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QL 638 .P4 S53 1999 Scientific Papers

Natural History Museum The University of Kansas Number 12:1-16 28 July 1999

A Reexamination of the Phylogenetic Relationships of the Sand Darters (Teleostei: Percidae)

Kate A. Shaw', Andrew M. Simons^ and E. O. Wiley'

'Division oflchthyologi/, Natural Histonj Museum, and Department of Ecology and Evolutionary Biology, The University of Kansas, Lawrence, Kansas 66045-2454, USA ami

^Bell Museum of Natural History and Depmrtment of Fisheries and Wildlife, The University of Minnesota, 1980 Fohvell Avenue, St. Paul, Minnesota 55108-6124, USA

CONTENTS ABSTRACT 1 INTRODUCTION 2 Acknowledgments 3 MATERIALS AND METHODS 3 RESULTS 6 DISCUSSION 10 LITERATURE CITED 12 APPENDIX 1. Specimens Examined 13 APPENDIX 2. Biochemical Data 14 APPENDIX 3. Character Matrix 15

ABSTRACT Phylogenetic relationships among the Boleosoma group of darters were examined for allozymic variation at 25 presumptive gene loci alone and in combination with 29 morphological characters. Qualitative analyses of allozymic and morphological— variation resulted in two most parsimonious trees showing the following relationships: Tree 1. (Etheostoma davisoni ((£. longimanum, E. nigrum)— (E. vitreum (£. clarum (£. pellucidum (E. meridianuni (E. vivax (E. bifascia, E. beanii)))))))); Tree 2. (E. davisoni (E. nigrum (E. longimanum (E. vitreum (E. clarum (E. pellucidum (E. meridianuni (E. z'iz'ax (E. bifascia, E. beanii))))))))). Quantitative analyses that incorporate an estimate of allelic frequencies were

QL 638 .P4 S53 1999 Scientific Papers

Natural History Museum The University of Kansas Number 12:1-16 28 July 1999

A Reexamination of the Phylogenetic Relationships of the Sand Darters (Teleostei: Percidae)

Kate A. Shaw', Andrew M. Simons^ and E. O. Wiley'

^Division Natural and of Ichthyology , History Musciiin, Department of Ecology and Evolutionary Biology, The University of Kansas, Lawrence, Kansas 66045-2454, USA and

'Be// Museum of Natural History and Department of Fisheries and Wildlife, The University of Minnesota, 1980 Fohvell Avenue, St. Paul, Minnesota 55108-6124, USA

CONTENTS ABSTRACT 1 INTRODUCTION 2 Acknowledgments 3 MATERIALS AND METHODS 3 RESULTS 6 DISCUSSION 10 LITERATURE CITED 12

APPENDIX 1. SPEaMENS Examined 13 APPENDIX 2. Biochemical Data 14 APPENDIX 3. Character Matrix 15

. — ous trees showing the following relationships: Tree 1 (Etheostoma davisoni ((£. lon^imanum, E. nigrum) (E. vitreum (£. claruin (£. peltucidum (£. meridiainim (£. znz'ax (£. hifascia, E. beanii)))))))); Tree 2. —(£. davisoni (E. nigrum (£. longimanum (£. vitreum (£. clarum {£. pellucidum (£. meridianum {£. vivax (E. hifascia, E. beanii))))))))). Quantitative analyses that incorporate an estimate of allelic frequencies were

used to discriminate among trees and showed that Tree 1 was 0.598 FREQPARS units shorter than Tree 2. There was both congruence and complementarity in the support provided by allozymic and morphological datasets. The sister-group relationship and allopatric distributions of £. beanii and £. bifascia are consistent with their origin resulting from vicariant speciation associated with the origin of the Mobile Basin.

