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BiologicalConservation 84 (1998) 107-I 17 0 1998 Elsevier Science Ltd

CONSERVATION PHYLOGENETICS OF OZARK CRAYFISHES: ASSIGNING PRIORITIES FOR AQUATIC HABITAT PROTECTION
Keith A. Crandall*
Department of Zoology and Monte L. Bean Museum, Brigham Young University,Provo, UT 84602-5255, USA

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

Abstract
A molecular phylogeny based on nucleotide sequence data from the 16s region of the mitochondrial genome was estimated for 20 species of crayfish native to the Ozark Plateaus region of Missouri and Arkansas, USA. With this phylogeny and information on geographic distribution, ecological specialization, and species abundance, species were assessed for their status as rare and endangered. Three species not previously listed, Orconectes nana, Orconectes macrus, and Cambarus maculatus, merit inclusion as rare in the Rare and Endangered Species of Missouri Checklist. Three other species merit inclusion with the status Watch List : Orconectes hylas, Orconectes medius, and Cambarus hubbsi. Additionally, the status of the following species merit changing from Watch List to Rare : Orconectes peruncus and Orconectes quadruncus. Fauna1 regions within the Ozark Plateaus were rankedfor conservation priority using species richness, taxonomic diversity, and phylogenetic diversity. The White River, Black River, and Mississippi River regions of the Ozarks were found to rank highest in terms of conservation priorities. 0 1998 Elsevier Science Ltd. All rights reserved. Keywords:

crayfish, phylogeny, endangered species, Ozarks, conservation phylogenetics, species richness.

INTRODUCTION For the planning of conservation policy and agendas at any level (local, state, national, or international), information is required on the relative importance of various habitats and species in the area under consideration. Typically, such information is sought in one of two complementary ways. One approach is to study a particular species of interest to define the endangered status *To whom correspondence should he addressed: 574 Widtsoe Building, Department of Zoology, Brigham Young University, Provo, UT 84602-5255, USA; Fax: (801) 378-7423; e-mail: keith_crandall@byu.edu
107

of that species and the importance of various population genetic and ecological parameters for the conservation of the species. This approach is labor-intensive, expensive, and time-consuming and the resulting information is typically relevant for only the single species under examination. Yet this approach provides excellent detailed information needed to maintain an endangered species. An alternative is to utilize taxonomic and/or phylogenetic richness as an indicator of important habitats which provide for a diversity of organisms and target such habitats in conservation policy. The weakness of this approach is that detailed population genetic data is not obtained for endangered species. The advantages to this approach are that it can identify whole regions of conservation interest as well as present preliminary information for directing further studies at the population genetic level. Many authors have emphasized the importance of systematics in the field of conservation biology (Templeton, 1991; Eldredge, 1992; Cracraft, 1994; Greene, 1994). Systematics and taxonomy define the groups of organisms for consideration as well as the diversity within and among these groups. Recently, methods have been developed to utilize the phylogeny of a species group to define a quantitative framework for assessing conservation priorities (May, 1990; Vane-Wright et al., 1991; Crozier, 1992; Faith, 1992, 1994). This approach allows for a preliminary assessment of conservation priorities and endangered species status without the need for time consuming and expensive population genetic and ecological surveys for each individual species and habitat region (Williams and Gaston, 1994). A systematic survey also identifies populations or species for which more detailed studies are needed. In this study, the phylogenetic approaches of Vane-Wright et al. (1991) and Faith (1992) are employed to determine priority rankings for the six ecological subdivisions within the Ozark Plateaus. Crayfish are well suited for the study of conservation biology of freshwater stream systems because these macroinvertebrates are an important part of freshwater ecosystems (Lodge, 1993) particularly in Ozark

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K. A. Crandall

streams (Rabeni et al., 1995). Of specific concern to conservationists are the streams of the Ozark Plateaus that harbor a number of endemic species that are on either state or federal rare and endangered species lists (e.g. Ozark hellbender Cryptobranchus bishopi; Neosho madtom Noturus placidus; Ozark cavefish Amblyopsis rosae; Neosho mucket Lampsilis rafinesqueana; Niangua darter Etheostoma nianguae; Big river crayfish Orconectes harrisonii). Because of this wealth of biodiversity, the stream systems of the Ozarks have been studied extensively. Pflieger (1989) and Matthews et al. (1992) have described many aquatic systems within the Ozarks as being ecologically, physiographically, and faunistitally distinct from one another. Using crayfish systematic relationships and geographic distributions, this study assigns conservation priorities to these ecologically important regions within the Ozark Plateaus. In addition, the study makes assessments of these crayfish populations for conservation considerations.

