Lewter, Jennifer A.

Szalanski, Allen L.

Yamashita, Tsunemi

UARK (University of Arkansas )

Journal of the Arkansas Academy of Science Volume 57 Article 34 2003 DNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalva: Unionidae) in Select Ozark and Ouachita Mountain Streams of Arkansas Jennifer A. Lewter University of Arkansas, Fayetteville Allen L. Szalanski University of Arkansas, Fayetteville Tsunemi Yamashita Arkansas Tech University Follow this and additional works at: https://scholarworks.uark.edu/jaas  Part of the Zoology Commons Recommended Citation Lewter, Jennifer A.; Szalanski, Allen L.; and Yamashita, Tsunemi (2003) "DNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalva: Unionidae) in Select Ozark and Ouachita Mountain Streams of Arkansas," Journal of the Arkansas Academy of Science: Vol. 57 , Article 34. Available at: https://scholarworks.uark.edu/jaas/vol57/iss1/34 This article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able to read, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking prior permission from the publisher or the author. This General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the Arkansas Academy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu. 216DNA Sequence Analysis of the Freshwater MusselLampsilis hydiana (Bivalvia: Unionidae) inSelect Ozark andOuachita Mountain Streams of ArkansasJennifer A.Lewter and Allen L.Szalanski Tsunemi YamashitaDepartment of EntomologyUniversity of ArkansasFayetteville, AR 72701Department ofBiologyArkansas Tech UniversityRussellville, AR 72801*Corresponding AuthorNearly 300 species of freshwater mussels are found inNorth America; this represents the greatest diversity offreshwater mussels in the world (Williams et al., 1993;Lydeard and Mayden, 1995). Although it is not clearlyunderstood which environmental and historical factors havefostered such diversity in North America, there is scientificevidence of anthropogenic factors causing the rapid declineof many species of freshwater mussels (Neves et al., 1997).The unionid mussels have recently become one of the mostendangered freshwater organisms in North America(Williams et al., 1993; Stein and Flack, 1997; Master et al.,1998). The primary threats to these mussels includepollution, increased sedimentation from stream alterations,loss of fish species that host the parasitic unionid larvae(glochidia), and the introduction of the exotic zebra mussel(Dreissena polymorpha) (Williams et al., 1993; King et al.,1999). To date, there has been very little research done onthe genetic diversity of unionid mussels (Liu et al., 1996;Roe and Lydeard, 1998). The traditional method ofclassifying taxa based on physical characteristics ischallenging with mussels because they do not have manydistinguishing features (Lydeard and Roe, 1998).Furthermore, knowing the exact geographical range of anendangered species (or sub-species) can provide insight as towhich anthropogenic factors may be the most detrimental tothe organism's survival (Roe et al., 2001). Finally,conservational laws and methods cannot be implementeduntil the endangered organism (s) is properly classified andits geographical range is known (Lydeard and Roe, 1998).The objective of this study was to examine a -300 bpregion of mitochondrial DNA in the 16s rRNA gene ofLampsilis hydiana and compare our results with those ofTurner et al. (2000). Previous to this study, the only geneticsresearch available for this species was done by Turner et al.(2000) who found five haplotypes using the 16S rRNA gene.Since L.hydiana can be found inmany different streamsin Arkansas, we hypothesized that the Lampsilis populationswould exhibit a population structure that reflects isolation-by-distance. For example, populations that are further apartgeographically have greater genetic variation thanpopulations that are closer together which could potentiallyinterbreed.Mussel Samples.— Lampsilis hydiana mussels werecollected from the North and Middle forks of the IllinoisBayou and the South Fourche La Fave River of Arkansas(Table 1.) Live specimens were brought back to theDepartment of Biology, Arkansas Tech Univ.inRussellville,AR and were killed by immersion in a jar of 95% ethanoland stored at -20° C.DNA Extraction, Sequencing and Sequencing Analysis.