Key words: Etheostoma, Ammociypta, allozymes, historical biogeography, phylogeny

INTRODUCTION

The sand darters are slender, translucent elongate, oi Ammocrypta within the genus Etheostoma and suggested darters known for their habit of themselves in burying that Etheostoma (loa) vitreum was the sister group to the substrates. Six of sand darters are sandy species currently subgenus Ammocrypta. This change resulted in four sub- classified in the Etheostoma subgenus Ammocri/pta: genera being included in the Boleosoma group of Etheostoma: E. beanii, E. vivax, E. claruni, E. and £. peUucidum, bifascia, Boleosoma, loa, Vaillantia, and Ammocrypta. Simons' hypoth- meridianum. esis of relationships within the subgenus Ammocrypta dif- The taxonomic history of Ammocrypta began with its fered somewhat from those postulated by Williams (1975). use as a generic name by Jordan (1877) with his descrip- Simons (1992) did find support for the £. peUucidum group; tion of y4. beanii. Three additional species now included in £. meridianum and £. peUucidum were sister taxa, and £. the subgenus Ammocrypta (originally described as vivax was the sister group to this pair. However, Simons Pleurolepis pellucida, A. vivax, and A. clam) were described (1992) did not find support for the £. beanii group; instead, by 1886 (Agassiz, in Putnam, 1863; Hay, 1883; and Jordan the species pair £. beanii-E. bifascia was the sister group to and Meek, 1885, respectively). Bailey and Gosline (1955) the £. peUucidum group and £. clarum was the sister group added Crystallaria asprelln to the genus Ammocrypta, but to all other members of the subgenus Ammocrypta. it in a placed monotypic subgenus Crystallaria. Although The relationships hypothesized by Simons (1989, 1991, and Gosline (1955) for a classification of Bailey argued 1992) and based on phylogenetic analyses, have not been darters three (Percina, and containing genera Anunocn/pta, widely accepted. For example, Etnier and Starnes (1993), Etheostoma) that was a few authors generally accepted, (e.g., Jenkins and Burkhead (1994), Mettee et al. (1996), and Moore, 1968; Miller and Robison, 1973) continued to rec- Pflieger (1997) all continued to recognize the genus as a Williams (1975) ognize Crystallaria monotypic genus. Ammociypta in their state guides (Tennessee, Virginia, Ala- described two additional within the species subgenus bama, and Missouri, respectively). Although Jenkins and Within this his A. beanii con- Ammocrypta. subgenus, group Burkliead (1993) alone justified their retention of the ge- sisted of the with a few scale rows (£. beanii, E. species nus Ammocrypta, in no case is an explicit alternative phy- and £. clariim) and his A. contained bifascia, pellucida group logenetic analysis or hypothesis presented. Wood and the to almost scaled (£. partially completely species Mayden (1997) however, found support for a sister-group E. meridianum, and £. vivax). peUucidum, relationship between Etheostoma beanii and Crystallaria in Williams (1975) considered the genus Ammocrypta maximum parsimony analyses and in their most parsi- more closely allied to the subgenus hnostoma of the genus monious FREQPARS tree, but they did not make any taxo- Percina than to Etiieostoma. However, Page and Whitt nomic recommendations based on this result. In the absence (1973a) argued that Percina was monophyletic and sug- of an explicit alternative phylogenetic analysis of more than gested that Ammocrypta was related to Etheostoma; this con- two taxa, we conrinue to consider members of Ammocrypta, clusion was based on a unique LDHB4 isozyme found only members of the genus Etheostoma, subgenus Ammocn/pta. in Perciim. (Their assessment of the of this charac- polarity The particular relationships of these species are of in- ter is unclear— in their 4, Etheostoma and Fig. p. 6, terest to biogeographers. Wiley and Mayden (1985) fol- are united a derived LDH B4 Ammocrypta by isozyme.) Page lowed the relationships suggested by Williams (1975) and and Whitt (1973b) stated that the TO shared £. isozyme by postulated that the distribution of members of the chlorosoma and A. indicated that (Vaillantia) pellucida Etheostoma beanii group was the result of a western was more related to Etheostoma than Ammocrypta closely vicariance event loosely associated with the Mississippi to Percina. River. Etheostoma clarum is found in the Mississippi River Simons (1989, 1991) removed Crystallaria from Basin and drainages to the west, and the presumed sister Ammocrypita and hypothesized that the phylogenetic posi- taxon (£. beanii-E. bifascia) is found east of the Mississippi tion of Crystallaria was basal to Etheostoma and Percina. River mainstem. Wiley and Mayden (1985) also suggested Simons (1989, 1992) subsumed the remaining six species that the distributions of two other groups could be attrib- Phylogenetic Relationships of Sand Darters uted to vicariance events involving the Mobile Bay Basin. Acknowledgments Ethcostoina beaiiii is found in the Mobile Basin and west- We thank G. Harp (Arkansas State University), L. Page, ward to the Mississippi River mainstem, whereas E. bifascia P. Ceas, and C. Johnston (Illinois Natural History Survey), is only found east of Mobile Bay. A similar situation is F. Pezold (Northeast Louisiana University), R. Jenkins found in the £. pelliicidiim group. Etheostoma meridiaiiiim is (Roanoke College), H. Bart and M. Taylor (Tulane Univer- a Mobile Basin whereas E. and E. vivax endemic, pellucidum R. sity), R. Mayden (University of Alabama), Cashner and are found in north and west of the Mobile Ba- drainages F. T. J. Grady (University of New Orleans), and Cross, sin. Simons' (1992) results did not substantiate the Schmidt, D. Siegel-Causey, and K. Toal, III (University of vicariance event associated with the Mississippi River or Kansas) for their help collecting specimens used in this a vicariance event between E. meridiniiuni and the species project. We also thank S. Layman for allowing us to use pair E. vivax-E. pellucidum. his unpublished results on tuberculation in darters and R. The main of this is to reexamine the rela- purpose study Wood for advice with the data analyses. This project was of members within the and tionships subgenus Aiumocrypta supported by grants from the General Research Fund, the controversial of the relationship subgenera Ammocnjpta University of Kansas (KU 3365) to E. O. Wiley, and the and loa new data from studies. by including electrophoretic National Science Foundation (BSR 8722562) to E. O. Wiley A of this is to reexamine the bio- secondary purpose study and D. Siegel-Causey. geographic liistory of the subgenus Ammocnjpta. MATERIALS AND METHODS

