8/12/2019 84(2)107-1998

1/11

ELSEVIER PI I: SOOO 6.3207 97)00112-2

Biol ogical onservat ion 4 1998) 107-I 170 1998 E lsevier Science L tdAll rights reserved. Printed in Great Britain

0006-3207/98 19.00+0.00

CONSERVATION PHYLOGENETICS OF OZARK CRAYFISHESASSIGNING PRIORITIES FOR AQUATIC HABITAT

PROTECTION

Keith A. Crandall*D epa r t men t o f Zoo l ogy and M on te L . Bean M useum, B r i gham Young U n iv er s i t y , r ovo , U T 84602 -5255, U SA

(Received 29 January 1996 ; revised version received 23 June 1997; accepted 7 July 1997)

AbstractA mol ecu la r ph y l ogeny based on nuc leo t i de sequence dat af r om the 16s r eg ion o f t he m i t ochond r i a l genome w ases t im a ted fo r 20 spec ies o f c ray f i sh na t i v e to the Oza r kP la t eaus reg ion o f M issou r i and A r kansas , U SA . W i t ht h i s phy l ogeny and n fo rm a t i on on geog raph i c d i s t r i bu t i on ,eco log i ca l spec ia l iza t i on , and spec ies abunda nce, spec iesw ere assessed for th ei r s t a t us as rare and endangered .Three spec ies not prev i ous ly l i s ted , Orconectes nana,Orconectes macrus, and Cambarus maculatus, me r i tin c lus io n as ra re in th e Rar e and Endangered Spec ies ofM issou r i Check l i s t . Th ree o the r spec ies mer i t i nc l us i onw i t h t h e s t a t u s W a t c h L i s t : Orconectes hylas, Orco-nectes medius, and Cambarus hubbsi. Add i t i o n a l l y , t h es ta t us o f t he fo l l ow ing spec ies m er i t chang ing f r omWa t c h L i s t t o Ra r e : Orconectes peruncus and Orco-nectes quadruncus. Fauna1 r eg i o n s w i t h i n t h e O za r kP la t eaus w ere ranked fo r conser va t i on p r i o r i t y us ing spec iesr i chness, t axo nom i c d i ve r si t y , and phy l ogenet i c d i v er s i t y .The W h i t e R i ve r , B lack R i ve r , and M iss i ssi pp i R i ve rr eg ions o f t he O za r ks w ere ound to rank h i ghes t i n te rmsof conserv a t io n pr i o r i t i es . 0 1998 E lsev i er Science Ltd .A l l r i gh ts reserv ed.

of that species and the importance of various populationgenetic and ecological parameters for the conservationof the species. This approach is labor-intensive, expen-sive, and time-consuming and the resulting informationis typically relevant for only the single species underexamination. Yet this approach provides excellentdetailed information needed to maintain an endangeredspecies. An alternative is to utilize taxonomic and/orphylogenetic richness as an indicator of importanthabitats which provide for a diversity of organism s andtarget such habitats in conservation policy. The weak-ness of this approach is that detailed po pulation geneticdata is not obtained for endangered species. Theadvantag es to this approach are that it can identifywhole regions of conservation interest as well as presentpreliminary information for directing further studies atthe population genetic level.

Keyw o rds : crayfish, phylogeny, endangered species,Ozarks, conservation phylogenetics, species richness.

INTRODUCTIONFor the plannin g of conservation policy and agenda s atany level (local, state, national, or international), infor-mation is required on the relative importance of varioushabitats and species in the area under consideration.Typically, such information is sought in one of twocomplementary ways. One approach is to study a parti-cular species of interest to define the endangered status

Many authors have emphasized the importance ofsystematics in the field of conservation biology (Tem-pleton, 1991; Eldredge, 1992; Cracraft, 1994; Greene,1994). Systematics and taxonomy define the groups oforganism s for consideration as well as the diversitywithin and among these groups. Recently, methodshave been developed to utilize the phylogeny of a spe-cies group to define a quantitative framework for asses-sing conservation priorities (M ay, 19 90; Vane-W right eta l . , 1991; Crozier, 1992 ; Faith, 1992, 1994). Thisapproach allows for a preliminary assessm ent of con-servation priorities and endangered species status with-out the need for time consuming and expensivepopulation genetic and ecological surveys for each indi-vidual species and habitat region (William s and Gaston,1994). A systematic survey also identifies populations orspecies for which more detailed studies are needed. Inthis study, the phylogenetic approaches of Vane-W rightet a l . (1991) and Faith (1992) are employed to determinepriority rankings for the six ecological subdivision swithin the Ozark Plateaus.

*To whom correspondence should he addressed: 574 Widtsoe Crayfish are well suited for the study of conservationBuilding, Departme nt of Zoology, Brigham Young Un iver- biology of freshwater stream system s because thesesity, Provo, UT 84602-5255, USA; Fax: (801) 378-7423; macroinvertebrates are an important part of fresh-e-mail: keith_crandall@byu.edu water ecosystems (Lodg e, 19 93) particularly in Ozark

107

8/12/2019 84(2)107-1998

2/11

108 K . A . C r a nda l lstreams (Rabeni et a l . , 1995). Of specific concern toconservationists are the streams of the Ozark P lateausthat harbor a number of endemic species that are oneither state or federal rare and endangered species lists(e.g. Ozark hellbender Cryp tob ranchus b i shop i ; Neoshomadtom No tu rus p lac idus ; Ozark cavefish Amb l y o p s i srosae; Neosho mucket Lam ps i l i s r a f i nesqueana ; Nianguadarter E theos toma n ianguae ; Big river crayfish Or co -nectes har r ison i i ) . Because of this wealth of biodiversity,the stream systems of the Ozarks have been studiedextensively. P flieger (1989) and Matthe ws et a l . (1992)have described many aquatic systems within the Ozarksas being ecologically, physiog raphically, and faunisti-tally distinct from one another. Using crayfish sys-tematic relationships and geographic distribution s, thisstudy assigns conservation priorities to these ecologi-cally important regions within the Ozark Plateau s. Inaddition, the study makes a ssessm ents of these crayfishpopulations for conservation considerations.METHODSPhylogeoetic approach es to conservation biologyPhylogenetic assessm ents of conservation priorities wereperformed using two distinct techn iques. The first, byVane-Wright et a l . (1991), quantified the amoun t oftaxonomic information for a particular species, X, in aphylogeny by counting the numb er of monophyleticgroups (or the number of nodes g oing back to the root)containing that taxon. This value, I,, was then dividedinto the sum of the Is over all taxa in the phylogenygiving the quotient of the total information for thattaxon. The quotient, QX , was then standardized by theminim um quotient value resulting in the taxonomicweight, W,, for each taxon with the weight of W, = 1.00assigned to the taxon of lowest rank. These weightswere summed over distinct habitats to quantitativelyrank these habitats for priority in conservation effortsbased on taxonomic diversity.