-DNA was extracted from a small piece of muscular foottissue (<1 cm3) using the following phenol/chloroformextraction protocol. Nine hundred |uL of Lifton Buffer (0.2Msucrose, 0.05M EDTA, 0.1M Tris, 0.5% SDS) was added toa 50 mL plastic tube with foot tissue. Forty-five (iL ofProteinase K (Promega, Madison, WI) were added and thetissue was homogenized with a glass stirring rod and placedon ice for one hour. One hunded uL of 8M potassiumacetate was added and the samples were vortexed brieflyand again chilled on ice for one hour. The solution wastransferred to a new microtube and centrifuged at 14000RPM for fifteen minutes. Supernatant was transferred to anew microtube, 2 uL of RNAse (Promega), were added tothe new tube, and itwas incubated at room temperature fortwenty minutes. One hundred uL chloroform/isoamylalcohol (CIA)and 300 uLof equilibrated phenol was added,samples were vortexed, incubated at room temp for fiveminutes, and centrifuged at 14000 RPM for five min. Theaqueous phase was pipetted to a new microtube, 400 jiLCIA were added, and the sample was vortexed andcentrifuged at 14000 RPM for five min. The aqueous phasewas pipetted to a new microtube, then 100 uLof 3Msodiumacetate were added. Three hunded uL of isopropanol wasadded, and sample was stored at -20°C for three days. Thesamples were then centrifuged at 14000 RPM for 30minutes, then 1mL of chilled 70% ETOH was added to theDNA pellet. The samples were spun at 14000 RPM for fiveminutes, supernatant removed and the DNA pellet wasrewashed with 1 mL chilled 70% ETOH and wascentrifuged for five min. Supernatant was removed and theDNA pellet was allowed to air dry for at least fifteenminutes. Finally, 200 uLof distilled H2O were added, DNAwas resuspended for two hours at room temperature andthen stored at 4°C.Journal of the Arkansas Academy of Science, Vol. 57, 2003216Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34Published by Arkansas Academy of Science, 2003217IDNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalvia: Unionidae) in Select Ozark andOuachita Mountain Streams of Arkansas—— ——^^—^—————tampans ovata AF385135————^————^—^^— LampSjfjs teres AF3851370^03—Haplotype 2| |—^ ™-Hap|otype 495B7JB5—Haplotype 1I Ifljgg Haplotype 3M1~5!067" M2M5M775M6M8———Haplotype 5M3Journal of the Arkansas Academy of Science, Vol.57, 2003217Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34https://scholarworks.uark.edu/jaas/vol57/iss1/34218Jennifer A.Lewter, Allen L. Szalanski and Tsunemi YamashitaTable 1. Specimens used in this study and observed haplotypes.MUSSEL DATE LOCATION Haplotype SEXID COLLECTEDMl 9-16-01 S. Fourche La Fave, AR 5 FM2 9-16-01 S. Fourche La Fave, AR 5 MM3 9-16-01 S. Fourche La Fave, AR 5 MM5 4-4-02 North Fork, IllinoisBayou, AR 5 ¥M6 4-21-02 Middle Fork, IllinoisBayou, AR 5 MM7 4-21-02 Middle Fork, IllinoisBayou, AR 5 FM8 4-21-02 Middle Fork, IllinoisBayou, AR 5 MApproximately 400 bp of the mtDNA 16S rRNA genewere amplified with the primers 16sint3-L (5'-TGAGCGTVCTAAGGTAGC- 3') (Turner et al., 2000)and 16sint4-H (5' -AKCCAACATCGAGGTCGCAA -3')(Turner et al., 2000). PCR was conducted using 9.25 ulDH2O, 3.0 ul of 10X Buffer (Promega), 0.5 ulDNTP, 0.5 ulBSA, 0.5 (ilDMSO, 0.5 ul formalin, and 0.4 units Thermusaquaticus (Taq) DNA polymerase (Promega). Primers wereadded at a final concentration of 20 fiM(5 ul each primer)and mixed with 5 ul DNA. The thermal cycler profileconsisted of 30 cycles of denaturation at 94°C for 30 sec,annealing at 50°C for 30 sec, and extension at 72°C for 1min.PCR products were sent to the Graduate Institute ofTechnology, Univ. of Arkansas, Little Rock for DNAsequencing. Mitochondrial DNA sequences from freshwatermussels Lampsilis ovata (GenBank accession AF385135) andL. teres (GenBank accession AF 385137) were added to theL. hydiana DNA sequences to act as outgroup taxa. DNAsequences were aligned using the PILEUP program inGCG (Genetics Computer Group, Madison, WI) andadjusted manually. The distance matrix option of PAUP*4.0b 10 (Swofford, 2001) was used to calculate geneticdistances according to the Kimura 2-parameter