Taxa examined.—All six members of the subgenus sources, and electrophoretic conditions are listed in Ap- Ammocnjpta were included in the ingroup. Outgroups were pendix 2. Twenty-seven presumptive gene loci were visu- chosen using the hypothesis of Simons (1992) as a guide. alized by histochemical staining. Electromorphs for each in of anodal Etheostoma nigrum and E. lo}igimaiutin were included as locus were coded a, b, c, etc., order increasing representatives of subgenus Boleosoma. These species have mobility. These designations are relevant to this study only. for all taxa the potential to act as the first (close) taxonomic outgroup. Two loci were fixed (M-Icdh-A and Ldh-A) Within Boleosoma, his clade consisting of £. nigrum, E. sampled and not included in the analyses. Two loci were olmstedi, and E. perlongum is represented here by E. nigrum. variable only within single species (Ldh-B within The species pair E. longimanum-E. podostcmone is repre- Etheostoma meridianum and Tpi-B within E. vivax), and these sented by E. longimaiium. Although E. )iigrum and £. autapomorphies were not considered further. The remain- longimanum were included in the analysis as taxa outside ing 23 variable loci were considered independent trans- the taxonomic group of interest {Ammocri/pta and E. vit- formation series (TS), and each allele was considered a reum), they were not designated as outgroups in the analy- unique state (TS 1-TS 23, Appendix 3). ses. The second (distant) taxonomic outgroup was £. Morphological characters.—Twenty-nine morpho- davisoni. This the that make species represents species pair logical transformation series originally described by the Vaillantia and was as the sole up subgenus designated Simons (1992) were included in this study. Many of the in the outgroup analyses. descriptions of these transformation series have been — and for this reason are Enzyme electrophoresis. Fishes were collected by slightly revised, they presented below. Transformation series and state numbers refer to seining, frozen immediately in liquid nitrogen, transported to the and stored at -70° C for to 5 (see those in 3 (TS 24-TS 52). laboratory,— up yr presented Appendix Appendix 1 Specimens Examined). Skeletal muscle, liver, TS 24.—Ascending process of the premaxilla: (0) per- tissues were dissected and and brain/eye homogenized pendicular to the alveolar process or (1 ) reclined posteriorly. in a 1:1 mixture of tissue and 0.01 M Tris, separately (v:v) TS 25.—Maxillary process of the premaxilla: (0) not 0.001 M and 0.001 M 6.8. EDTA, mercaptoethanol, pH elongate or (1) elongate and enlarged. at for 10 min at were centrifuged 15,000x ^^ Homogenates TS 26. —Premaxillary socket of the maxilla: (0) V- 5° C. Within 72 h, the supernatant fractions were electro- shaped, with the lateral and medial walls approximately phoresed at 5° C on horizontal starch gels composed of equal in length or (1) U-shaped, with the lateral wall curv- 12% hydrolyzed potato starch (Starch Art Corp.). His- ing medially and longer than the medial wall. tochemical staining protocols did not differ substantially TS 27.—Palatine teeth: (0) present or (1) absent. from those of Murphy et al. (1990). TS 28.—Notch posteroventral to the articular process Enzyme nomenclature follows the recommendations of the quadrate: (0) shallow to absent or (1 ) cut deeply into of the International Union of Biochemistry Nomenclature the body of the quadrate. Committee (1984), and locus nomenclature follows the rec- TS 29.— of the (0) rounded, with a ommendations of Buth (1983). Enzymes, loci, tissue Body quadrate: SciENTinc Papers, Natural History Museum, The University of Kansas — notch between the body and the posterior process of the TS 45. —Postcleithrum 2: (0) present or (1) absent. quadrate or (1) rectangular, without a notch. TS 46. Longitudinal struts on the anal proximal TS 30.—Hyomandibular struts: (0) present as cruci- pterygiophores: (0) present or (1) absent. within the or (1) form thickenings hyomandibula extremely TS 47.—Process for the insertion of the m. infracarijinlis reduced to absent. medius on the anterior face of the first anal pterygiophore: TS 31.—Descending process of the hyomandibula: (0) (0) present or (1) absent. This character was considered because in his it long, extending beyond the preopercular groove or (1) homoplastic by Simons (1992), analysis short, terminating at the end of the preopercular groove. supported a group consisting of Etheostoma bennii, E. chlorosoma, E. davisoiii, E. and E. TS 32.—Hyomandibular spur: (0) absent or (1) present. stigmaeum, jessiae. TS 48.