Specific areas of endemism were then ranked for con-servation p riorities b ased on the proportion of weightsfound in a particular area. The weight for a given areawas calculated by summ ing the weigh ts for each taxonpresent in that area an d dividing by the total w eight forall taxa. These percentages will not add up to 100unless all taxa are allopatric. The region w ith the highestpercentage score was then considered of highest priorityin terms of preserving taxonomic diversity. Next orderpriorities were determined by eliminating all taxa foundin the highest priority region and repeating the analysisof the remaining regions. This approach takes intoaccount the complementarity of species distributions forsubsequent rankings (Vane-Wright et al . , 1991).

The Vane-Wright et a l . (1991) approach has theadvantage of incorporating information on phyloge-netic relatedness, but does not incorporate informationon genetic distinctiveness of a particular species. The

second phylogenetic approach was therefore employedtaking into accou nt the branch lengths of the phylogenyand thereby weighting groups according to geneticdiversity, as well as their taxonomic diversity (Faith,1992). The phylogenetic diversity index, PD , was calcu-lated for a group of taxa by simply sum ming the branchlengths of the branches interconnecting that group.Crozier (1992) has developed a similar technique forincorporating branch length information. Faith (1994)has shown that these methods are monotonically rela-ted, therefore only the method of Faith (1992) wasemployed in this study.Nonphylogenetic assess men t of rarityAn alternative approach for determining species pro-tection status was an assessment of rarity of that spe-cies. Degree of rarity was assessed for each crayfishspecies using geographic range, habitat specificity, andlocal population size following Rabino witz et a l . (1986).Wide or narrow distribution was determined relative tothe Ozark crayfish species in this study, so, widespreadspecies are still somew hat limited in distribution,e.g. O rconectes lu t eus is found only in the northernOzarks, the Current River, and in prairie stream s ofnortheastern Miss ouri (Pfheger, 1987). Geographic rangeswere calculated from species distribution maps providedby Plheger (1987), Williams (1954), and Page (1985).The ranges were dichotomized using a cluster analysisof Km eans in the statistics software package SYSTA T.Habitat specificity and local population size were deter-mined from literature accounts, mainly Pllieger (1987)and Williams (1954) and personal observations.Study areaThe Ozark fauna1 region is located in the central UnitedStates and encompasses much of southern Missouri an dnorthern Arkan sas (Fig. 1). The region largely overlapswith the Ozark Plateaus Phy siographic Province, char-acterized by older bedrocks, higher elevations, andgreater local relief than s urrounding areas (Pflieger, 1989).The streams of the Ozarks are typically narrow, steep-sided valleys, frequently bordered by high bluffs. Thedistributions of native fish fauna show a strong corre-spondence with variations in certain physical features ofstreams (such as turbidity, substrate, base flow, andgradient), makin g fish distribution a useful criterion fordescribing aquatic habitat regions (Pflieger, 1971;Matthews and Robison, 1988; Ptlieger, 198 9; Matthewset al . , 1992). Substrates in these streams include coarsegravel, rubble, boulders, and bedrock. Stream gradientsare generally high and the channels consist of a series ofwell-defined riffles and pools. T he Ozarks have beensubdivided into six regions characterized by the majorriver drainages within each region, including Neosho,Wh ite, Black (including the Black, Current, andEleven-Point rivers), Southeast (including the St. Fran-cis, Castor, and Wh itewater rivers), Miss issippi (theMeramec river system), and Missouri (including the

8/12/2019 84(2)107-1998

3/11

Conser va t i on phy l ogenet i cs o f Oza r k c ray f i shes 109

38

96 94 92 90

40d

38

36

34

94 92 90

Fig. 1. Physiographic and faunistic sub-regions of the OzarkPlateaus: A-Neosho, &W hite, C-Black, & Southeast,E-M ississippi, F-M issouri. Major river drainages and tri-butaries are as follows: l- Mis sissippi, 2-M issouri, 3 Osage,4-Niangua, S-Gasconade, bMeramec, 7-Spring, &Elk,9-White, IO-North Fork, 1 -Eleven Point, 12--Current,13-Black, l&S t Francis, 1S-Castor, lbwhitewater.

Niang ua, Osage , and Gasconade rivers). These regionsof the Ozark Plateaus will be assessed for conservationpriorities based on crayfish taxonomic and phylogeneticdiversity.Samples and dataCray f i sh samp lesThe crayfish used in this study were collected by handduring 1989-1 992 (Table 1). Upon capture, the abdo-men and gill tissues were dissected from the crayfish, p utinto cryotubes, and frozen imm ediately in liquid nitro-gen. The tissues were then returned to the lab and storedat -80C to await DN A extraction. A form I male anda female were put directly into 70 EtOH for preser-vation a s voucher specimens when available; male cray-fish of the family Camb aridae are sexually dimorphicwith the form I male being sexually active show ingsclerotized, corneous, an d lengthened terminal elementsof at least the first pleopod while form II males, thenonbreeding form, possess terminal elemen ts that arenot well differentiated and are never comeous. Identifi-cations were verified by J. F. Fitzpatrick Jr of the Uni-versity of South Alab ama. The voucher specimens hav ebeen deposited in the University of Alaba ma DecapodCollection and are available upo n request.

Phy logeny recons t r uc t i onNucleotide sequence data were obtained from the 16sregion of the mitochondrial DNA (mtDNA) using PCRamplification and cycle sequencing as described inCranda ll and Fitzpatrick (1996). DN A sequences werealigned using the program CLUSTALV (Higgins et al . ,1992). Insertion and deletion gaps were coded as miss-ing characters and recoded as single events for phylo-genetic analysis using parsimony, as suggested bySwofford (1 993). Phylogenetic signal within this datasetwas assessed using the gl, statistic of the random treedistribution (Hillis and Huelsenbeck, 1992), using theRAN DOM TREE option in PAUP (Swofford, 19 93).Significance levels for the gl, statistic were obtainedfrom Table 3.2 in Hillis and Huelsenbeck (1992).