model(Kimura, 1980) of sequence evolution. Maximumlikelihood and unweighted parsimony analysis on thealignments were conducted using PAUP* 4.0b 10 (Swofford,2001). Gaps were treated as missing data or as a 5thcharacter state and a random addition sequence wasemployed. The reliability of trees was tested with abootstrap test (Felsenstein, 1985). Parsimony bootstrapanalysis included 1,000 resamplings using the Branch andBound algorithm of PAUP*. For maximum likelihoodanalysis, the default likelihood parameter settings were used(HKY85 6-parameter model of nucleotide substitution, andempirical base frequencies), with the exception of thetransition/transversion ratio which was set to 2.345. Theseparameters were used to carry out a heuristic search usingPAUP*, using either the single most parsimonious tree asthe starting tree, or step-wise addition.PCR products were approximately 400 bp in length.One hundred eighteen nucleotide characters were excludedin the PILEUP analysis, and out of the 309 remainingcharacters, 251 were constant. Allseven samples of Lampsilishydiana matched the 'haplotype 5' sequence (GenBankaccession AF191569) previously published by Turner et al.,(2000).Thirty-one characters were variable and parsimony-uninformative, while 27 characters were parsimony-informative. Pairwise genetic distances ranged from 0.00 to0.07 (excluding outgroup taxa) (Table 2). Nucleotidefrequencies were A=0.38817, C =0.20102, G =0.18934 andT=0.22147. The maximum parsimony analysis resulted inatree with a length of 64 and a consistency index (CI) of0.969 (Fig. 1). Haplotype 5 formed a distinct clade relativeto the four other haplotypes. No difference was foundbetween excluding gaps and treating gaps as a 5th characterstate. The maximum likelihood tree, -In L= 690.49790, wasidentical to the maximum parsimony tree (Fig. 1).We have confirmed that there are L. hydiana of'haplotype 5' located in the South Fourche La Fave River,and this same haplotype is found in the North and Middleforks of the Illinois Bayou. Since sample sizes were small,future research should be conducted including moreJournal of the Arkansas Academy of Science, Vol. 57, 2003218Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34Published by Arkansas Academy of Science, 2003219DNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalvia: Unionidae) in Select Ozark andOuachita Mountain Streams of ArkansasTable 2. Pairwise genetic distances among the 5 haplotypes Lampsilis hydiana and 2 Lampsilis outgroup taxa.1123 4 5 6 7L. ovataL. teres 0.044Haplotype2 0.124 0.112Haplotype4 0.124 0.112 0.007Haplotype 1 0.121 0.108 0.010 0.010Haplotype3 0.117 0.104 0.007 0.007 0.010Haplotype 5 0.124 0.107 0.038 0.038 0.041 0.031specimens from each site. The geographical range of L.hydiana is not clearly defined, so itwould also be beneficialto sample as many different Arkansas streams and rivers aspossible. Other molecular markers such as the nuclear ITSregion may also be used to detect sequence diversity.Nuclear protein genes are believed to evolve at a slower ratethan mitochondrial genes, so it is important to note thatnuclear markers may reflect different stages of evolutionaryhistory than mitochondrial markers (Machordom et al.,2003). Other researchers working with freshwater musselssuggest that if different populations within the same speciesare found (i.e., no gene exchange is possible between them),they should be managed as separate conservation units,particularly by avoiding relocating the mussels (King et al.,1999). It is essential to protect the genetic lineages ofunionids that have been evolving over thousands of years.IAcknowledgments.—We thank Jobi Brown andndall Moles for collecting samples. This project wasided inpart by the Arkansas Tech University Physical ande Sciences Endowment Fund.Literature Citedrenstein, J. 1985. Confidence limits on phylogeneties:An approach using the bootstrap. Evolut. 39:783-791.riura, M. 1980. A simple method for estimatingevolutionary rate of base substitutions throughcomparative study of nucleotide sequences. J. Molec.Evolut. 