— thickened of fin and TS 33.—Ventral plate of the urohyal: (0) flattened, with Swollen, tips spines rays absent or such that the the insertion for the urohyal-hypohyal ligaments directed of breeding males: (0) (1) present, the of the is covered a knob anteriorly from the anterior surface or (1 ) curved, with tip pelvic spine by large fleshy the ventralmost and are swol- insertion sites directed anteroventrally from the ventral and pectoral rays pelvic rays thickened at the surface. Simons (1992) considered this character homoplas- len and tips. it the of tic, because in his analysis supported monophyly TS 49.—Body squama tion: (0) almost complete or (1) the Boleosoma and loa. subgenera reduced laterally to a few rows of scales. TS 34.—Articular for the on the process interhyal pos- TS 50.—Tubercles on pelvic fins of breeding males: (0) terior (0) or (1) absent. ceratohyal:— present present or (1) absent. This transformation series and the TS 35. Posterior margin of the preopercle: (0) smooth next one were both part of a single transformation series or (1) serrate with a few points projecting beyond the mar- in Simons' (1992) analysis. Although tuberculation is usu- gin of the bone. ally considered a single multistate variable, the presence of tubercles areas of the occur TS 36.—Notch in the anterior angle of the preopercle: on many body independently across all taxa of darters and are considered different trans- (0) present, roofing the articulation for the interhyal or (1) absent. formation series in this study. That is, the presence of tu- — bercles on the pelvic fins does not show a one-to-one cor- TS 37. Opercular spine: (0) present or (1) absent. — respondence with the presence of tubercles on the anal fin. TS 38. Opercular strut: (0) strong, extending from the Tubercles on the pelvic fins of breeding males have been hyomandibular articulation to or almost to the posterior observed for darters, Percina evides, P. less many including: pal- margin of the opercle or (1 ) greatly reduced, extending mnris, P. shumardi, P. vigil, P. auraiitiaca, P. copelandi, than half the distance to the margin. Crystallaria asprella and, within the genus Etheostoma, all TS 39.—Posterodorsal extension of the (0) subopercle: members of the subgenera Allohistium, Ammocrypta, elongate and filamentous or (1) truncated near the dorsal Doration, and loa, and E, chlorosoma, E. punctulatum, E. of the margin opercle. E. E. E. E. boschtiiigi, cragini, pallididorsum , aiistrale, hopkinsi, TS 40.—Mesethmoid: (0) thick and anteri- expanded E. spectabile, E. luteovinctum, E. serrifer, E. gracile, E. zonifer, the lateral ethmoids or orly, extending anteriorly beyond E. fiisiforme, E. sahidae, E. collis, E. pweliare, E. microperca, (1) thin and concave not lat- anteriorly, extending beyond and £. fonticola (Collette, 1965; Bailey and Etnier, 1988; eral ethmoids. This character was considered homoplastic Jenkins, 1971; and S. R. Layman, pers. comm.). by Simons (1992), because in his analysis it supported group- TS 51. —Tubercles on the anal fins of breeding males: ing together the subgenera Vailhmtin, Boleosoma, and Ion. (0) absent or (1) present (Collette, 1965; Bailey and Etnier, TS 41.—Maxillary ligament insertion: (0) on two dorso- 1988; Jenkins, 1971; and S. R. Layman, pers. comm.). Anal lateral projections of the mesethmoid, (1) on a single fin tubercles have been observed in Etheostoma parvipinne, dorsomedian knob of the mesethmoid, or (2) on two E. fricksium, E. radiosiim, E. whipplei, and E. trisella; these dorsomedian ridges of the mesethmoid. Simons (1992) con- five taxa lack pelvic fin tubercles. Of the taxa that exhibit sidered this character homoplastic because In his analysis it pelvic fin tubercles (listed above), only E. pellucidum, E. the Boleosoma and Ion. supported grouping together subgenera meridinniim, and E. vitreum lack anal fin tubercles. All mem- TS 42.—Vomerine teeth: or usu- (0) always present (1) bers of the subgenera Etheostoma, Ulocentra (sensu Bailey absent. ally and Etnier, 1988), and Boleosoma lack anal (and pelvic) fin TS 43.—Membrane bone on the lateral margin of the tubercles, as does E. davisoiii. nasal: (0) extensive, the or (1) — overlying olfactory capsule TS 52. Shape of the female genital papilla: (0) coni- reduced to a thin the canal. slip along cal, (1) cupped, or (2) flat and bilobed. Variation in the 44. —Remnant of the lateral line canal of the TS shape of the female genital papilla is widespread among supracleithrum: (0) present or (1) absent. members of Etheostoma; however, most species have short. Phylogenetic Relationships of Sand Darters