second phylogenetic approach was therefore employed taking into account the branch lengths of the phylogeny and thereby weighting groups according to genetic diversity, as well as their taxonomic diversity (Faith, 1992). The phylogenetic diversity index, PD, was calculated for a group of taxa by simply summing the branch lengths of the branches interconnecting that group. Crozier (1992) has developed a similar technique for incorporating branch length information. Faith (1994) has shown that these methods are monotonically related, therefore only the method of Faith (1992) was employed in this study. Nonphylogenetic assessment of rarity An alternative approach for determining species protection status was an assessment of rarity of that species. Degree of rarity was assessed for each crayfish species using geographic range, habitat specificity, and local population size following Rabinowitz et al. (1986). Wide or narrow distribution was determined relative to the Ozark crayfish species in this study, so, widespread species are still somewhat limited in distribution, e.g. Orconectes luteus is found only in the northern Ozarks, the Current River, and in prairie streams of northeastern Missouri (Pfheger, 1987). Geographic ranges were calculated from species distribution maps provided by Plheger (1987), Williams (1954), and Page (1985). The ranges were dichotomized using a cluster analysis of Kmeans in the statistics software package SYSTAT. Habitat specificity and local population size were determined from literature accounts, mainly Pllieger (1987) and Williams (1954) and personal observations. Study area The Ozark fauna1 region is located in the central United States and encompasses much of southern Missouri and northern Arkansas (Fig. 1). The region largely overlaps with the Ozark Plateaus Physiographic Province, characterized by older bedrocks, higher elevations, and greater local relief than surrounding areas (Pflieger, 1989). The streams of the Ozarks are typically narrow, steepsided valleys, frequently bordered by high bluffs. The distributions of native fish fauna show a strong correspondence with variations in certain physical features of streams (such as turbidity, substrate, base flow, and gradient), making fish distribution a useful criterion for describing aquatic habitat regions (Pflieger, 1971; Matthews and Robison, 1988; Ptlieger, 1989; Matthews et al., 1992). Substrates in these streams include coarse gravel, rubble, boulders, and bedrock. Stream gradients are generally high and the channels consist of a series of well-defined riffles and pools. The Ozarks have been subdivided into six regions characterized by the major river drainages within each region, including Neosho, White, Black (including the Black, Current, and Eleven-Point rivers), Southeast (including the St. Francis, Castor, and Whitewater rivers), Mississippi (the Meramec river system), and Missouri (including the

METHODS Phylogeoetic approaches to conservation biology Phylogenetic assessments of conservation priorities were performed using two distinct techniques. The first, by Vane-Wright et al. (1991), quantified the amount of taxonomic information for a particular species, X, in a phylogeny by counting the number of monophyletic groups (or the number of nodes going back to the root) containing that taxon. This value, I,, was then divided into the sum of the I s over all taxa in the phylogeny giving the quotient of the total information for that taxon. The quotient, QX, was then standardized by the minimum quotient value resulting in the taxonomic weight, W,, for each taxon with the weight of W, = 1.00 assigned to the taxon of lowest rank. These weights were summed over distinct habitats to quantitatively rank these habitats for priority in conservation efforts based on taxonomic diversity. Specific areas of endemism were then ranked for conservation priorities based on the proportion of weights found in a particular area. The % weight for a given area was calculated by summing the weights for each taxon present in that area and dividing by the total weight for all taxa. These percentages will not add up to 100% unless all taxa are allopatric. The region with the highest percentage score was then considered of highest priority in terms of preserving taxonomic diversity. Next order priorities were determined by eliminating all taxa found in the highest priority region and repeating the analysis of the remaining regions. This approach takes into account the complementarity of species distributions for subsequent rankings (Vane-Wright et al., 1991). The Vane-Wright et al. (1991) approach has the advantage of incorporating information on phylogenetic relatedness, but does not incorporate information on genetic distinctiveness of a particular species. The

Conservation phylogenetics of Ozark crayfishes

96 94
92

109

90

Phylogeny reconstruction

40d

38

38

36

34

94

92

90

Fig. 1. Physiographic and faunistic sub-regions of the Ozark

Plateaus: A-Neosho, &White, C-Black, &Southeast, E-Mississippi, F-Missouri. Major river drainages and tributaries are as follows: l- Mississippi, 2-Missouri, 3%Osage, 4-Niangua, S-Gasconade, bMeramec, 7-Spring, &Elk, 9-White, IO-North Fork, 1l-Eleven Point, 12--Current, 13-Black, l&St Francis, 1S-Castor, lbwhitewater.