16:111-120.King, T.L.,M.S. Eackles, B.Gjetvaj, W. R. Hoeh. 1999.Intraspecific phylogeography of Lasmigona subviridis(Bivalvia: Unionidae): Conservation implications ofrange discontinuity. Molec. Ecol. 8:S65-S78.Liu,HP.,J. B. Mitton,and S.J. Herrmann. 1996. Geneticdifferentiation in and management recommendationsfor the freshwater mussel Pyganodon grandis. Amer.Malacolog. Bull. 13:117-124.Lydeard, C, and K.J. Roe. 1998. Phylogentic Systematics:The missing ingredient in the conservation offreshwater unionid bivalves. Fisheries 23: 16-17.Lydeard, C, and R.L. Mayden. 199.5. A diverse andendangered aquatic ecosystem of the Southeast UnitedStates. Conser. Biol. 9:800-805.Machordom, A., R. Araujo, D. Erpenbeck, and M.Ramos. 2003. Phylogeography and conservationgenetics of endangered European Margaritiferidae(Bivalvia:Unionoidea.) Biol.J. Linn. Soc. 78:235-252.Master, L.L.,S. R. Flack, and B. A. Stein. 1998. Rivers ofLife: Critical Watersheds for Protecting FreshwaterBiodiversity. The Nature Conservancy, Arlington, VA.Neves, R.J., A.E. Bogan, J. D.Williams, S. A.Ahlstedt,and P. Hartfield. 1997. Status of aquatic mollusks in theSoutheastern United States: A downward spiral ofdiversity. In: Aquatic fauna in Peril: the SoutheasternPerspective (eds Benz, G.W., Collins, D.E.), pp.43-85.Special publication 1. Southeast Aquatic ResearchInstitute, Lenz Design and Communications, Decatur,GA.Roe, K. J., P. D. Hartfield, and C. Lydeard. 2001.Phylogeographic analysis of the threatened andendangered superconglutinate-producing mussels of thegenus Lampsilis (Bivalvia: Unionidae.) Molec. Ecol.10:2225-2234.Roe, K.J., and C. Lydeard. 1998. Molecular systematics ofthe freshwater mussel genus Potamilus (Bivalvia:Unionidae.) Malacologia 39:195-205.Stein, B. A., and S. R. Flack. 1997. 1997 species report card:the state of U.S. plants and animals. The NatureJournal of the Arkansas Academy of Science, Vol. 57, 2003219Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34https://scholarworks.uark.edu/jaas/vol57/iss1/34220Jennifer A.Lewter, Allen L.Szalanski and Tsunemi YamashitaConservancy, Arlington, VA.Swofford, D.L.2001. PAUP*: Phylogeny Analysis UsingParsimony (* and other methods), version 4.0b 10.Sinauer Associates, Sunderland, MA.l\irner,T.F.,J. C.Trexler,J. L.Harris, and J. L.Haynes.2000. Nested cladistic analysis indicates populationfragmentation shapes genetic diversity in a freshwatermussel. Genetics 154:777-785.T\irner, T.F.,J. C. Trexler,J. L.Harris, and J. L.Haynes.2000. Nested cladistic analysis indicates populationfragmentation shapes genetic diversity in a freshwatermussel. Genetics 154:777-785.Williams,J. D., M.L. Warren Jr., K.S. Cummings,J. L.Harris, and R.J. Neves. 1993. Conservation status offreshwater mussels of the United States and Canada.Fisheries 18:6-22.Yang, Z. 1994. Maximum likelihood phylogeneticestimation from DNA sequences with variable ratesover sites: Approximate methods. J. Molec. Evol.39:306-314.Journal of the Arkansas Academy of Science, Vol 57, 2003220Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34Published by Arkansas Academy of Science, 2003

English

DNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalva: Unionidae) in Select Ozark and Ouachita Mountain Streams of Arkansas

ScholarWorks@UARK

Journal of the Arkansas Academy of ScienceVolume 57 Article 342003DNA Sequence Analysis of the Freshwater MusselLampsilis hydiana (Bivalva: Unionidae) in SelectOzark and Ouachita Mountain Streams ofArkansasJennifer A. LewterUniversity of Arkansas, FayettevilleAllen L. SzalanskiUniversity of Arkansas, FayettevilleTsunemi YamashitaArkansas Tech UniversityFollow this and additional works at: http://scholarworks.uark.edu/jaasPart of the Zoology CommonsThis article is available for use under the Creative Commons license: Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0). Users are able toread, download, copy, print, distribute, search, link to the full texts of these articles, or use them for any other lawful purpose, without asking priorpermission from the publisher or the author.This General Note is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Journal of the ArkansasAcademy of Science by an authorized editor of ScholarWorks@UARK. For more information, please contact scholar@uark.edu.Recommended