of all the data will deter- conical papillae. Of the taxa included in this analysis, fe- most parsimonious arrangement is sister to one of the male £. davisoni have cup-like papillae (Howell, 1968), fe- mine whether E. vitreum the group or com- male £. longimanum and £. nigrum have bilobed, flattened following: the subgenus Annnocrypta, any species to? bination of of the Boleosoma, papillae (Cole, 1967), and all females of the subgenera species Ammociypta, subgenus of the Boleosoma and and Atiiiuocrifpta have conical papillae. a clade consisting subgenera the £. the Some of the transformation series used by Simons Ammocn/pta, species pair nigrum-E. longimanum, E. or the £. longimanum. This tests (1992) are not included here, because they are uninforma- species nigrum, species Simons' (1992) assertion that E. vitreum is the sister group tive in this study. They were either synapomorphic for the to the subgenus Ammocrypta. entire Boleosotna group (his characters 3 and 4), provided data were coded each locus as the support for groups within the subgenus Boleosouia (his Allozyme using transformation series and the allelic as the charac- characters 7, 24, 25, and 32), supported the subgenus arrays ter states. The order or cost of transformation from one Vaillantia (his characters 10, 35, 36, and 38 [in part]), or were state to another was directed matrices autapomorphic for Etheostoma vitreiiin (his characters 29 character by step (Mabee and 1993). Two kinds of and 37 [in part]). Humphries, step-matrix files (available from the authors upon request) were con- Phylogenetic analyses.—The underlying methodol- structed. In the first, allozyme transformation series were ogy for this study is phylogenetic parsimony analysis directed by a series of step matrices that contained only (Hennig, 1966; Kluge and Farris, 1969; Wiley, 1981; Farris, character states observed in the study taxa. This allows 1983; Churchill et al., 1985; Farris and Kluge, 1985, 1986). coding of all observed allelic combinations; hypothetical The precept that all available evidence must be brought to ancestral character states at interior nodes of resulting bear on any statement about relationships (Kluge, 1989) is phylogenetic trees were limited to character states observed also adhered to. The outgroup comparison method has in study taxa. The second step-matrix file contained a been shown to be the most comprehensive for polarizing states observed in single, large step matrix of character hypotheses of transformation (Stevens, 1980; Farris, 1982; extant taxa as well as all other possible allelic arrays. Be- Kluge, 1984, 1985; Brooks and Wiley, 1985), and conse- cause of restrictions on the number of characters allowed quently, it was used in this study. by PAUP, this approach can only accommodate five or A of were on the allozyme variety analyses performed fewer alleles (31 states) per locus. Therefore the original data alone and on the data in with allozyme conjunction data matrix had to be reduced in size. This reduction was the data: (1) analyses of allozyme data us- morphological accomplished by deleting autapomorphic alleles from the BIOSYS-1 (Swofford and Selander, 1981), (2) ing qualita- data matrix for those taxa in which they occurred. For tive of and combined allozyme and analyses allozyme PAUP analyses the most parsimonious and several near data PAUP 3.1.1 (Swofford, 1993), and morphological using most parsimonious topologies were saved for FREQPARS (3) quantitative analyses of the topologies produced from analysis. the preceding BIOSYS-1 and PAUP 3.1.1 analyses utilizing Two groups of FREQPARS analyses were performed. FREQPARS (Swofford and Berlocher, 1987; Swofford, 1988). The first included only the allozyme frequencies. The sec- For BlOSYS-1 from the analyses, genotype frequencies ond included both allozyme frequencies and morphologi- data were used to construct follow- allozyme topologies cal characters. Morphological characters were treated as if a of methods: (1972) distance, ing variety Rogers genetic they were fixed electromorphs for the taxa in which they modified distance 1978), Prevosti distance Rogers (Wright, occur. That is, the presence of a particular morphological 1978), Cavalli-Sforza and Edwards (1967) chord (Wright, character state in a particular taxon was considered ho- Cavalli-Sforza and Edwards (1967) arc distance, distance, mozygous and present in 100% of the population sampled. and Edwards (1971, 1974) "E" distance. These dis- genetic All topologies produced using BIOSYS-1 and PAUP analy- tances were summarized in distance- trees (Farris, Wagner ses were tested using FREQPARS. Simons' (1992) tree was 1972) Swofford's (1981) Addition Criterion using Multiple also tested in this fashion. Tree lengths resulting from these Procedure and were rooted with the single outgroup, FREQPARS analyses were used as the final arbiter between Etheostoma davisoni. The resultant were then topologies competing hypotheses of phylogenetic relationships of the used as trees for the third of input group analyses ingroup taxa. This is because these analyses include all (FREQPARS). available data pertaining to these species (i.e., both character Two groups of PAUP analyses were performed. Any allozyme frequency data and morphological transformation series with three or more character states states) and they provided greater discrimination among Etheostoma was polarized if possible, but unordered. Only competing hypotheses. davisoni was designated as an outgroup, because then the Scientific Papers, Natural History Museum, The University of Kansas