Given significant phylogenetic signal, phylogenyreconstruction was carried out using parsimony analy-ses by performing the heuristic search option in PAU Pwith random addition of taxa (Swofford, 1993). Tocheck for the possibility of multiple tree islands (Hendyet a l . , 1988; Maddison, 1991) at least 100 random addi-tion searches were made for each alternative weightingscheme. A stepmatrix based on empirically derivedprobabilities was used to weight mutational changes.This stepmatrix resulted in a weighting of transversionsto transitions of 2:l. A transition bias in mtD NA iscommonly encountered (Moritz et a l . , 1987) and a 2:lweighting of transversions to transitions better reflectsthe molecular biology of the DN A region. For opera-tional taxonomic units (OTUs) that differed by fewerthan 10 mutational steps, the method of Templeton eta l . (1992) was used to assign most parsimonious rela-tionships. Crandall (1994) has demon strated the effec-tiveness of this statistical parsimony procedure and itscombined use with traditional methods of phylogenyestimation (Crandall and Fitzpatrick, 1996). All treeswere rooted with Procamba rus acu tus serving as theoutgroup to the Ozark end emic species. A heuristicassessm ent of confidence in the resulting relationshipswas performed by using the bootstrap procedure (Fel-senstein, 1985, 1988) for all methods except the Tem-pleton et a l . method. Here probabilities were assignedusing equation (8) of Templeton et a l . (1992). Thebootstrap percentile v alues were based on 200 bootstrapreplications and shou ld be taken only as a heuristicguide to the confidence in a particular node (Hillis andBull, 1993).

Alternative tree topologies were tested for significantdifferences usin g the nonparam etric sign test (Pragerand Wilso n, 1988). This test counts the number ofmutational changes supporting topology A vs thosesupporting topology B and compares the relative levelsof support against a binomial distribution with the nullhypothesis of p=O .50, i.e. equal su pport for eithertopology. Unlike Prager and Wilson (1988) I used atwo-tailed test as a more conservative test of compatibletree topology. When the total number of characters dif-ferentiating two topologies was >2 5, the large sample

8/12/2019 84(2)107-1998

4/11

110 K. A. Crandall

approximation was used to test alternatives. This teststatistic has an asymptotic N(0, 1) distribution (Hollan-der and Wolfe, 1973). This test is an unweighted versionof the Wilcoxon signed rank test presented by Temple-ton (1983). The number of mutational changes su p-porting one topology relative to another were calculatedusing the COM PARE TWO TREES option in Mac-Clade (Maddison and Maddison, 1992).

R SULTS

Phylogeny reconstructionA heuristic search resulted in one most parsim onioustree (Fig. 2). There was no evidence of multiple treeislands within the dataset. These data were significantlyskewed indicating s ignificant phylogenetic signal(gi = -0.594, p < O-01). Restricting the dataset to include

only those OTU s that differed by 210 nucleotide sub-stitutions resulted in a gl score of -0.565, once againindicating significant phylogenetic signal (p < 0.01).Those O TUs that differed by < 10 nucleotide substitu-tions were connected using the procedure in Templetonet al. (1992). To perform the Vane-W right et al (1991)procedure, a tree with terminal taxa equivalent to spe-cies is required. After asse ssing the monophyly of eachspecies (see below), the tree was pruned so that each tiptaxon represented a unique taxon (e.g. 0. luteus l-4 inFig. 2 to 0. luteus Fig. 3). In some instances, non-monophyletic taxa were pruned as well. The justificationfor pruning these taxa is given in the Species Statussection below.Taxonomic and phylogenetic diversityThe taxonomic diversity index of Vane-W right et al.(1991) which ranked for priority in conservation efforts

SpeciesTable 1. Specimens utilized in this study, listed in alphabetical order

Label0 County, State Major drainageCambaru s Erebicambar us) hubbsiC. E.) maculatusOrconectes Billecambarus) harrisonii0. Buannult fi ctus) meeki 10. B.) meeki 20. B.) palm eri longimanus0. Cro ckeri nus) eupunctus0. Gremicambarus) vi ri li s 10. G.) vi ri li s 20. Procericambar us) hyl as 10. P.) hy las 20. P.) longidi gitus 10. P.) longidi gitus 20. P.) luteus I0. P.) lu t eus 20. P.) l ut eus 30. P.) lu t eus0. P.) macru s 10. P.) macrus 20. P .) medius 10. P.) medius 20. P.) nana 10. P.) nana 20. P.) neglectus chaenodacty l us 10. P. neglectus chaenodacty l us 20. P.) negl ect us neglectus 30. P.) ozar kae 10. P.) ozark ae 20. P.) ozar kae 30. P.) 1eruncus0. P.) 2eruncus0. P.) puncti manus 10. P.) puncti manus 20. P.) puncti manus 30. P.) puncti manus 40. P.) quadruncus 10. P.) quadr uncus 20. P.) w ii hamsi0. Tragulicambarus) lanciferProcambarus Ortmannicus) actus

hubb 122macu 63harr 139meek 205meek 230palm 457eupu 120viri 143viri 95hyla 103hyla 105long 212long 213lute 96lute 23lute 13lute 112macr 234macr 218medi 151medi 77nana 192nana 193negl 181negl 132negl240ozar 172ozar 170ozar 127pent 137per-u 138punt 119punt 160punt 164punt 114quad 185quad 186will 221lane 513acut 102

Oregon, MOCrawford, MOWashington, MOMadison, ARWashington, ARLeFlore, OKOregon, MOM OCrawford, MOIron, MOIron, MOMadison, ARMadison, ARCrawford, MOCrawford, MOCrawford, MOTexas, MOLawrence, MOMcDonald, MOCrawford, MOCrawford, MOBenton, ARBenton, AROzark, MOOzark, MOLawrence, MOOzark, MOFulton, AROregon, MOWayne, MOWayne, MOOregon, MOPhelps, MOTexas, MOTexas, MOMadison, MOMadison, MOBarry, MOAlexander, ILSt Charles, MO

Eleven Point RiverMeramec RiverBig RiverWhite RiverWhite RiverCedar LakeEleven Point RiverNiangua RiverMeramec RiverBlack RiverBlack RiverWhite RiverWhite RiverMeramec RiverMeramec RiverMeramec RiverBig Piney RiverSpring RiverElk RiverMeramec RiverMeramec RiverWhite RiverWhite RiverLittle North ForkNorth ForkSpring RiverNorth ForkSpring RiverSpring RiverSt.Francis RiverSt.Francis RiverEleven Point RiverLittle Piney RiverBig Piney RiverBig Piney RiverSt.Francis RiverSt.Francis RiverWhite RiverMississippi RiverMississippi River

uRepresents numerical identification label used in tissue collections with the first four letters being that of the species name and theunique number identifying the animal.

