HISTORICAL AFFINITIES AND POPULATION BIOLOGY OF THE BLACK WARRIOR WATERDOG ( ALABAMENSIS)

FINAL REPORT

FY 1998

FEB Craig Guyer - 4 1999 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: ) are paedomorphic, stream-dwelling 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 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 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 to have diverged relatively recently from their common ancestor. Waterdogs from the lower Coastal Plains of Alabama represent two species. Those from the Mobile drainages form an apparently monophyletic group that is more closely related to theN. alabamensis-maculosus clade tl1an it is to waterdogs from drainages farther east. The latter form a monophyletic group indicating that they are an evolutionarily distinctive unit. Because previous electrophoretic analysis found no protein differences between animals from the Mobile drainage and drainages in Louisiana known to harbour N. beyeri (Guttman et al. 1990), Alabama waterdogs from the Mobile drainage should be referred to as N. beyeri. Specimens from drainages east of the Mobile drainage have been referred to by Hecht (1958) as N. lodingi. The phylogenetic tree generated in this study justifies the use of this name for specimens from Alabama, Georgia, and Florida. Based on results of this study and previously published data, the state of Alabama has a total of four diagnosible species of Necturus. Necturus maculosus should be used for those forms from the Tennessee River drainages of the nortl1ern part of the state. This species has a boldly striped larva, a flattened head and body, and lives in rocky rivers and streams. Necturus alabamensis should be used for those forms from the Sipsey, Locust, and Mulberry Forks of the Black Warrior River. This species, like N. macu/osus, has a boldly striped larva, lives in rocky streams, and has a flattened head and body. All other waterdogs in the state have unstriped larvae, live in slow, sandy-bottomed streams and have shorter bodies and rounder heads (Bart et al. 1997). There is no known morphological feature that will separate N. beyeri from N. lodingi. However, the forrner may have more bold spotting on the tail than the latter. Additionally, N. /odingi has a scalloped border to the coloration of the tail, a feature created by numerous small white to yellow spots. N. beyeri lacks this color pattern. The systematic relationships recovered in this study have two important consequences for conservation efforts. First, clariflcation of the taxonomy ofwaterdogs within the state of Alabama now

3 allows legislation to be drafted that includes identification of taxa with validated scientific names. Because survey data for the Black Warrior waterdog (N a/abamensis) documents the extreme rarity of this species, efforts at the State level should be made to protect this taxon. At a mirtimum, collection of this species should be prohibited, except for scientific studies that will allow conservation or recovery of this taxon. Because this species overlaps the distribution of the flattened musk turtle (Sternotherus depressus) and is apparently as rare as that species, protection of the Black Warrior waterdog as threatened nuder the Act is warranted. The second conservation consequence of this study stems from identification of the sister species of the Black Warrior waterdog. If aspects oflife history of Necturus alabamensis are to be inferred from comparative methods, then N maculosus is the best source for such comparative data. The close evolutionary ties between the two explains their similar habitat requirements and body shape. Additionally, this finding indicates two possible scenarios that explain the current distribution of N a/abamensis and N maculosus. One scenario is that some event caused the divergence of N. maculosus from N beyeri and that N maculosus latter crossed from the Tennessee River system into the Black Warrior system leading to the divergence of N alabamensis. Alternatively, N. alabamensis and N. beyeri might have diverged, the former above and the latter below the fall line of the Mobile drainages. This might have been followed by invasion of an alabamensis-lilce ancestor into the Tennessee River drainage followed by subsequent speciation of N. maculosus and expansion into its current wide distribution in the Mississippi River drainages. Trapping success was poor. Careful placement of traps avoided loss due to theft, a problem noted last year. However, in 168 trap days only four individuals were captured (Table 2). None possessed a PIT tag from previous samples, so each was implanted with a tag and released at the point of capture. I have no explanation for the reduced trapping success. Traps were placed in areas that appeared to be similar to sites that were successful in previous years. Leaf packs seemed to be less abundant here (and elsewhere in AL) than in previous years. Altered patterns of rainfall may have resulted in more rapid transport ofleaves down stream, reducing the number ofleafpacks and their duration at a particular site. This might have reduced activity patterns ofwaterdogs associated with reproduction. It is my impression that waterdogs are available for capture (either in minnow traps or by dip net) only when they move from non-reproductive sites of activity to leaf packs (where they reproduce). In the coming field season, both minnow traps and dipnerting will be used to capture animals. I avoided dipnetting in 1998 because minnow traps appeared to be adequate for sampling adults and because dipnetting might alter habitat associated with reproduction. Given results from last year, it appears better to attempt to mark the largest possible number ofwaterdogs even if reproduction might be disrupted for one season. Also, rather than placing traps at standardized locations, they will be placed at pools knowu to harbor adults and tl1ese pools will form the focus of future resampling efforts. The picture that emerges from the accumulating population data is that individuals are extremely clumped in distribution and that these clumps are widely spaced. Therefore, even in the very best known sites, sampling is difficult.