RESULTS

Observed genotypic frequencies are provided in Table alone, with the changes in transformation series directed 1. All taxa were polymorphic for at least two loci, most by a single, large step matrix produced a single most par- taxa were polymorphic at 6-8 loci, and Etheostoma beanii simonious tree (TL =132 steps) with a monophyletic sub- was polymorphic at 10 loci. genus Ammocrypta that was the sister group to a group of £. vitreum and the £. Analysis of the allozyme frequency data using BIOSYS- consisting species pair longimanum- E. This tree has the same as that in 1 produced two trees. These differed from each other only nigrum. topology Fig- ure 3. in the placement of Etheostoitm nigrum. Rogers genetic distance, modified Rogers distance, Prevosti distance, and Total evidence PAUP analysis using a series of step = Edwards "E" distance methods all resulted in a topology matrices found a single most parsimonious tree (TL 207) wherein £. nigrum was the sister taxon to the subgenus with a monophyletic subgenus Ammoeri/pta as the sister the sister Ammocrypta (Fig. 1). The Cavalli-Sforza and Edwards chord taxon to Etheostoma vitreum; these taxa formed distance and Cavalli-Sforza and Edwards arc distance taxon to the species pair, £. longimanum-E. nigrum (Fig. 5). methods resulted in identical topologies, with £. nigrum Total evidence PAUP analysis using the single, large step = the sister taxon to the species pair £. longimnnum-E. vit- matrix resulted in two most parsimonious trees (TL 174) reum (Fig. 2). with a monophyletic subgenus Ammocrypta that was the sister to £. vitreum. In one tree 6), £. is PAUP analysis of the allozyme data alone, using a se- group (Fig. nigrum the sister to a £. ries of step matrices that only allowed character states ob- group monophyletic group containing E. vitreum, and the In served in the study taxa, produced two equally parsimo- longimainim, subgenus Amnwcn/pta. = the other tree 5), the £. nious trees (TL 166 steps). Each contained a monophyl- (Fig. species pair longimanum-E. form a sister to £. vitreum and etic subgenus Ammocrypita that was the sister group to nigrum monophyletic group the Etheostoma nigrum, E. longimanum, and E. vitreum. In one subgenus Anunocrypta. tree (Fig. 3), £. vitreum was the sister group to the species The most parsimonious tree examined in the pair £. longimanum-E. nigrmn, and in the other (Fig. 4), £. FREQPARS analyses based on the allozyme data alone was nigrum was the sister group to the species pair £. 113.442 FREQPARS units long. This tree is isomorphic with longimanum-E. vitreum. PAUP analysis of the allozyme data one of the two most parsimonious trees based on the PAUP