8/12/2019 84(2)107-1998

5/11

Conser va t i on phy l ogenet i cs o f Oza r k c ray f i shes 111the six geographic regions of the Ozark Plateaus (Pl- fourth. The Southeast and Misso uri River regionsP5; Fig. 3), placed highest priority on the White River ranked fifth and sixth, respectively.region, with the Mis sissipp i River region close behind The PD index of Faith (1992) ranked the Black River(Pl an d P2; Fig. 3). The Neosho River region wa s region as most worthy of conservation efforts (Table 2)ranked third in priority using this index having the with the White River region and the Mississippi Riverhighest score at the P3 level. In the first priority rank- close in priority. The values for the remaining regions ofings (Pl), the Black River region had a much higher the Neosho River, the Southeast, and the Misso uripercentile value th an the Neosho region, but the River were much lower. Simple species count per areaNeosho region ranked higher than the Black River and a count of rare species (those falling into the var-region at the P3 level. This is because many of the spe- ious rare categories as defined below , Table 3) resultedties distributed in the Black River region are also dis- in similar rankings. The PD index was also used to ranktributed in the White River region. Thu s when species of individual species with values show n in Fig. 2. Thus 0.the Wh ite River region were removed for the calculation h y l a s would have the lowest relative priority amongof P3 values, the priority of the Black River dropped to crayfishes examined b ased on phylogenetic diversity

15 036 d 0

bngidigitus 11 longidigtus

Phylogenetic Diversity

012

1El11

II

16

Fig. 2 Maximum parsimony tree inferred from 16s nucleotide sequence data from the mitochondrial genome. Numb ers in bold/italics on the nodes are the bootstrap percentile values based on 200 bootstrap replications. An (*) indicates a connection using theprocedure in Templeton et al. (1992) that was supported at a 95 confidence level (equation (8) in Templeton et al. (1992)).Numbers along branches indicate the number of unambiguous changes along that branch as calculated using MacClade (Maddisonand Maddison, 1992). Phylogenetic diversity for each population as calculated by the method of Faith (1992) is shown in a box formultiple individuals or standing alone for single individuals.Table 2. Pbylogenetic diversity associated with tbe Ozark regions calculated by the method of Faith 1992)

Ozark regionsNeosho White Black Southeast Mississippi Missouri

No. species 5 9 3 6 4No. rare species 3 7 1 4 1Phylogenetic diversity 68 115 117 48 111 18

8/12/2019 84(2)107-1998

6/11

2 K . A . C r a nda l l

w 1NO Wh BI SE MS MOI .0 llltells0. medius0 ne@ctus 1

l _ 12 0 quadruncus

415 0 longidigitus

1

1 4

1- 3

5r 10 0 meeki2

4

12 0 wil l iamsi

P acutus

1.181.181.301.441.631.632.172.172.17

2.602.604.334.334.33

f

f

t

Total: 41. 12 8.04 18. 45 12. 98 3.36 15. 52 2.18Pl : 19. 6 44. 9 31. 6 8.2 37. 7 5.3P2: 10. 6 - 12. 1 5.3 34. 9 2.4P3: 10. 6 - 6.8 2. 9 - 0.0P4: 6 8 2.9 - 0.0P5: _ 2.9 - 0.0

Fig. 3. Maximum parsimony tree with duplicate individuals of the same species eliminated from the analysis and branch lengthsshown along the branches. W represents the taxonomic weights calculated by the method of Vane-Wright et al. (1991). The aquaticsubdivisions within the Ozark Plateaus are as follows; Ne = Neosho , Wh = White, Bl= B lack, SE = Southeast, MS = Mississippi, andMO = Missouri. Pl-P5 give the percentage diversity scores working through the five levels of priority ranking. The region with thehighest value at a particular priority level receives that priority, e.g. the White R iver region has the highest score at the Pl level, thusit is highest in priority.

Table 3. Rarity of C anrbarus, Orconectes, and procom barus crayl hes baaed on geographical distribution (km*), habitat specificity,and local population size in Ozark Pla teaus (after Rabinowitz et al., 1 986)Geographic distribution Wide NarrowHabitat specificity Broad Restricted Broad RestrictedPopulation sizeSomewhere large

Everywhere small

0. luteus (251,360)0. neglectus neglectus(200,275)0. palmeri (250,500)0. puntimanus (215,995)0. virilis (350,000)P. ucutus (300,000)

C. hubbsi 0. macrus143,950) 22,791)0. longidigitus 0. medius170,015) (11,457)0. ozarkae 0. nana 16,942)

0 ancifeP275,000)0. meeki220,000)

C. maculatus 23,040)0. eupunctuf 10,300)0. harri sonif 8,500)0. hylu s 23,040)0. neglectuschaenodactylus 28,610)0. peruncuP 3,225)0. quadruncuf 5,616)0. will iamsFb 5,547)

YSpecies currently listed in the Rare and Endangered Species of Missouri Checklist.bSpecies currently listed as candidate species by the US Fish and Wildlife Service.The total distributional area for 0. ozarkae was estimated to be 118,905 km2, but if one divides up the area represented by the twophylogenetically distinct populations (Fig. 3) then each population is placed in the narrowly distributed group with distributionalareas of 43,079 km* for 0. ozarkae 1 and 75,826 km2 for 0. ozarkae 2.