CitationLewter, Jennifer A.; Szalanski, Allen L.; and Yamashita, Tsunemi (2003) "DNA Sequence Analysis of the Freshwater Mussel Lampsilishydiana (Bivalva: Unionidae) in Select Ozark and Ouachita Mountain Streams of Arkansas," Journal of the Arkansas Academy of Science:Vol. 57 , Article 34.Available at: http://scholarworks.uark.edu/jaas/vol57/iss1/34216DNA Sequence Analysis of the Freshwater MusselLampsilis hydiana (Bivalvia: Unionidae) inSelect Ozark andOuachita Mountain Streams of ArkansasJennifer A.Lewter and Allen L.Szalanski Tsunemi YamashitaDepartment of EntomologyUniversity of ArkansasFayetteville, AR 72701Department ofBiologyArkansas Tech UniversityRussellville, AR 72801*Corresponding AuthorNearly 300 species of freshwater mussels are found inNorth America; this represents the greatest diversity offreshwater mussels in the world (Williams et al., 1993;Lydeard and Mayden, 1995). Although it is not clearlyunderstood which environmental and historical factors havefostered such diversity in North America, there is scientificevidence of anthropogenic factors causing the rapid declineof many species of freshwater mussels (Neves et al., 1997).The unionid mussels have recently become one of the mostendangered freshwater organisms in North America(Williams et al., 1993; Stein and Flack, 1997; Master et al.,1998). The primary threats to these mussels includepollution, increased sedimentation from stream alterations,loss of fish species that host the parasitic unionid larvae(glochidia), and the introduction of the exotic zebra mussel(Dreissena polymorpha) (Williams et al., 1993; King et al.,1999). To date, there has been very little research done onthe genetic diversity of unionid mussels (Liu et al., 1996;Roe and Lydeard, 1998). The traditional method ofclassifying taxa based on physical characteristics ischallenging with mussels because they do not have manydistinguishing features (Lydeard and Roe, 1998).Furthermore, knowing the exact geographical range of anendangered species (or sub-species) can provide insight as towhich anthropogenic factors may be the most detrimental tothe organism's survival (Roe et al., 2001). Finally,conservational laws and methods cannot be implementeduntil the endangered organism (s) is properly classified andits geographical range is known (Lydeard and Roe, 1998).The objective of this study was to examine a -300 bpregion of mitochondrial DNA in the 16s rRNA gene ofLampsilis hydiana and compare our results with those ofTurner et al. (2000). Previous to this study, the only geneticsresearch available for this species was done by Turner et al.(2000) who found five haplotypes using the 16S rRNA gene.Since L.hydiana can be found inmany different streamsin Arkansas, we hypothesized that the Lampsilis populationswould exhibit a population structure that reflects isolation-by-distance. For example, populations that are further apartgeographically have greater genetic variation thanpopulations that are closer together which could potentiallyinterbreed.Mussel Samples.— Lampsilis hydiana mussels werecollected from the North and Middle forks of the IllinoisBayou and the South Fourche La Fave River of Arkansas(Table 1.) Live specimens were brought back to theDepartment of Biology, Arkansas Tech Univ.inRussellville,AR and were killed by immersion in a jar of 95% ethanoland stored at -20° C.DNA Extraction, Sequencing and Sequencing Analysis.-DNA was extracted from a small piece of muscular foottissue (<1 cm3) using the following phenol/chloroformextraction protocol. Nine hundred |uL of Lifton Buffer (0.2Msucrose, 0.05M EDTA, 0.1M Tris, 0.5% SDS) was added toa 50 mL plastic tube with foot tissue. Forty-five (iL ofProteinase K (Promega, Madison, WI) were added and thetissue was homogenized with a glass stirring rod and placedon ice for one hour. One hunded uL of 8M potassiumacetate was added and the samples were vortexed brieflyand again chilled on ice for one hour. The solution wastransferred to a new microtube and centrifuged at 14000RPM for fifteen minutes. Supernatant was transferred to anew microtube, 2 uL of RNAse (Promega), were added tothe new tube, and itwas incubated at room temperature fortwenty minutes. One