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1 . Tree for ten of Etheostoma Fig. topeilogy species resulting from Fig. 2. Tree topology for ten species of Etheostoma resulting from BIOSYS-1 analysis of the allozyme data alone using Rogers genetic dis- BIOSYS-1 analysis of the allozyme data alone using Cavalli-Sforza and tance, modified Rogers genetic distance, Prevosti distance and Edwards Edwards chord distance and the Cavalli-Sforza and Edwards arc dis- "E" distance methods. This topology is 116.006 freqpars units when only tance methods. This topology is 115.606 freqpars units when only the the allozyme data are considered and 21 6.004 freqpars units for the total- allozyme data are considered and 211.604 freqpars units for the total- evidence data. evidence data. Phylogenetic Relationships of Sand Darters

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trees for ten Fig. 3. The first of two most parsimonious (166 steps) data species of Etheostoma resulting from PAUP analyses of the allozyme alone using a series of step matrices. This is also the single most parsimonious tree (132 steps) resulting from PAUP analyses of the allozyme data alone using a single large step matrix. This topology is 113.442 freqpars units when only the allozyme data are considered and 205.441 5. The most tree (207 for ten freqpars units for the total-evidence data. Fig. single parsimonious steps) species of Etheostoma resulting from PAUP analyses of the total-evidence data using a series of step matrices. This is also the first of two most parsimonious of the trees (174 steps, the second is Fig. 6) resulting from PAUP analyses total-evidence data using a single large step matrix. This topology is 117.376 193.375 freqpars units when only the allozyme data are considered and .<^ for the total evidence. .<^ c>y freqpars units p.^' .<^ Unambiguous character-state support (see text) resulting from PAUP /^^ analysis of the total-evidence data using a series of step matrices for each of the labeled nodes is as follows: 1.—TS 1 (presence of c and loss of b), TS 13 (loss of d), TS 33:1 (curved ventral plate of the urohyal), TS 48:1 (thick- flat ened fin tips of breeding males), and TS 52:2 (female genital papillae and bilobed). 2.—TS 15 (loss of a), TS 19 (presence of c and loss of b), TS 22 matrix was TS 24:1 (loss of d or f, depending on which step used), (posteriorly reclined ascending process of the premaxUla), TS 27:1 (palatine teeth absent), TS 29:1 (rectangular body of the quadrate), TS 30:1 (hyomandibular struts extremely reduced to absent), TS 34:1 (articular process for the interhyal on the posterior ceratohyal absent), TS 42:1 (vomerine teeth usu- the ally absent), TS 44:1 (remnant of the lateral-line canal on supracleithrum absent), and TS 50:0 (tubercles on pelvic fins of breeding males present). 3.—TS 7 (presence of b and loss of a), TS 1 1 (presence of d and loss of a), TS d or 13 (presence of e and loss of b), TS 14 (loss of c and presence of f, 22 of b and loss depending on which step matrix was used), TS (presence of e), TS 31:1 (descencling process of hyomandibula short), TS 32:1 of (hyomandibular spur present), TS 36:1 (notch in the anterior angle the preopercle absent), TS 40:0 (mesethmoid thick and expanded anteriorly), and TS 43:1 (membrane bone on the lateral margin of the nasal reduced). — of the serrate) and TS 39:1 4. TS 35:1 (posterior margin preopercle 5.— 1 4 (posterodorsal extension of the subopercle truncated). TS (presence matrix was usecf) and TS of d and loss of e or f, depending on which step matrix 23 (presence of c and/or the loss of b, depending on which step was used). 6.—TS 22 (presence of a and/or loss ofb, depending on which used) and TS 51:1 (tubercles on the anal fins of step-matrix was breeding — 2 of f males present). 7. TS 1 (presence of c and loss of b), TS (presence of and g and loss of d), TS 4 (presence of b and loss of c), TS 21 (presence c), TS 26:1 (premaxillary socket of the maxilla U-shaped), TS 28:1 (notch trees for to the articular of the quadrate deeply cut), TS 35:0 Fig. 4. The second of two most parsimonious (166 steps) posteroventral process of the smooth), TS 38:1 strut greatly ten of Etheostoma from PAUP of the (posterior margin preopercle (opercular species resulting analyses allozyme struts on reduced), TS 45:1 (postcleithrum 2 absent), TS 46:1 (longitudinal data alone using a series of step matrices. This topology is 1 1 3.840 freqpars and TS 49:1 the anal proximal pterygiophores absent), (body squamation units the data are considered and 209.839 — that the when only allozyme freqpars reduced laterally). A. allozymic character states support node, units for the total-evidence data. M.—morphological character states that support the node. Scientific Papers, Natural History Museum, The University of Kansas