LITERATURE CITED

Ashton, R.E., Jr. and J. Peavy. 1986. Black Warrior waterdog, Necturus sp. In R.H. Mount (ed.), Artimals of Alabama in Need of Special Attention, pp. 63-65, Alabama Agri. Exp. Sta., Auburn Urtiv., Alabama. Bailey, K.A. and C. Guyer. 1998. Demography and population status of the flattened musk turtle (Sternotherus depressus) in the Black Warrior River basin of Alabama. Chelortian Conserv. and Bioi. 3:77-83. Bart, H.L., Jr., M.A. Bailey, R.E. Aston, Jr., and P.E. Moler. 1997. Taxonomic and nomenclatural status of the upper Black Warrior River waterdog. J. Herpetol. 31:192-201. Bishop, S.C.1943. Handbook of . Comstock Pub!. Co. Inc.,Ithaca,NewYork. Brode, W.E. 1969. A systematic study of salamanders in the genus Necturus, Rafinesque. Unpubl. PhD dissertation, Univ. Southern Mississippi, Hattiesburg.

4 Conant, R. 1958. A field guide to reptiles and of the United States and Canada east ofthe 100th meridian. Houghton-Mifflin Co., Boston, Massaclmcets. Conant, R. 1975. A field guide to reptiles and amphibians of eastern and central North America, 2nd. ed. Houghton Mifflin Co., Boston, Massachucets. Conant, R. and J.T. Collins. 1998. A field guide to reptiles and amphibians: Eastern and central North America. 3rd ed. Houghton Mifflin Co., Boston, Massachucets. Guttman, S.I., L.A Weight, P.E. Moler, R.E. Aston, B.W. Mansell, and J. Peavy. 1990. An electrophoretic analysis ofNecturusfrom the south-eastern United States. J. Herpetol. 24:163- 175. 7 Guyer, C. 1998. A status survey of the Black Warrior waterdog (Necturus sp.). Final report submitted to the Alabama Department of Conservation, Dec. 20, 1997. Hecht, M.K. 1958. A synopsis of the mudpuppies of eastern North America. Proc. Staten Island Inst. Arts Sci. 21:1-38. Maxson, L.R., P.E. Moler, and B.W. Mansell. 1988. Albumin evolution in salamanders of the genus Necturus (Amphibia: Proteidae). J. Herpetol. 22:231-235. Moritz, C., C.J. Schneider, and D.B. Wake. 1992. Evolutionary relationships within the Ensatina eschscholtzii complex confirm the ring species interpretation. Syst. Biol41:273-291. Mount, R.H. 1975. The reptiles and amphibians of Alabama. Agric. Exp. Sta., Auburn Univ., Auburn, Alabama. Neill, W.T. 1963. Notes on the Alabama waterdog, Necturus alabamensis Viosca. Herpetologica 19:166- 174. Schmidt, K.P. 1953. A checklist of North American amphibians and reptiles, 6th e., Amer. Soc. Ichthyologists and Herpetologists. Viosca, P., Jr. 1937. A tentative revision of the genusNecturus, with descriptions of three new species from the southern Gulf drainage area. Copeia 1937:120-138.

5 Table 1. Collecting localities and disposition of voucher materials used in this stndy. Auburn University Museum (ADM) numbers are tag numbers or field tag (FT numbers). Taxon is genus or specific epithet (within Necturus).