Niangua, Osage, and Gasconade rivers). These regions of the Ozark Plateaus will be assessed for conservation priorities based on crayfish taxonomic and phylogenetic diversity. Samples and data
Crayfish samples

The crayfish used in this study were collected by hand during 1989-1992 (Table 1). Upon capture, the abdomen and gill tissues were dissected from the crayfish, put into cryotubes, and frozen immediately in liquid nitrogen. The tissues were then returned to the lab and stored at -80C to await DNA extraction. A form I male and a female were put directly into 70% EtOH for preservation as voucher specimens when available; male crayfish of the family Cambaridae are sexually dimorphic with the form I male being sexually active showing sclerotized, corneous, and lengthened terminal elements of at least the first pleopod while form II males, the nonbreeding form, possess terminal elements that are not well differentiated and are never comeous. Identifications were verified by J. F. Fitzpatrick Jr of the University of South Alabama. The voucher specimens have been deposited in the University of Alabama Decapod Collection and are available upon request.

Nucleotide sequence data were obtained from the 16s region of the mitochondrial DNA (mtDNA) using PCR amplification and cycle sequencing as described in Crandall and Fitzpatrick (1996). DNA sequences were aligned using the program CLUSTALV (Higgins et al., 1992). Insertion and deletion gaps were coded as missing characters and recoded as single events for phylogenetic analysis using parsimony, as suggested by Swofford (1993). Phylogenetic signal within this dataset was assessed using the gl, statistic of the random tree distribution (Hillis and Huelsenbeck, 1992), using the RANDOM TREE option in PAUP (Swofford, 1993). Significance levels for the gl, statistic were obtained from Table 3.2 in Hillis and Huelsenbeck (1992). Given significant phylogenetic signal, phylogeny reconstruction was carried out using parsimony analyses by performing the heuristic search option in PAUP with random addition of taxa (Swofford, 1993). To check for the possibility of multiple tree islands (Hendy et al., 1988; Maddison, 1991) at least 100 random addition searches were made for each alternative weighting scheme. A stepmatrix based on empirically derived probabilities was used to weight mutational changes. This stepmatrix resulted in a weighting of transversions to transitions of 2:l. A transition bias in mtDNA is commonly encountered (Moritz et al., 1987) and a 2:l weighting of transversions to transitions better reflects the molecular biology of the DNA region. For operational taxonomic units (OTUs) that differed by fewer than 10 mutational steps, the method of Templeton et al. (1992) was used to assign most parsimonious relationships. Crandall (1994) has demonstrated the effectiveness of this statistical parsimony procedure and its combined use with traditional methods of phylogeny estimation (Crandall and Fitzpatrick, 1996). All trees were rooted with Procambarus acutus serving as the outgroup to the Ozark endemic species. A heuristic assessment of confidence in the resulting relationships was performed by using the bootstrap procedure (Felsenstein, 1985, 1988) for all methods except the Templeton et al. method. Here probabilities were assigned using equation (8) of Templeton et al. (1992). The bootstrap percentile values were based on 200 bootstrap replications and should be taken only as a heuristic guide to the confidence in a particular node (Hillis and Bull, 1993). Alternative tree topologies were tested for significant differences using the nonparametric sign test (Prager and Wilson, 1988). This test counts the number of mutational changes supporting topology A vs those supporting topology B and compares the relative levels of support against a binomial distribution with the null hypothesis of p=O.50, i.e. equal support for either topology. Unlike Prager and Wilson (1988) I used a two-tailed test as a more conservative test of compatible tree topology. When the total number of characters differentiating two topologies was >25, the large sample

110

K. A. Crandall

approximation was used to test alternatives. This test statistic has an asymptotic N(0, 1) distribution (Hollander and Wolfe, 1973). This test is an unweighted version of the Wilcoxon signed rank test presented by Templeton (1983). The number of mutational changes supporting one topology relative to another were calculated using the COMPARE TWO TREES option in MacClade (Maddison and Maddison, 1992).

RESULTS

Phylogeny reconstruction A heuristic search resulted in one most parsimonious tree (Fig. 2). There was no evidence of multiple tree islands within the dataset. These data were significantly skewed indicating significant phylogenetic signal (gi = -0.594, p < O-01). Restricting the dataset to include

only those OTUs that differed by 210 nucleotide substitutions resulted in a gl score of -0.565, once again indicating significant phylogenetic signal (p < 0.01). Those OTUs that differed by < 10 nucleotide substitutions were connected using the procedure in Templeton et al. (1992). To perform the Vane-Wright et al. (1991) procedure, a tree with terminal taxa equivalent to species is required. After assessing the monophyly of each species (see below), the tree was pruned so that each tip taxon represented a unique taxon (e.g. 0. luteus l-4 in Fig. 2 to 0. luteus Fig. 3). In some instances, nonmonophyletic taxa were pruned as well. The justification for pruning these taxa is given in the Species Status section below. Taxonomic and phylogenetic diversity The taxonomic diversity index of Vane-Wright et al. (1991) which ranked for priority in conservation efforts