hundred uL chloroform/isoamylalcohol (CIA)and 300 uLof equilibrated phenol was added,samples were vortexed, incubated at room temp for fiveminutes, and centrifuged at 14000 RPM for five min. Theaqueous phase was pipetted to a new microtube, 400 jiLCIA were added, and the sample was vortexed andcentrifuged at 14000 RPM for five min. The aqueous phasewas pipetted to a new microtube, then 100 uLof 3Msodiumacetate were added. Three hunded uL of isopropanol wasadded, and sample was stored at -20°C for three days. Thesamples were then centrifuged at 14000 RPM for 30minutes, then 1mL of chilled 70% ETOH was added to theDNA pellet. The samples were spun at 14000 RPM for fiveminutes, supernatant removed and the DNA pellet wasrewashed with 1 mL chilled 70% ETOH and wascentrifuged for five min. Supernatant was removed and theDNA pellet was allowed to air dry for at least fifteenminutes. Finally, 200 uLof distilled H2O were added, DNAwas resuspended for two hours at room temperature andthen stored at 4°C.Journal of the Arkansas Academy of Science, Vol. 57, 2003216Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34Published by Arkansas Academy of Science, 2003217IDNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalvia: Unionidae) in Select Ozark andOuachita Mountain Streams of Arkansas—— ——^^—^—————tampans ovata AF385135————^————^—^^— LampSjfjs teres AF3851370^03—Haplotype 2| |—^ ™-Hap|otype 495B7JB5—Haplotype 1I Ifljgg Haplotype 3M1~5!067" M2M5M775M6M8———Haplotype 5M3Journal of the Arkansas Academy of Science, Vol.57, 2003217Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34http://scholarworks.uark.edu/jaas/vol57/iss1/34218Jennifer A.Lewter, Allen L. Szalanski and Tsunemi YamashitaTable 1. Specimens used in this study and observed haplotypes.MUSSEL DATE LOCATION Haplotype SEXID COLLECTEDMl 9-16-01 S. Fourche La Fave, AR 5 FM2 9-16-01 S. Fourche La Fave, AR 5 MM3 9-16-01 S. Fourche La Fave, AR 5 MM5 4-4-02 North Fork, IllinoisBayou, AR 5 ¥M6 4-21-02 Middle Fork, IllinoisBayou, AR 5 MM7 4-21-02 Middle Fork, IllinoisBayou, AR 5 FM8 4-21-02 Middle Fork, IllinoisBayou, AR 5 MApproximately 400 bp of the mtDNA 16S rRNA genewere amplified with the primers 16sint3-L (5'-TGAGCGTVCTAAGGTAGC- 3') (Turner et al., 2000)and 16sint4-H (5' -AKCCAACATCGAGGTCGCAA -3')(Turner et al., 2000). PCR was conducted using 9.25 ulDH2O, 3.0 ul of 10X Buffer (Promega), 0.5 ulDNTP, 0.5 ulBSA, 0.5 (ilDMSO, 0.5 ul formalin, and 0.4 units Thermusaquaticus (Taq) DNA polymerase (Promega). Primers wereadded at a final concentration of 20 fiM(5 ul each primer)and mixed with 5 ul DNA. The thermal cycler profileconsisted of 30 cycles of denaturation at 94°C for 30 sec,annealing at 50°C for 30 sec, and extension at 72°C for 1min.PCR products were sent to the Graduate Institute ofTechnology, Univ. of Arkansas, Little Rock for DNAsequencing. Mitochondrial DNA sequences from freshwatermussels Lampsilis ovata (GenBank accession AF385135) andL. teres (GenBank accession AF 385137) were added to theL. hydiana DNA sequences to act as outgroup taxa. DNAsequences were aligned using the PILEUP program inGCG (Genetics Computer Group, Madison, WI) andadjusted manually. The distance matrix option of PAUP*4.0b 10 (Swofford, 2001) was used to calculate geneticdistances according to the Kimura 2-parameter model(Kimura, 1980) of sequence evolution. Maximumlikelihood and unweighted parsimony analysis on thealignments were conducted using PAUP* 4.0b 10 (Swofford,2001). Gaps were treated as missing data or as a 5thcharacter state and a random addition sequence wasemployed. The reliability of trees was tested with abootstrap test (Felsenstein, 1985). Parsimony bootstrapanalysis included 1,000 resamplings using the Branch andBound algorithm of PAUP*. For maximum likelihoodanalysis, the default likelihood parameter settings were used(HKY85 6-parameter model of nucleotide substitution, andempirical base frequencies), with the exception of thetransition/transversion ratio which was set to 2.345. Theseparameters were used to carry out a heuristic search usingPAUP*, using either the single most parsimonious tree asthe starting tree, or step-wise addition.PCR products were approximately 