AUM# Taxon State County Locality

35000 lodingi AL Covington Camp Creek at crossing with FSR 304

35004 beyeri AL Perry Beaverdam Creek

35006 beyeri AL Perry Beaverdam Creek

35007 alabamensis AL Winston Brushy Creek

35012 /odingi AL Escambia Miller Creek at crossing with FSR 311

35013 lodingi AL Escambia Miller Creek at crossing with FSR 311

35014 Siren AL Perry Beaverdam Creek at

35018 beyeri MS Noxubee Noxubee River

35019 beyeri AL Mobile Mobile River drainage

35021 maculosus AL Lauderdale Cedar Creek

35101 puncta/us GA Richmond Fort Gordon

FT 51 alabamensis AL Winston Sipsey Fork

FT 52 beyeri AL Tuscaloosa Big Sandy Creek

FT 53 beyeri AL Perry Beaverdam Creek

FT54 beyeri AL Perry Beaverdam Creek

FT 55 beyeri AL Perry Beaverdam Creek

FT56 lodingi FL Calhoun Appalachicola River

FT57 /odingi FL Calhoun Appalachicola River

FT 58 lodingi FL Calhoun Appalachicola River

6 Table 2. Waterdogs captured during tbe 1998 field season (Jan. 10- Apr. 19, 1998).

SITE SEX SVL DATE TAG NUMBER

Sipsey Fork M 145 Feb 7 8g8a087929

Sipsey Fork M 170 Feb 7 8g8a090629

Sipsey Fork F 150 Feb21 8g8a08774d

Brushy Creek M 195 Feb 21 8g8a087f0a

7 Figure 1. Evolutionary relationships of the genus Necturus. Specimen numbers refer to animals listed in Table 1. Numbers at interior nodes indicate the number of unequivocal changes in character states supporting a node.

'co------Siren (AUM35014) ~------N. punctatus (AUM 35101) N. lodingi (AUM 35012) N. lodingi (AUM 350!3) N. lodingi (AUM 35000) N. lodingi (FT 56) N. lodingi (FT 57) N. lodingi (FT 58)

N. alabamensis (AUM35007) N. macula sus (AUM 3502!) N. alabamensis (FT 51)

N. beyeri (AUM35018) N. beyeri (AUM35019) N. beyeri (FT 52) N. beyeri (FT 53) N. beyeri (FT 54) N. beyeri (FT 55) N. beycri (AUM 35004) N. beyeri (A UM 35006)

8 Appendix 1. DNA sequence data from 346 informative sites of the cytochrome b gene of Siren and Necturus. Each sequence is identified by an Auburn University Musemn (AUM) tag nmnber or field tag number; see Table 1 for taxon and locality. Sequence data are A~ adenine, C ~ cytosine, G ~ guanine, T ~ thymine, ? ~ unknown.

AUM35000

ATTTTAAJu\ATCTTCATTTATTTTGTTATATTTTTTTAATACACCCCATATGCTATTAGA?CATT AACCCTAGCTTCATATCAGTAGACATTTCTTACTTTTTCTGTTAGCTTATTTTTTATAGCCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACCTGCAACTCTGGGCTTAATCAATTATT TAGCTACCTTGAGGGACTATTA?TTGGTCTGCGACCGCTGGACAGCGTTCACTCT?T?AATATT AGCACTGA TTAACTTCTCATTT ACCTAAATT? ATAATATTT ACC???????????????????????????????? ??????????????

AUM35004

GTCTTAAJu\ATCTTCATTTATCTCATTATATCTTTTTAACACACCCCATATGCTATTAGAGCAT TAACCCTAGCTTTATATCAGTAGACATTTCTCACTTGTTTTGTTAGCTTATTTTTTATAGTCAT AGAATTCTACTTCTCCTTGTGAAAGCTG?GTACACACATGCAACTCTGGGCTTAATCAGTTATT TAGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTCACTTTATCTCTCTTTTTATTTCT TAAGTTCATATTGCACCTTCCATTGT

AUM35006

GTCTTAAAAATCTTCATTTATCTCATTATATCTTTTTAACACACCCCATATGCTATTAGA?CAT TAACCCTA?CTTTATATCAGTAGACATTTCTCACTTTTTTTGTTAGCTTATTTTTTATA?TCATA GAATTC?ACTTCTCCTTGTGAAAGCTGAGTACACACATGCAACTCTGGGCTTAATCAGTTATTT AGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTA?TAACA?TA GCAC?GATTAACCTCTCATTTACTT?AACTTATAATATTCACTTCA?CTCTCTTTTTA?TTCTTAT GTTC???????????????????