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

Species
Cambarus (Erebicambarus) hubbsi C. (E.) maculatus Orconectes (Billecambarus) harrisonii 0. (Buannultfictus) meeki 1 0. (B.) meeki 2 0. (B.) palmeri longimanus 0. (Crockerinus) eupunctus 0. (Gremicambarus) virilis 1 0. (G.) virilis 2 0. (Procericambarus) hylas 1 0. (P.) hylas 2 0. (P.) longidigitus 1 0. (P.) longidigitus 2 0. (P.) luteus I 0. (P.) luteus 2 0. (P.) luteus 3 0. (P.) luteus 0. (P.) macrus 1 0. (P.) macrus 2 0. (P.) medius 1 0. (P.) medius 2 0. (P.) nana 1 0. (P.) nana 2 0. (P.) neglectus chaenodactylus 1 0. (P. j neglectus chaenodactylus 2 0. (P.) neglectus neglectus 3 0. (P.) ozarkae 1 0. (P.) ozarkae 2 0. (P.) ozarkae 3 0. (P.) peruncus 1 0. (P.) peruncus 2 0. (P.) punctimanus 1 0. (P.) punctimanus 2 0. (P.) punctimanus 3 0. (P.) punctimanus 4 0. (P.) quadruncus 1 0. (P.) quadruncus 2 0. (P.) wiihamsi 0. (Tragulicambarus) lancifer Procambarus (Ortmannicus) actus

Label0 hubb 122 macu 63 harr 139 meek 205 meek 230 palm 457 eupu 120 viri 143 viri 95 hyla 103 hyla 105 long 212 long 213 lute 96 lute 23 lute 13 lute 112 macr 234 macr 218 medi 151 medi 77 nana 192 nana 193 negl 181 negl 132 negl240 ozar 172 ozar 170 ozar 127 pent 137 per-u 138 punt 119 punt 160 punt 164 punt 114 quad 185 quad 186 will 221 lane 513 acut 102

County, State Oregon, MO Crawford, MO Washington, MO Madison, AR Washington, AR LeFlore, OK Oregon, MO MO Crawford, MO Iron, MO Iron, MO Madison, AR Madison, AR Crawford, MO Crawford, MO Crawford, MO Texas, MO Lawrence, MO McDonald, MO Crawford, MO Crawford, MO Benton, AR Benton, AR Ozark, MO Ozark, MO Lawrence, MO Ozark, MO Fulton, AR Oregon, MO Wayne, MO Wayne, MO Oregon, MO Phelps, MO Texas, MO Texas, MO Madison, MO Madison, MO Barry, MO Alexander, IL St Charles, MO

Major drainage Eleven Point River Meramec River Big River White River White River Cedar Lake Eleven Point River Niangua River Meramec River Black River Black River White River White River Meramec River Meramec River Meramec River Big Piney River Spring River Elk River Meramec River Meramec River White River White River Little North Fork North Fork Spring River North Fork Spring River Spring River St.Francis River St.Francis River Eleven Point River Little Piney River Big Piney River Big Piney River St.Francis River St.Francis River White River Mississippi River Mississippi River

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

Conservation phylogenetics of Ozark crayfishes

111

the six geographic regions of the Ozark Plateaus (PlP5; Fig. 3), placed highest priority on the White River region, with the Mississippi River region close behind (Pl and P2; Fig. 3). The Neosho River region was ranked third in priority using this index having the highest score at the P3 level. In the first priority rankings (Pl), the Black River region had a much higher percentile value than the Neosho region, but the Neosho region ranked higher than the Black River region at the P3 level. This is because many of the speties distributed in the Black River region are also distributed in the White River region. Thus when species of the White River region were removed for the calculation of P3 values, the priority of the Black River dropped to

fourth. The Southeast and Missouri River regions ranked fifth and sixth, respectively. The PD index of Faith (1992) ranked the Black River region as most worthy of conservation efforts (Table 2) with the White River region and the Mississippi River close in priority. The values for the remaining regions of the Neosho River, the Southeast, and the Missouri River were much lower. Simple species count per area and a count of rare species (those falling into the various rare categories as defined below, Table 3) resulted in similar rankings. The PD index was also used to rank individual species with values shown in Fig. 2. Thus 0. hylas would have the lowest relative priority among crayfishes examined based on phylogenetic diversity
Phylogenetic Diversity

12 0 1

El
11 16

II
36 d 15 . 0. bngidigitus 1 0. longidigtus 2

Fig. 2. Maximum parsimony tree inferred from 16s nucleotide sequence data from the mitochondrial genome. Numbers in bold/ italics on the nodes are the bootstrap percentile values based on 200 bootstrap replications. An (*) indicates a connection using the procedure 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 (Maddison and Maddison, 1992). Phylogenetic diversity for each population as calculated by the method of Faith (1992) is shown in a box for multiple individuals or standing alone for single individuals.
Table 2. Pbylogenetic diversity associated with tbe Ozark regions calculated by the method of Faith (1992)

Ozark regions Neosho No. species No. rare species Phylogenetic diversity 5 3 68 White 9 7 115 Black Southeast 3 1 48 Mississippi 6 4 111 Missouri 4 1 18

117

112

K. A. Crandall

w
0. llltells

1NO

Wh

BI
I

SE
??