8/12/2019 84(2)107-1998

7/11

Conser va t i on phy lo genet i cs o f Oza r k c ray f i shes 113because it has the lowest PD score. 0. macrus and0. pa lme r i share the highest score.Rarity of way&& speciesThe first component of the rarity analysis is the distri-butional area. Cluster an alysis of species ranges resultedin two statistically significant clusters (p < 0.001,F-ratio), corresponding to species distributed over largeor small areas. Those species within the wide geogra-phical distribution cluster had a minim um range of143,950 km2 and a maximum range of 350,000 km2 witha mean of 225,23 0 km2 (Table 3 ). Narrowly distributedspecies had a minim um distributional range of3,225 km2 (0. peruncus), a maximum range of75,826 km2 with a mean of 21,380 km2 . Further differ-entiation based on local population sizes and habitatspecificity are given in Table 3. Species marked super-script a are currently listed in the Rare an d End an-gered Species of Misso uri Checklist (1991). All speciesin the most restricted definition of rarity (narrow distri-bution, restricted habitat specificity, and local popula-tion size everywhere small) are currently listed by thestate of Missouri as rare. O nly half of the species listedin the next priority category (narrow distribution,restricted hab itat specificity, and local population sizesomewhere large) are currently listed by Misso uri. Thestate of Arkan sas has no endangered species checklist.Only Orconect es w i l l i a m s i was listed on the US FederalEndangered Species list as a candidate species.

DISCUSSIONSystematic implicationsThe determination of species bound aries is perhaps themost important area of application of phylogeny toconservation biology. M any species concepts have phy-logenetic relatedness as the central focus in the deter-mination of species, e.g. phylogenetic species concept(Cracraft, 1983), cohesion species concept (Templeton,1989), evolutionary species concept (Wiley, 197 8). Fur-thermore, phylogeny can be used to assess the degree ofgenetic isolation betw een two populations (Slatkin andMad dison, 1989). It is of primary importance to estab-lish whether or not distinct populations are distinct

species within an hypothesis testing framework (Sitesand Cranda ll, 1997). This determination greatly influ-ences conservation policy in terms of introductions andlistings as rare and endangered. In the following section,I explore the species status of various crayfish popu la-tions based on the phylogenetic information available.

The phylogeny determined by 16s mtDNA shown inFig. 2 places a number of crayfish species in non-monophyletic groups. Und er certain species concepts(e.g. Cracraft, 1983), species must form monophyleticgrouping s. Other concepts (e.g. Templeton, 1989) allowfor non-monoph yletic relationships within a species. Totest the monophyly of these species, an alternativetopology with the species in question as a monophyleticgroup was compared to the tree in Fig. 2 using thesign test. The results of these comparison s are shownin Table 4, indicating that both 0. quadruncus and0. oza r kae form statistically significant non-monophy-letic relationships.One explanation to the non-monophyly of mtD NAhaplotypes within a species is the problem of gene treesreflecting species (or population) trees. The results oftheoretical population genetic work indicate that forsome time after the divergence of two or more popula-tions, there is a high probability that populations mayshow non-monoph yletic relationships for a specificgene; therefore, the gene genealogy may not accuratelyreflect the population divergence. Neigel and Avise(1986) and Takahata and Slatkin (1990) have shownthat, in fact, the expected progression of gene phyloge-nies following divergence is polyphyletic + paraphyletic+ mo nophyletic. The probability of monophyly is sig-nificantly reduced by the presence of gene flow (Taka-hata and Slatkin, 1990) increases with smallerpopulation size (Padm adisastra, 1987) and is affectedby the geographic distribution of the populations (Nei-gel and Avise, 1986). Given these results an d theapparent recent divergence of the crayfish populationsin this study (as evidenced by the relative lack of geneticdivergence amon g individuals) patterns of nonmono -phyly may reflect this recent divergence and the discor-dance between 16s mtDNA genealogy and the speciesphylogeny.

Althoug h monophyly is a requirement for a limitednumb er of species concepts (e.g. Cracraft, 1983), it is ofTable 4. Testing monophyletic groups: Orconectes species

Taxonomic group Nonmonophyly Monophyly B p value Bf. p0. quadruncus 30 2 4.95 < 0.0002 < 0~00020. neglect us 9 3 0.073 0.2920. ozarkae 15 0 3.87 < 0~0002 * < 04008 0. punctimanus 6 0 0.0156 0.062The values given under nonmonophyly and monophyly indicate the number of characters for that particular hypothesis was shortercompared to the alternative hypothesis. B is the large sample approximation for the sign test given in equation (2). When B con-tains no value, the B statistic wa s used which is simply the value under Maximum Parsimony. p values for the B statistic are one-sided from a normal distribution (Hollander and Wolfe, 1973) and those for B are one sided from a binomial distribution withp = 0.5 (Hollander and Wolfe, 1973). Bf. p are the p values with the Bonferroni correction for multiple com parisons (Neter et al.,1985), *p < 0.05; *p < 0401.

8/12/2019 84(2)107-1998

8/11

114 K. A . C r anda l linterest to examine the particular cases of non-mono-phyly. W e reject the hypothesis of species monophylyfor both 0. oza r kae and 0. quadruncus. The distinctpositioning of two populations of 0. oza r kae corre-sponds to isolated drainages with ozar 127 and 170 fromthe Spring River drainage in Arkansas and ozar 172from the North Fork of the Wh ite. These two popula-tions have been identified as having two distinctmorphologies (Pflieger, pers. comm.) a nd are geneticallydifferentiated (p < 0@ 08, Table 4). Furthermore, thissame geographic separation and degree of genetic isola-tion has been observed in the hellbender salamand erCryp tob ranchus b i shop i (Routman et al. , 1994), addingsupport to a hypothesis of vicariant biogeographic iso-lation of population s. Finally, Pflieger (1989) and Mat-thews et a l . (1992) have suggested these drainages areecologically distinct from one another, but detailedecological studies are needed to confirm ecologicaldivergence for these two populations of crayfish. Giventhis suggestive ecological, phylogenetic, and morpholo-gical differentiation, these two populations of 0. oza r kaemay pass species criteria for the phylogenetic, evolu-tionary, and cohesion concepts of species. The speciesstatus via the recognition concept and the biologicalspecies concept will require further study.

An alternative explanation is that this particularindividual (ozar 172) is the result of a hybridizationevent between an 0. oza r kae male and an 0. pun c t ima -nus/v i r i f i s see below) female. This hypothesis is sup-ported by the low amoun t of variation between ozar 172and punt 119, 160, and 164. Punt 160 and 164 haveidentical nucleotide sequences for the 16s region. Punt119 is two nucleotide substitution s different from punt160/l 64 and three nucleotide substitution s differentfrom ozar 172 . Because mtDN A is maternally inherited,a single successful hybridization of a female pun c t ima -n u s l v i r i l i s with an oza r kae male would result in offspringwith the pun c t imanu s l v i r i l i s mtDNA genotype. Hybridi-zation h as been documented in crayfishes, bu t is con-sidered to be a very rare phenomen on (Capelli andCapelli, 1980; Smith, 1981; Berrill, 1985; Cesaroni et al . ,1992). Once again , this result requires a further exami-nation of the species status of 0. oza r kae using moredetailed population genetic information.