AUM35007

GTCTTAAAAATCTTCATTTGTTTCATTATATCTTTTTAATACACCCTATATGCTATTAGAACAT TAACCCTAGCTTTATATCAGTA?ACATTTCCCACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTAAAAGCTGAGT?CACACCTAGCTCCTGGGGCTTAATCAGTTATTT CGCTACATCGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATATT AGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTCACTTTACCTCTCTTTTCATTTCTT AAGTTT???????????????????

AUM35012

ATTTTAAAAATCTTCATTTATTTTGTTATATTTTTTTAATACACCCCATATGCTATTAGA?CATT AACCCTAGCTTTATGTCAATAGACATTTCTTACTTTTTCTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACCTGCAACTCTGGGCTTAATCAATTATT TAGCTACATTGGGGGACTACTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTTACTTCCCCTCTCTTTTATTTGCT AAAGTT????????????????????

9 AUM35013

ATTTTAAAAATCTTCATTTATTTTGTTATATTTTTTTAATACACCCCATATGCTATTAGA?CATT GACCCTAGCTTCATATCAGTAGACATTTCTTACTTTTTCTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACCTAGAACTCTGGGCTTAATCCATTATT TACCCACCTTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAA?ATATTTACTTCCT?CCTCTTTT?A?C?CCT TAGTTC?TATTGC????????????

AUM35014

ATTCTATGTATCCTCCATGATCTTTATACTTATATTCAATATCTATAATACACCATCTCTGCCC TATATTTAGCTTTATACTGACAATTACCTCCTAATTTTTTTACCTGCATTTTATCTACAGCTGC AGTATTCTACTATTACTTGTGTAGGCCGAATATCTACATGGATATTTGAGGCTTATTACCTATA TGGCCACATTAAGGAATCCCTAGTAAATTTATGATAGATATACATTGTCTCATTAAGCTATGC AAGTCCTATCCTCATTTATAAAAATACTTACT?GAAACCCATGCATCATCTCTCATCCTCCTTT TCAGTGATAGCACATATATAACTATGA

AUM35018

GTCTTAAAAATCTTCATTTATTTCATTATATCTTTTTAACACACCCCATATGCTATTAGA?CATT AACCCTAGCTTTATATCAGTAGACATTTCTCACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCC?TGTGAAAGCTGAGT?CACACATGCAACTCTGGG?TTA?TCCATTTTTCA CCTACATTGAGGGACTATTA?TTGGTCTGCGACCGCTGGACAGCGTTCACTTTACTAATATTAG CACTGATTAACTTCTCATTTACTTAAATTTAA?ATATTTACTTTATCTCTCTTTTTATTTCTTAA GTTCATA?TGCACCTTCC?TCGT

AUM35019

GCCTTAAAAATCTTCATTTATTTCATTATATCTTTTTAACACACCCCATATGCTATTAGAGCAT TAACCCTAGCTTTATATCAGTAGACATTTCTCACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACATGCAACCCTGGGCTTTATCACTTATTT AGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTACTAATATT AGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTTACTTTATCTCTCTTTTTATTTCTT AAGTTCATATTGCACCTTCCATTG

AUM35021

GTCTTAAAAATCTTCAT?TGTTTCATTATATCTTTTTAATACACCCTATATGCTATTAGA?CATT AACCCTAGCTTTATATCAGTAGACATTTCCCACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGT?AAAGCTGAGTACACACCTGCAACTCTGGGCTTAATCAATTATTT CCGCACATCGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATATT AGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTCACTTTATCTCTCTTTTCATTTCTT AAGTTTATATTACACCTTCCATTGT

10 AUM35101

ATTTTAAGCTTCTCCACTTATTTTATTATATCIIIITAGAATACCCCATATACGATCAGAGCTT CAACCCGAGCCTTATATCTATAGAAATTTTATCCI'l I I I ITGTTAGCTTATTTGTTATAGTCAT AGAATTCTACTTCTCCTTGT?AAGGCTGAGTACACACAAGCATGTCTAGGCTTAATCAGTTATT TAGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACATCGTTTACTTTATTAATGC TAGCACTGATTAACTCCTTATCCACTTAAATTTAAAATCTTTACTTTTTCTCTCTTCCTGTTCCT TAACTTTATATTATTTAGTCTGTTGT