400 bp in length.One hundred eighteen nucleotide characters were excludedin the PILEUP analysis, and out of the 309 remainingcharacters, 251 were constant. Allseven samples of Lampsilishydiana matched the 'haplotype 5' sequence (GenBankaccession AF191569) previously published by Turner et al.,(2000).Thirty-one characters were variable and parsimony-uninformative, while 27 characters were parsimony-informative. Pairwise genetic distances ranged from 0.00 to0.07 (excluding outgroup taxa) (Table 2). Nucleotidefrequencies were A=0.38817, C =0.20102, G =0.18934 andT=0.22147. The maximum parsimony analysis resulted inatree with a length of 64 and a consistency index (CI) of0.969 (Fig. 1). Haplotype 5 formed a distinct clade relativeto the four other haplotypes. No difference was foundbetween excluding gaps and treating gaps as a 5th characterstate. The maximum likelihood tree, -In L= 690.49790, wasidentical to the maximum parsimony tree (Fig. 1).We have confirmed that there are L. hydiana of'haplotype 5' located in the South Fourche La Fave River,and this same haplotype is found in the North and Middleforks of the Illinois Bayou. Since sample sizes were small,future research should be conducted including moreJournal of the Arkansas Academy of Science, Vol. 57, 2003218Journal of the Arkansas Academy of Science, Vol. 57 [2003], Art. 34Published by Arkansas Academy of Science, 2003219DNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalvia: Unionidae) in Select Ozark andOuachita Mountain Streams of ArkansasTable 2. Pairwise genetic distances among the 5 haplotypes Lampsilis hydiana and 2 Lampsilis outgroup taxa.1123 4 5 6 7L. ovataL. teres 0.044Haplotype2 0.124 0.112Haplotype4 0.124 0.112 0.007Haplotype 1 0.121 0.108 0.010 0.010Haplotype3 0.117 0.104 0.007 0.007 0.010Haplotype 5 0.124 0.107 0.038 0.038 0.041 0.031specimens from each site. The geographical range of L.hydiana is not clearly defined, so itwould also be beneficialto sample as many different Arkansas streams and rivers aspossible. Other molecular markers such as the nuclear ITSregion may also be used to detect sequence diversity.Nuclear protein genes are believed to evolve at a slower ratethan mitochondrial genes, so it is important to note thatnuclear markers may reflect different stages of evolutionaryhistory than mitochondrial markers (Machordom et al.,2003). Other researchers working with freshwater musselssuggest that if different populations within the same speciesare found (i.e., no gene exchange is possible between them),they should be managed as separate conservation units,particularly by avoiding relocating the mussels (King et al.,1999). It is essential to protect the genetic lineages ofunionids that have been evolving over thousands of years.IAcknowledgments.—We thank Jobi Brown andndall Moles for collecting samples. This project wasided inpart by the Arkansas Tech University Physical ande Sciences Endowment Fund.Literature Citedrenstein, J. 1985. Confidence limits on phylogeneties:An approach using the bootstrap. Evolut. 39:783-791.riura, M. 1980. A simple method for estimatingevolutionary rate of base substitutions throughcomparative study of nucleotide sequences. J. Molec.Evolut. 16:111-120.King, T.L.,M.S. Eackles, B.Gjetvaj, W. R. Hoeh. 1999.Intraspecific phylogeography of Lasmigona subviridis(Bivalvia: Unionidae): Conservation implications ofrange discontinuity. Molec. Ecol. 8:S65-S78.Liu,HP.,J. B. Mitton,and S.J. Herrmann. 1996. Geneticdifferentiation in and management recommendationsfor the freshwater mussel Pyganodon grandis. Amer.Malacolog. Bull. 13:117-124.Lydeard, C, and K.J. Roe. 1998. Phylogentic Systematics:The missing ingredient in the conservation offreshwater unionid bivalves. Fisheries 23: 16-17.Lydeard, C, and R.L. Mayden. 199.5. A diverse andendangered aquatic ecosystem of the Southeast UnitedStates. Conser. Biol. 9:800-805.Machordom, A., R. Araujo, D. Erpenbeck, and M.Ramos. 2003. 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DNA Sequence Analysis of the Freshwater Mussel Lampsilis hydiana (Bivalva: Unionidae) in Select Ozark and Ouachita Mountain Streams of Arkansas

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