0. quadruncus and 0. peruncus are hypothesized tohave allopatric distributions within the St. Francis R iverdrainage in Misso uri. 0. quadruncus is distributed in theSt. Francis River drainage exclusive of Big Creek, thedrainage supporting populations of 0. peruncus (Wil-liams, 1954 ; Pflieger, 1987). Because of the extensivedifferentiation between quad 185 and 1 86, two indivi-duals foun d at the same locality, it is suggested here thatquad 186 is really a sample of 0. peruncus. The 16ssequence obtained from q uad 186 is only 1 nucleotidedifferent from peru 137 and 2 substitution s differentfrom peru 138. Because n o form I male was collected forthis population, positive identification was not possible.This evidence does suggest, however, that these species

do occur sympatrically, at least in Cedar Bottom Creekin Ma dison County, Miss ouri. This result warrants fur-ther investigation into the range of 0. peruncus.

Althoug h the monophyly of the species O rconectesneglectus cannot be rejected, it is interesting to note thatthe two populations of this species that reside in differ-ent areas of the phylogeny represent two subspeciesfrom isolated drainages: negl 240 O rconectes neglectusneglectus) is from the Spring River d rainage in WesternMissouri and Eastern Kansas, and negl 132 and 181O rconectes neg lec tus chaenoda cty lu s) are from the

North Fork of the Wh ite River drainage. Their place-ment in two distinct areas in the phylogeny sugges ts thepossibility of sufficient divergence to be considered dis-tinct species. Obviously, population genetic analyses ofthese two subspecies is warranted to determine speciesstatus.

Implications for conservation biologySpecies statusThe status of species currently listed by the State ofMisso uri in the Rare and Endangered Species of Mis-souri Check list (199 1) is supported by this study. T able 3show s that the majority of those species listed fall withinthe most threatened category: narrowly distributed,restricted in habitat with small population sizes. Twospecies, O rconectes la nc i fe r and O rconectes meek i , arewidely distributed, but with restricted h abitat specificityand small population sizes. These species are narrowlydistributed within the state of Misso uri resulting in theirinclusion in the checklist. 0. har r ison i i , 0 . eupunct us ,and 0. w i l l i a m s i are also listed by the state; they havenarrow distribution s and restricted habitat specificitybut large local populations. The latter two have a highphylogenetic diversity ind ex and all have a moderatetaxonomic diversity ind ex (Fig. 2). Based on this infor-mation, I recommend changes in conservation status forseveral species. Two crayfish species, 0. peruncus and0. quadruncus, are currently listed by the state with thestatus of Watch List,

i.e. not currently rare or endangered, but has arestricted distribution or has experienced sufficientdecline to indicate it may soon becom e Rare orEndang ered would be changed to Rare,where Rare indicates present in small numbers. Ifenvironment worsens, status in Misso uri coulddeteriorate to Endang ered.These species are narrowly distributed, with restricted

habitat and small population sizes. Three other speciesnot yet listed by the state of Misso uri, 0. nana, 0.macrus , and C. macu la t us , are recommended to beadded as Rare. These species are locally abunda nt, butshow narrow geographic ranges and habitat specificity.Furthermore, each species ranks extremely high forboth phylogenetic and taxonomic diversity. I further

8/12/2019 84(2)107-1998

9/11

Conser va t i on phy l ogenet i cs o f Oza r k c ray f i shes 115Table 5. Current and recomm ended status of Ozark crayiishes

SpeciesCambarus hubbsiC. maculatusOrconectes eupunctus0. harrisonii0. hylas0. lanctjier0. longidigitus0. luteus0. macrus0. medius0. meeki0. nana0. neglectus0. ozarkae0. palmeriO .peruncus0. punctimanus0. quadruncus0. v i r i l i s0. wi l l iams i

Current status?NoneNoneRWLNoneENoneNoneNoneNoneENoneNoneNoneNoneWLNoneWLNoneR

Suggested statusWLRRWLWLENoneNoneRWLERNoneNoneNoneRNoneRNoneR

Rarity TD PDW:R:L 4.33 14N:R:L 4.33 18N:R:L 1.63 14N:R:L 1.63 7N:R:L 1.18 6W:R:S 2.60 20W:R:L 144 15W:B:L 1.00 12N:B:L 2.17 26N:B:L 1.00 12W:R:S 2.17 11N:B:L 2.17 21W:B:L 1.26 16W:R:L 1.24 14W:B:L 2.60 26N:R:S 1.18 9W:B:L 1.18 12N:R:S 1.18 11W:B:L 1.18 4N:R:L 4.33 12

TD is taxonomic diversity; PD is phylogenetic diversity; Rarity: geographic distribution-W = wide, N = narrow; Habitat specifi-city-B = broad, R = restricted; local population size-L = large, S = small.?jtatus as listed in the Rare and Endangered Species of Missouri Checklist (1991).recommend three additional species be listed as WatchList (C. hubbsi, 0. hyl as, and 0. medius) based mainlyon the restricted distribution of the O rconectes speciesand the high taxonomic and phylogenetic diversityindices for C. hubbsi. The designation of 0. hylas and 0.medius as Watch List species is recommended princi-pally on the rarity criterion. Table 3 show s both of thesespecies as narrowly distributed with habitat specializa-tion, but with large population sizes. Neither speciesranks h igh in terms of taxonomic or phylogeneticdiversity resulting in their Watch List status. C. hubbsi,on the other han d, is widely distributed with largepopulations but with a restricted habitat. The listing ofthis species as Watch List is due to its high taxonomicdiversity index. A summ ary of these recommended list-ings is given in Table 5, as well as associated data forcategorizations.Habitat pr ior i t iesThe rankings of the six Ozark regions are summarizedin Table 6. Notice that no matter what the measure, it isclear that the White, Black, and Mississippi regions areof higher priority than the Neosho, Southeast, and