MS *

MO .

0. medius 0. ne@ctus 1
l_ 12

0. quadruncus

4
15 1

0. longidigitus
1.18 1.18

1.30 1.44 *

1 5 r 2

3 10

1.63 1.63

0. meeki

2.17 2.17 2.17 *

??

2.60 2.60 12

0. williamsi

4.33 4.33 4.33

P. acutus Total: Pl: P2: P3: P4: P5:

41.12 8.04 19.6 10.6 10.6 18.45 44.9 12.98 31.6 12.1 6.8 3.36 8.2 5.3 2.9 2.9 2.9 15.52 2.18 37.7 34.9 5.3 2.4 0.0 0.0 0.0

. .

. .

6.8 _

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

Table 3. Rarity of Canrbarus, Orconectes, and procombarus crayl%hes baaed on geographical distribution (km*), habitat specificity, and local population size in Ozark Plateaus (after Rabinowitz et al., 1986) Geographic distribution Habitat specificity Population size Wide Broad 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) Restricted
C. hubbsi (143,950) 0. longidigitus (170,015) 0. ozarkae

Narrow Broad
0. macrus (22,791) 0. medius

Restricted
C. maculatus (23,040) 0. eupunctuf (10,300) 0. harrisonif (8,500) 0. hylus (23,040) 0. neglectus chaenodactylus (28,610) 0. peruncuP(3,225) 0. quadruncuf (5,616) 0. williamsFb (5,547)

Somewhere large

(11,457)
0. nana (16,942)

Everywhere small

0.IancifeP
(275,000) 0. meeki (220,000)

YSpecies currently listed bSpecies currently listed The total distributional phylogenetically distinct areas of 43,079 km* for

in the Rare and Endangered Species of Missouri Checklist. as candidate species by the US Fish and Wildlife Service. area for 0. ozarkae was estimated to be 118,905 km2, but if one divides up the area represented by the two populations (Fig. 3) then each population is placed in the narrowly distributed group with distributional 0. ozarkae 1 and 75,826 km2 for 0. ozarkae 2.

Conservation phylogenetics of Ozark crayfishes

113

because it has the lowest PD score. 0. macrus 0. palmeri share the highest score.

and

Rarity of way&& species The first component of the rarity analysis is the distributional area. Cluster analysis of species ranges resulted in two statistically significant clusters (p < 0.001, F-ratio), corresponding to species distributed over large or small areas. Those species within the wide geographical distribution cluster had a minimum range of 143,950 km2 and a maximum range of 350,000 km2 with a mean of 225,230 km2 (Table 3). Narrowly distributed species had a minimum distributional range of 3,225 km2 (0. peruncus), a maximum range of 75,826 km2 with a mean of 21,380 km2. Further differentiation based on local population sizes and habitat specificity are given in Table 3. Species marked superscript a are currently listed in the Rare and Endangered Species of Missouri Checklist (1991). All species in the most restricted definition of rarity (narrow distribution, restricted habitat specificity, and local population size everywhere small) are currently listed by the state of Missouri as rare. Only half of the species listed in the next priority category (narrow distribution, restricted habitat specificity, and local population size somewhere large) are currently listed by Missouri. The state of Arkansas has no endangered species checklist. Only Orconectes williamsi was listed on the US Federal Endangered Species list as a candidate species.

DISCUSSION

Systematic implications The determination of species boundaries is perhaps the most important area of application of phylogeny to conservation biology. Many species concepts have phylogenetic relatedness as the central focus in the determination of species, e.g. phylogenetic species concept (Cracraft, 1983), cohesion species concept (Templeton, 1989), evolutionary species concept (Wiley, 1978). Furthermore, phylogeny can be used to assess the degree of genetic isolation between two populations (Slatkin and Maddison, 1989). It is of primary importance to establish whether or not distinct populations are distinct