AUMFIELD TAG 51

GTCTTAAAAATCTTCATTTGTTTCATTATATCTTTTTAATACACCCTATATGCTATTAGA?CATT AACCCTAGCTTTATATCAGTAGACATTTCCCACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACCTGCAACTCTGGGCTTAATCAATTATT TCGCTACATCGAGGGA?TATTA?TTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATATT AGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTCACTTTATCTCTCTTTTCATTTCTT AAGTTTATATTACACCTTCCATTGT

AUMFIELD TAG 52

GCCTTAAAAATCTTCATTTATTTCATTATATCTTTTTAACACACCCCATATGGTATTAGAGCAT CAACCCTAGCTTTATATCAGTAGACATTTCTTACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTAAAAGCTGAGTACACACATGCAACTCTGGGCTTAATCAGTTATT TAGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTACTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTTACTTTATCTCTCTTTTTATTTCT TAAGTTCATATTGCACCTTCCATTGT

AUM FIELD TAG 53

GTCTTAAAAATCTTCATTTATCTCATTATATCTTTTTAACACACCCCATATGCTATTAGAGCAT TAACCCTAGCTTTATATCAGTAGACATTTCTCACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACATGCAACTCTGGGCTTAATCAGTTATT TAGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTTACTTTATCTCTCTTTTTATTTCT TAAGTTCATATTGTACCTTCCATTGT

AUMFIELD TAG 54

GTCTTAAAAATCTTCATTTATCTCATTATATCTTTTTAACACACCCCATATGCTATTAGAGCAT TAACCCTAGCTTTATATCAGTAGACATTTCTCACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACATGCAACTCTGGGCTTAATCAGTTATT TAGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTTACTTTATCTCTCTTTTTATTTCT TAAGTTCATATTGCACCTTCCATTGT

11 AUMFIELD TAG 55 GTCTTAAAAATCTTCATTTATCTCATTGTATCTTTTTAACACACCCCATGTGCTATTAGAGCAT TAACCCTAGCTTTATATCAGTAGACATTTCTCACIIIIIIIGTTAGCTTAIIIIIIATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACATGCAACTCTGGGCTTAATCAGTTATT TAGCTACATTGAGGGACTATTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTTACTTTATCTCTCTTTTTATTTCT TAAGTTCATATTGCACCTTCCATTGT

AUMFIELD TAG 56 ATTTTAAAAATCTTCATTTATTTTATTATATTTTTTACATACACCCCATATGCTATTAGAGCAT TAACCCTAGCTTTATATTGGTAGACATTTCTAACTTTTTTTGTTAGCTTATTTTTTATAGTCATA GAATTCTACTTCTCCTTGTGAAAGCTGAGTACACACCTGCAACTCTGGGCTTAATCAGTTATT TAGCTACATTGAGGGACTACTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATAT TAGCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTCACTTCCTCTCTCTCTTTATTTCT TAAGTTCGTATTACACCTTCCATTAT

AUM FIELD TAG 57 ATTTTAAAAATCTTCATTTATTTTATTATATTTTTTACATACACCCCATATGCTATTAGAGCAT TAACCCTAGCTTTATATTGGTAGACATTTCTAACTT?TTTTGTTAGCTTATTGTTTATAGTCATG ATAATCCA?CTCC?CCTG?AACACCGGTGGTATAAACCAGCTCCCCGGGCGGATCCCATTTTTC CCC?T?CCTAGTAGGGCATACAGTGGTCTGCGACCGCTGGACAGCGTTCACTT????????????????? ???????????????????????????????????????????????????????????????????????????????????

AUM FIELD TAG 58 ATTTTAAAAATCTTCATTTATTTTATTATATTTTTTACATACACCCCATATGCTATTAGA?CATT AACCCTAGCTTTATATTGGTAGACATTTCTAACTTTTTTTGTTAACTTATTTTTTATA?TCATAA TATTCCA?CTCC?CCTGAACAACCGTAGTACACACCTGCAACTCTGGGCTTAATCAGTTATTTA GCTACATTGAGGGACTACTAGTTGGTCTGCGACCGCTGGACAGCGTTCACTTTATTAATATTA GCACTGATTAACTTCTCATTTACTTAAATTTAAAATATTCACTTCCTCTCTCTCTTTATTTCTTA AGTTCGTATTACACCTTCCATTAT

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