Misso uri regions. It is also interesting to note that a llmeasures give nearly equivalent rankings of regionswith the notable exception of the taxonomic diversityindex. Here, the Black River region receives a muchlower ranking than from other m ethods. This is due tothe complementarity aspect of the TD index (Vane-Wright et al., 1991). The TD measure initially ranks theBlack region very high, but because many of the speciesin this region overlap with those in the White Riverregion, which is ranked higher, subsequ ent ranking ofthe Black River region is reduced. I do not claim thatone measure is better or more meaning ful than ano ther,but any differences presum ably reflect different biologi-cal aspects un der co nsideration as well as differentmethodological philosophies (K rajewski, 1994). Theadvantage to these approaches is that they offer harddata upon which one can base sound decisions. Ofcourse, this ranking scheme is primarily for the primarybenefit of crayfish. It would be worthwh ile to perform asimilar exam ination of these fauna1 regions using a dif-ferent taxonomic group. However, because theseregions are based on distributional data from manyspecies including fish and have been shown to correlate

Table 6. A compa rison of various ranking criteria for faunistic regions of the OzarksOzark regions

Neosho White Black Southeast Mississippi MissouriSR Rank 4.5 1 2 4.5 3 6RS Rank 4 1 2 5.5 3 5.5TD Rank 3 1 4 5 2 6PD Rank 4 2 1 5 3 6StlIll 15.5 5 9 20 11 23.5SR-Species Richness, RS-Rare Species, TD-Taxonomic Diversity, and PD-Phylogenetic Diversity.

8/12/2019 84(2)107-1998

10/11

116 K. A . C r anda l l

with a numb er of ecological attributes, it is expectedthat the present assessm ent w ill have broad applicationover these freshwater ecosystems.The phylogenetic perspectiveThe strength of the phylogenetic approach is in theassessm ent of species status as independent lineages andidentifying imp ortant areas for further populationgenetic studies. Furthermore, the phylogenetic informa-tion adds additional parameters (taxonomic diversityand phylogenetic diversity represented by a species) tothe assessment of species rarity status and assessmentsof habitat priorities. Indeed, a s preserving geneticdiversity is often a goal in conservation biology (Tem-pleton, 1991; Crozier, 1992), it seems pertinent toinclude some measure of genetic distinctiveness into aweighting scheme for habitat preservation. The infor-mation gained from this assessm ent of habitat prioritiescan be an important additional source of data, yet theconclusions drawn from these data are similar to thosedrawn from species richness assessments. These quanti-tative measures of taxonomic and phylogenetic distinc-tiveness combined with ecological considerations ofabundan ce and geographic distribution allow for accu-rate assessmen ts of the endangered status of the speciesof crayfish examined herein and the ranking of subre-gions in the Ozarks for conservation priorities. As morestudies are performed that include a phylogenetic com-ponent to the assessm ent of species rarity and habitatpriority, a better picture of the various utilities of thephylogenetic approach will emerge.

CKNOWLEDGEMENTSI would like to thank Cindy and Kyle Crandall, Ma r-shal and Jennifer Hedin, Chris Phillips, and Eric Rout-man for assistance in field collections of these and manyother crayfishes. Joy Bergelson, Laurie Dries, NickGeorgiadis, Anne Gerber, Delbert Hutchison, ChrisPhillips, Ow en Sexton, and Alan Templeton providedhelpful comm ents on the manus cript, as well as livelyand enlightening discussion s in the area of conservationbiology. Jo e Fitzpatrick and Bill Pflieger providedinvaluable support and encouragement with my learn-ing of the crayfish taxonomy. The Missouri Departmen tof Conservation and the National Science Foundation(BSR-9 11200 0) provided funding for this research.

REFERENCESBerrill, M. (1985) Laboratory induced hybridization of twocrayfish species, Orconect es rusti cus and 0. propinquus. J.

Crust , Bi ol . 5, 347-349.Capelli, G. M. and Capelli, J. F. (1980) Hybridization betweencrayfish of the genus Orconectes: Morphological evidence(Decapoda, Cambaridae). Crustaceana 39, 121-132.

Cesaroni, D., Allegrucci, G. and Sbordoni, V. (1992) A nar-row hybrid zone between two crayfish species from a Mexi-can cave. J. Evol . Bi ol . 5, 643659.Cracraft, J. (1983) Species concepts and speciation analysis.Current Orni thology 1 159-187.Cracraft, J. (1994) Species diversity, b iogeography, and theevolution of biotas. Amer. 2001. 34, 33-47.

Crandall, K. A. (1994) Intraspecific cladogram estimation:Accuracy at higher levels of divergence. Syst. Bi ol. 43, 222-235.Crandall, K. A. and Fitzpatrick Jr, J. F. (1996) Crayfishmolecular systematics: using a combination of procedures toestimate phylogeny. Syst . Biol . 45, l- 26.Crozier, R. H. (1992) Genetic diversity and the agony ofchoice. Bi ol . Conserv . 61, 1 -l 5.Eldredge, N. (1992) Systemati cs, Ecology, and the Biodi versit yCrisis. Columbia University Press, New York.Faith, D . P. (1992) Conservation evaluation and phylogeneticdiversity. Bi ol. Conserv. 61, l-10.Faith, D. P. (1994) Genetic diversity and taxonomic prioritiesfor conservation. Bi ol. Conserv . 68, 69-14.Felsenstein, J. (1985) Confidence limits on phylogenies: anapproach using the bootstrap. Evol uti on 39, 783-791.Felsenstein, J. (1988) Phylogenies from molecular sequences:inference and reliability. Annu. Rev. Genet. 22, 521-565.Greene, H. W. (1994) Systematics and natural history, foun-dations for understanding and conserving biod iversity.Amer. Zool . 34, 48-56.Hendy, M. D., Steel, M. A., Penny, D . and Henderson, I. M.(1988) Families of trees and consensus. In ClassiJicati on andRelat ed Methods of Dat a Analy sis, ed. H. H. Bock, pp. 3362. Elsevier, Amsterdam.Higgins, D. G., Bleasby, A. J. and Fuchs, R. (1992) CLUS-TAL V : improved software for multiple sequence alignment.Comput . Appl ic. Biosci. 8, 189-191.Hillis, D. M. and Bull, J. J. (1993) An empirical test of boot-strapping as a method for assessing confidence in phyloge-netic analysis. Syst. Bi ol . 42, 182-192.Hillis, D. M. and Huelsenbeck, J. P. (1992) Signal, noise, andreliability in molec ular phylog enetic analyses. J. Hered. 83,189-195.Hollander, M. and Wolfe, D. A. (1973) Nonparametric Statis-ti cal Methods. John Wiley and Sons, New York.Krajewski, C. (1994) Phylogenetic measures of biodiversity: Acomparison and critique. Bi ol. Conserv . 69, 33-39.Lodge, D . M. (1993) Biological invasions: lessons for ecology.Trends in Ecology and Evol uti on 8, 133-137.Maddison, D. R. (1991) The discovery and importance ofmultiple islands of most-parsimonious trees. Syst. Zool . 40,315-328.Maddison, W. P. and Maddison, D. R. (1992) MacClade:Anal ysis of Phylogeny and Character Evoluti on. SinauerAssociates, Sunderland, M A.Matthew s, W. J. and Robison, H. W. (1988) The distributionof the fishes of Arkansas: A multivariate analysis. Copeia1988, 358-374.Matthew s, W. J., Hough, D. J. and Robison, H. W. (1992)Similarities in fish distribution and wat er quality pat terns instreams of Arkansas: Congruence of multivariate analyses.Copeia 1992, 296-305.May, R. M. (1990) Taxonomy as destiny. Nat ure 347, 129-130.Moritz, C., Dowling, T. E. and Brown, W. M. (1987) Evolu-tion of animal mitochondrial DNA : R elevance for popula-tion biology and systematics. Annu. Rev. Ecol. Syst. 18,269-292.Neigel, J. E. and Avise, J. C. (1986) Phylogenetic relationshipsof mitochondrial DNA under various demographic modelsof speciation. In Evolutionary Processes and Theory. eds,