species within an hypothesis testing framework (Sites and Crandall, 1997). This determination greatly influences conservation policy in terms of introductions and listings as rare and endangered. In the following section, I explore the species status of various crayfish populations based on the phylogenetic information available. The phylogeny determined by 16s mtDNA shown in Fig. 2 places a number of crayfish species in nonmonophyletic groups. Under certain species concepts (e.g. Cracraft, 1983), species must form monophyletic groupings. Other concepts (e.g. Templeton, 1989) allow for non-monophyletic relationships within a species. To test the monophyly of these species, an alternative topology with the species in question as a monophyletic group was compared to the tree in Fig. 2 using the sign test. The results of these comparisons are shown in Table 4, indicating that both 0. quadruncus and 0. ozarkae form statistically significant non-monophyletic relationships. One explanation to the non-monophyly of mtDNA haplotypes within a species is the problem of gene trees reflecting species (or population) trees. The results of theoretical population genetic work indicate that for some time after the divergence of two or more populations, there is a high probability that populations may show non-monophyletic relationships for a specific gene; therefore, the gene genealogy may not accurately reflect the population divergence. Neigel and Avise (1986) and Takahata and Slatkin (1990) have shown that, in fact, the expected progression of gene phylogenies following divergence is polyphyletic + paraphyletic + monophyletic. The probability of monophyly is significantly reduced by the presence of gene flow (Takahata and Slatkin, 1990) increases with smaller population size (Padmadisastra, 1987) and is affected by the geographic distribution of the populations (Neigel and Avise, 1986). Given these results and the apparent recent divergence of the crayfish populations in this study (as evidenced by the relative lack of genetic divergence among individuals) patterns of nonmonophyly may reflect this recent divergence and the discordance between 16s mtDNA genealogy and the species phylogeny. Although monophyly is a requirement for a limited number of species concepts (e.g. Cracraft, 1983), it is of

Table 4. Testing monophyletic groups: Orconectes species

Taxonomic group
0. 0. 0. 0. quadruncus neglectus ozarkae punctimanus

Nonmonophyly
30 9 15 6

Monophyly
2 3 0 0

B 4.95 3.87

p value < 0.0002 0.073 < 0~0002 * 0.0156

Bf. p
< 0~0002 0.292 < 04008 0.062

The values given under nonmonophyly and monophyly indicate the number of characters for that particular hypothesis was shorter compared to the alternative hypothesis. B is the large sample approximation for the sign test given in equation (2). When B contains no value, the B statistic was used which is simply the value under Maximum Parsimony. p values for the B statistic are onesided from a normal distribution (Hollander and Wolfe, 1973) and those for B are one sided from a binomial distribution with p = 0.5 (Hollander and Wolfe, 1973). Bf. p are the p values with the Bonferroni correction for multiple comparisons (Neter et al., 1985), *p < 0.05; ?? **p < 0401.

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K. A. Crandall do occur sympatrically, at least in Cedar Bottom Creek in Madison County, Missouri. This result warrants further investigation into the range of 0. peruncus. Although the monophyly of the species Orconectes neglectus cannot be rejected, it is interesting to note that the two populations of this species that reside in different areas of the phylogeny represent two subspecies from isolated drainages: negl 240 (Orconectes neglectus neglectus) is from the Spring River drainage in Western Missouri and Eastern Kansas, and negl 132 and 181 (Orconectes neglectus chaenodactylus) are from the North Fork of the White River drainage. Their placement in two distinct areas in the phylogeny suggests the possibility of sufficient divergence to be considered distinct species. Obviously, population genetic analyses of these two subspecies is warranted to determine species status. Implications for conservation biology
Species status

interest to examine the particular cases of non-monophyly. We reject the hypothesis of species monophyly for both 0. ozarkae and 0. quadruncus. The distinct positioning of two populations of 0. ozarkae corresponds to isolated drainages with ozar 127 and 170 from the Spring River drainage in Arkansas and ozar 172 from the North Fork of the White. These two populations have been identified as having two distinct morphologies (Pflieger, pers. comm.) and are genetically differentiated (p < 0@08, Table 4). Furthermore, this same geographic separation and degree of genetic isolation has been observed in the hellbender salamander Cryptobranchus bishopi (Routman et al., 1994), adding support to a hypothesis of vicariant biogeographic isolation of populations. Finally, Pflieger (1989) and Matthews et al. (1992) have suggested these drainages are ecologically distinct from one another, but detailed ecological studies are needed to confirm ecological divergence for these two populations of crayfish. Given this suggestive ecological, phylogenetic, and morphological differentiation, these two populations of 0. ozarkae may pass species criteria for the phylogenetic, evolutionary, and cohesion concepts of species. The species status via the recognition concept and the biological species concept will require further study. An alternative explanation is that this particular individual (ozar 172) is the result of a hybridization event between an 0. ozarkae male and an 0. punctimanus/virifis (see below) female. This hypothesis is supported by the low amount of variation between ozar 172 and punt 119, 160, and 164. Punt 160 and 164 have identical nucleotide sequences for the 16s region. Punt 119 is two nucleotide substitutions different from punt 160/l 64 and three nucleotide substitutions different from ozar 172. Because mtDNA is maternally inherited, a single successful hybridization of a female punctimanuslvirilis with an ozarkae male would result in offspring with the punctimanuslvirilis mtDNA genotype. Hybridization has been documented in crayfishes, but is considered to be a very rare phenomenon (Capelli and Capelli, 1980; Smith, 1981; Berrill, 1985; Cesaroni et al., 1992). Once again, this result requires a further examination of the species status of 0. ozarkae using more detailed population genetic information. 0. quadruncus and 0. peruncus are hypothesized to have allopatric distributions within the St. Francis River drainage in Missouri. 0. quadruncus is distributed in the St. Francis River drainage exclusive of Big Creek, the drainage supporting populations of 0. peruncus (Williams, 1954; Pflieger, 1987). Because of the extensive differentiation between quad 185 and 186, two individuals found at the same locality, it is suggested here that quad 186 is really a sample of 0. peruncus. The 16s sequence obtained from quad 186 is only 1 nucleotide different from peru 137 and 2 substitutions different from peru 138. Because no form I male was collected for this population, positive identification was not possible. This evidence does suggest, however, that these species

