. 2) To mark and recapture adult waterdogs in Sipsey Fork and Brushy Creek. METHODS Eighteen live specimens of Necturus, collected from 11 localities and representing all known and suspected species within Alabama, served as the ingroup and one live Siren served as the outgroup (Table I). These animals were dipnetted from leaf packs in streams and either were preserved in buffer or returned to the lab where they were killed by emersion in chloretone (all protocol approved by Auburn University IACUC; PRN 9812-R-0835). Fresh or buffer-preserved tissues (a rear leg oflarge specimens; the tail of small specimens) were excised, frozen, and used for extraction of DNA. Isolation of total genomic DNA was performed with QIAamp tissue kits (QIAGEN Inc., 28159 Stanford Ave., Santa Clarita CA 91355-1106). Then a double-stranded, 840 base pair section of the cytochrome b gene was amplified via the polymerase chain reaction (PCR) as suggested by Saiki et al. (1988) followed by amplification of single-stranded DNA via the protocol of Allard et a!. (1990). Sequencing of this gene was performed with the dideo>.-y-chain-termination method of Sanger eta!. (1977) and was done in two segments corresponding to MVZ 15 and MVZ 16 primers (Moritz eta!. 1992). Sequencing occurred in both directions assisted by Taq polymerase in a thermal cycler. Denaturation was performed at 94 °C, primer annealing at 55 °C, and primer extension at 72 °C, each for one minute and replicated for 34 cycles. PCR product was purified using QIAquick kits (QIAGEN, Inc.) and analyzed on a Perkin Elmer 373A automated sequencer operated by the Nucleic Acid Facility of the Scott-Ritchey Research Center at 2 the Auburn University School of Veterinary Medicine. Primers MVZ 15, MVZ 16, and cyt-b2 (Moritz et al. 1992) were used for sequencing. The DNA sequence data were used to make a character matrix for phylogenetic analysis. The matrix consisted of the sequence of the cytochrome b gene of individual specimens. These were aligned by eye prior to analysis (Appendix 1). The Hennig-86 computer program was used to estimate the most parsimonious set of historical relationships for the 18 sequenced individuals. The character states (one of four possible bases in DNA) were considered to be unpolarized and the primitive state at each locus on the DNA chain was determined by the outgroup method. An exhaustive tree-generating algorithm was nsed, thus guaranteeing that the shortest possible tree was found. To assess population biology of the Black Warror waterdog, two lOOm transects were established, one in Sipsey Fork (site 116 of Guyer 1997) and one in Brushy Creek (site 38 of Guyer 1997). Trap stations were positioned at 10m intervals (0-lOOm, inclusive) along each trap line. Four minnow traps, each baited with canned cat food, were placed at each trap station. Traps were placed in deep pools along the stream edge and tied together with a string leader that was placed so that the traps could be relocated but remain hidden from view. These traps were examined every two weeks from Jan. 10 to Apr. 19, 1998. RESULTS AND DISCUSSION A total of346 sites (out of 840) were informative (differed from the outgroup in two or more individuals of the ingroup). A single most-parsimonious tree was generated from these data (Fig. 1). This tree had a consistence index of. 76, indicating that sufficient phylogenetic signal was present in the data set to justify interpreting the evolutionary relationships implied by the tree. In this tree the Black Warrior waterdog (Necturus alabamensis) was more closely related to the mudpuppy (Necturus maculosus) than it was to waterdogs from anywhere else in the Gulf Coastal Plains. In fact, the sequence data for N. alabamensis were indistinguishable from those of the single specimen of N. macu/osus analyzed in this study. N. alabamensis differs electrophoretically from N. macu/osus (Guttman et al. 1990). Additionally, both electrophoretic (Guttman et al. 1990) and DNA sequence data (this study) document derived characters shared by these taxa and not found in any other waterdog . Therefore, waterdogs referred to as N. alabamensis and N. maculosus represent two distinct, diagnosable species that appear