8/12/2019 84(2)107-1998

11/11

Conser va t i on phy l ogenet i cs o f Oza r k c ray f i shes 117S. Karlin and E. Nevo. pp. 515-534. Academic Press, Inc.,New York.

Neter, J., Wasserman, W. and Kutner, M. H. (1985) AppliedLi near Stati stical Models. Richard D. Inrwin, Inc., Home-wood, IL.Padmadisastra, S. (1987) The genetic divergence of threepopulations. Theor. Popn. Biol. 32, 347-365.

Page, L. M. 1985) The crayfishes and shrimps (Decapoda) ofIllinois. Il li nois Nat ural H istor y Survey Bul leti n 33, 335448.Pflieger, W. L. (1971) A distributional study of Missourifishes. Uni versity of Kansas, M useum of Natural Hi story,Publ icat io n Seri es 20, 226570.Pllieger, W. L. (1987) An introduction to the crayfishes ofMissouri. M issouri Conservat ioni st 48, 17-3 1.Pflieger, W. L. (1989) Aquatic Communit y Classtfi cati on Sys-tem for M issouri. Missouri Department of Conservation,Jefferson City, MO.Prager, E. M. and Wilson, A. C. (1988) Ancient origin of lac-talbumin from Iysozyme: Analysis of DNA and amino acidsequences. J. M ol . Evol . 21, 326335.Rabeni, C. F., Gossett, M. and McClendon, D. D. (1995)Contribution of crayfish to benthic invertebrate productionand trophic ecology of an Ozark stream. Freshwat er Cruy -fish 10, 163-173.Rabinowitz, D., Cairns, S. and Dillon, T. (1986) Seven formsof rarity and thei r frequency in the flora of the British Isles.In Conserva ti on Biol ogy: The Sci ence of Scarcit y and D i ver-sit y, ed. M. Soule, pp. 182-204. Sinauer A ssociates, Sun-derland, MA.Routman, E., Wu, R . and Templeton, A. R. (1994) Parsi-mony, molecular evolution, and biogeography: The case ofthe North American Giant Salamander. Evolut ion 48, 1799-1809.Sites Jr, J. W. and Crandall, K. A. (1997) Testing speciesboundaries in biodiversity studies. Conservat ion Bi ology, 11,in press.Slatkin, M. and Maddison, W. P. (1989) A cladistic measureof gene flow from the phylogenies of alleles. Genet ics 123,603-613.

Smith, D. G. (1981) Evidence for hybridization between tw ocrayfish species (Decapoda: Cambaridae: Orconectes) w ith acomment on the phenomenon in Cambarid crayfish. Amer.M id l. Na t. 105,405-407.Swofford, D. L. 1993 PAVP: Phyl ogeneti c Anal ysis Usi ngParsimony. 3.1.1. Smithsonian Institution, Washington,DC.Takahata, N. and Slatkin, M . (1990) Genealogy of neutralgenes in two partially isolated populations. Theor. Popn.Biol. 38, 331-350.Templeton, A. R. (1983) Convergent evolution and non-parametric inferences from restriction data and DNAsequences. In Stat isti cal Anal ysis of DNA Sequence Dat a,ed. B. S. Weir, pp. 151-17 9. Marcel Dekker, Inc., NewYork.Templeton, A. R. (1989) The meaning of species and specia-tion: a genetic perspe ctive. In Speciat io n and It s Consequen-ces, eds D. Otte and J. A. Endler, pp. 3-27. SinauerAssociates, Inc., Sunderland, MA.Templeton, A. R. (1991) Genetics and conservation biology.In Species conservat ion: A Popul ati on-biol ogical A pproach,ed. A. Seitz and V. Loeschcke, pp. 15-29. Birkhauser Ver-lag, Basel.Templeton, A. R., Crandall, K. A. and Sing, C. F. (1992) Acladistic analysis of phenotypic associations with haplotypesinferred from restriction endonuclease mapping and DNAsequence data. III. Cladogram estimation. Geneti cs 132,619633.Vane-Wright, R. I., Humphries, C. J. and Williams, P. H.(1991) What to protect?-Systematics and the agony ofchoice. Biol. Conserv. 55, 235-254.Wiley, E. 0. (1978) The evolutionary species concept recon-sidered. Syst. Zool . 27, 17-26.Williams, A. B. (1954) Speciation and distribution of thecrayfishes of the Ozark Plateaus and Ouachita Provinces.Uni v. Kansas Sci . Bul l . 36, 803-918.Williams, P. H. and Gaston, K. J. (1994) Measuring more ofbiodiversity: Can higher-taxon richness predict wholesalespecies richness? Biol. Conserv. 67, 211-217.