The status of species currently listed by the State of Missouri in the Rare and Endangered Species of Missouri Checklist (199 1) is supported by this study. Table 3 shows that the majority of those species listed fall within the most threatened category: narrowly distributed, restricted in habitat with small population sizes. Two species, Orconectes lancifer and Orconectes meeki, are widely distributed, but with restricted habitat specificity and small population sizes. These species are narrowly distributed within the state of Missouri resulting in their inclusion in the checklist. 0. harrisonii, 0. eupunctus, and 0. williamsi are also listed by the state; they have narrow distributions and restricted habitat specificity but large local populations. The latter two have a high phylogenetic diversity index and all have a moderate taxonomic diversity index (Fig. 2). Based on this information, I recommend changes in conservation status for several species. Two crayfish species, 0. peruncus and 0. quadruncus, are currently listed by the state with the status of Watch List , i.e. not currently rare or endangered, but has a restricted distribution or has experienced sufficient decline to indicate it may soon become Rare or Endangered would be changed to Rare , where Rare indicates present in small numbers. If environment worsens, status in Missouri could deteriorate to Endangered. These species are narrowly distributed, with restricted habitat and small population sizes. Three other species not yet listed by the state of Missouri, 0. nana, 0. macrus, and C. maculatus, are recommended to be added as Rare . These species are locally abundant, but show narrow geographic ranges and habitat specificity. Furthermore, each species ranks extremely high for both phylogenetic and taxonomic diversity. I further

Conservation phylogenetics of Ozark crayfishes Table 5. Current and recommended status of Ozark crayiishes Species
Cambarus hubbsi

115

Current status? None None R WL None E None None None None E None None None None WL None WL None R

Suggested status WL R R WL WL E None None R WL E R None None None R None R None R

Rarity W:R:L N:R:L N:R:L N:R:L N:R:L W:R:S W:R:L W:B:L N:B:L N:B:L W:R:S N:B:L W:B:L W:R:L W:B:L N:R:S W:B:L N:R:S W:B:L N:R:L

TD 4.33 4.33 1.63 1.63 1.18 2.60 144 1.00 2.17 1.00 2.17 2.17 1.26 1.24 2.60 1.18 1.18 1.18 1.18 4.33

PD 14 18 14 7 6 20 15 12 26 12 11 21 16 14 26 9 12 11 4 12

C. maculatus
Orconectes eupunctus 0. harrisonii 0. hylas 0. lanctjier 0. longidigitus 0. luteus 0. macrus 0. medius 0. meeki 0. nana 0. neglectus 0. ozarkae 0. palmeri O.peruncus 0. punctimanus 0. quadruncus 0. virilis 0. williamsi

TD is taxonomic diversity; PD is phylogenetic diversity; Rarity: geographic distribution-W 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 Watch List (C. hubbsi, 0. hylas, and 0. medius) based mainly on the restricted distribution of the Orconectes species and the high taxonomic and phylogenetic diversity indices for C. hubbsi. The designation of 0. hylas and 0.
medius

= wide, N = narrow; Habitat specifi-

as Watch List species is recommended principally on the rarity criterion. Table 3 shows both of these species as narrowly distributed with habitat specialization, but with large population sizes. Neither species ranks high in terms of taxonomic or phylogenetic diversity resulting in their Watch List status. C. hubbsi, on the other hand, is widely distributed with large populations but with a restricted habitat. The listing of this species as Watch List is due to its high taxonomic diversity index. A summary of these recommended listings is given in Table 5, as well as associated data for categorizations.
Habitat priorities

The rankings of the six Ozark regions are summarized in Table 6. Notice that no matter what the measure, it is clear that the White, Black, and Mississippi regions are of higher priority than the Neosho, Southeast, and

Missouri regions. It is also interesting to note that all measures give nearly equivalent rankings of regions with the notable exception of the taxonomic diversity index. Here, the Black River region receives a much lower ranking than from other methods. This is due to the complementarity aspect of the TD index (VaneWright et al., 1991). The TD measure initially ranks the Black region very high, but because many of the species in this region overlap with those in the White River region, which is ranked higher, subsequent ranking of the Black River region is reduced. I do not claim that one measure is better or more meaningful than another, but any differences presumably reflect different biological aspects under consideration as well as different methodological philosophies (Krajewski, 1994). The advantage to these approaches is that they offer hard data upon which one can base sound decisions. Of course, this ranking scheme is primarily for the primary benefit of crayfish. It would be worthwhile to perform a similar examination of these fauna1 regions using a different taxonomic group. However, because these regions are based on distributional data from many species including fish and have been shown to correlate

Table 6. A comparison of various ranking criteria for faunistic regions of the Ozarks Ozark regions Neosho SR Rank RS Rank TD Rank PD Rank StlIll SR-Species 4.5 4 3 4 15.5 Richness, RS-Rare White 1 1 1 2 5 Species, TD-Taxonomic Black 2 2 4 1 9 Southeast 4.5 5.5 5 5 20 Diversity, and PD-Phylogenetic Mississippi 3 3 2 3 11 Diversity. Missouri 6 5.5 6 6 23.5

116

K. A. Crandall Cesaroni, D., Allegrucci, G. and Sbordoni, V. (1992) A narrow hybrid zone between two crayfish species from a Mexican cave. J. Evol. Biol. 5, 643659. Cracraft, J. (1983) Species concepts and speciation analysis. Current Ornithology 1, 159-187. Cracraft, J. (1994) Species diversity, biogeography, and the evolution of biotas. Amer. 2001. 34, 33-47. Crandall, K. A. (1994) Intraspecific cladogram estimation: Accuracy at higher levels of divergence. Syst. Biol. 43, 222235.

with a number of ecological attributes, it is expected that the present assessment will have broad application over these freshwater ecosystems.
The phylogenetic perspective

The strength of the phylogenetic approach is in the assessment of species status as independent lineages and identifying important areas for further population genetic studies. Furthermore, the phylogenetic information adds additional parameters (taxonomic diversity and phylogenetic diversity represented by a species) to the assessment of species rarity status and assessments of habitat priorities. Indeed, as preserving genetic diversity is often a goal in conservation biology (Templeton, 1991; Crozier, 1992), it seems pertinent to include some measure of genetic distinctiveness into a weighting scheme for habitat preservation. The information gained from this assessment of habitat priorities can be an important additional source of data, yet the conclusions drawn from these data are similar to those drawn from species richness assessments. These quantitative measures of taxonomic and phylogenetic distinctiveness combined with ecological considerations of abundance and geographic distribution allow for accurate assessments of the endangered status of the species of crayfish examined herein and the ranking of subregions in the Ozarks for conservation priorities. As more studies are performed that include a phylogenetic component to the assessment of species rarity and habitat priority, a better picture of the various utilities of the phylogenetic approach will emerge.

Crandall, K. A. and Fitzpatrick Jr, J. F. (1996) Crayfish molecular systematics: using a combination of procedures to estimate phylogeny. Syst. Biol. 45, l-26. Crozier, R. H. (1992) Genetic diversity and the agony of choice. Biol. Conserv. 61, 1l-l 5. Eldredge, N. (1992) Systematics, Ecology, and the Biodiversity Crisis. Columbia University Press, New York. Faith, D. P. (1992) Conservation evaluation and phylogenetic diversity. Biol. Conserv. 61, l-10. Faith, D. P. (1994) Genetic diversity and taxonomic priorities for conservation. Biol. Conserv. 68, 69-14. Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 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, foundations for understanding and conserving biodiversity.
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ACKNOWLEDGEMENTS

Hillis, D. M. and Bull, J. J. (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42, 182-192. Hillis, D. M. and Huelsenbeck, J. P. (1992) Signal, noise, and reliability in molecular phylogenetic analyses. J. Hered. 83,
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I would like to thank Cindy and Kyle Crandall, Marshal and Jennifer Hedin, Chris Phillips, and Eric Routman for assistance in field collections of these and many other crayfishes. Joy Bergelson, Laurie Dries, Nick Georgiadis, Anne Gerber, Delbert Hutchison, Chris Phillips, Owen Sexton, and Alan Templeton provided helpful comments on the manuscript, as well as lively and enlightening discussions in the area of conservation biology. Joe Fitzpatrick and Bill Pflieger provided invaluable support and encouragement with my learning of the crayfish taxonomy. The Missouri Department of Conservation and the National Science Foundation (BSR-9 112000) provided funding for this research.

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