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CO Phylogenetic Relationships of Sand Darters

a. 10 Scientific Papers, Natural History Museum, The University of Kansas

when directed a matrix 3). For .<^ by single, large step (Fig. ^o^ ./..<^ allozyme data alone, the tree presented by Simons (1992; = ^ ^ TL 121.208) was considerably longer than the most parsimonious FREQPARS trees, as were the two trees found = <<> ^ <(> <0- <0^ <

Unambiguous character-state support (i.e., character states that support the monophyly of the group in both ACCTRAN and DELTRAN optimizations) for the total- evidence tree shown in Figure 5 is extensive. Five derived character states support the monophyly of the subgenus Bolcosoum. Eleven unambiguous synapomorphies support Etheostoma vitretim as the sister group to the subgenus 6. The second of two most trees Fig. parsimonious (174 steps, the Ammoctypta, and 10 support monophyly of the subgenus first is 4) for ten of Ethcostonm from PAUP Fig. species resulting analyses Ammocn/ptn. Two unique, unambiguous character-state of the total-evidence data using a single large step matrix. This topology transformations each three clades: is 115.974 freqpars units when only the allozyme data is used and 193.973 support monophyletic E. E. freqpars units for the total-evidence data. peUnciditm, meridianum, E. vivax, E. bifascia, and E. beanii; E. meridimuim, E. vivax, E. bifascia, and £. beaiiii; and analysis of the allozyme data alone when directed by a E. vivax, E. bifascia, and E. beanii. The E. bifascia-E. beanii series of step matrices and to the single most parsimoni- species pair is supported by 11 unambiguous ous tree from the PAUP analysis of the allozyme data alone synapomorphies.

DISCUSSION

The most parsimonious FREQPARS total-evidence tree the analysis of morphological data alone. Figure 5 differs (Fig. 5) differs from the morphology-only tree of Simons from Simons (1992) in the placement of Etheostoma vivax, (1989, 1992) and the allozyme-only trees (Figs. 3, 4). This E. meridianum, and E. pellucidum. In our total-evidence result strongly indicates that both types of data contrib- FREQPARS analysis, these three taxa are sequentially basal to the uted shortest topology. For three branches the con- to the E. beanii-E. bifascia species pair not the monophyl- tribution of each of is type data essentially equal and there- etic sister group to the species pair. Optimization of our fore congruent. These branches subtend the following total-evidence data onto the tree topology reported by clades: subgenus Boleosoma (2 allozymic [A], 3 morpho- Simons (1992) results in a FREQPARS tree length of 195.207 logical [M]), subgenus Ammocrijpia (5A, 5M), and steps; this result is 1 .832 FREQPARS steps longer than our Etheostoma vivax, E. bifascia, and E. beanii (lA, IM). Two most parsimonious total-evidence tree (Fig. 5). Moreover, clades are best supported by the morphological data, but relationships found in this study do not support either of allozymic character states also support the relationships the species groups proposed by Williams (1975) on the basis of loa and Ammocrypta (3A, 8M), and the species pair E. of external morphology. Optimization of our total-evidence beanii-E. bifascia (4A, 7M). Two clades are supported by data data onto the tree implied by Williams (1975) was not pos- that show complementarity (i.e., branches that would lack sible, because he did not include all the taxa used in our unambiguous support in analyses that have only one type study. We are therefore unable to provide a comparable of data). These include the clade consisting of £. FREQPARS length for our total-evidence data optimized meridianum, E. vivax, E. bifascia, and E. beanii (2A, OM) and onto Williams' (1975) set of relationships. Ammocrypta, exclusive of E. claritm (OA, 2M). The analysis of both allozymic data and the morpho- of The addition allozymic data to the morphological logical data together also results in a different set of rela- data compiled by Simons (1992) results in a different set of tionships within Aunnocrypta than those resulting from the relationships within Ammocn/pto than those resulting from analysis of the allozyme data alone. Within the subgenus Phylogenetic Relationships of Sand Darters 11

Ammociyptn, the allozyme-only analyses always placed shows three allozymic character states (TS 15: loss of a, TS Etht'ostonm pdlucidum as the most basal member of the 19: presence of c and loss of b, and TS 22: loss of d or f clade, whereas the total-evidence analyses placed E. claniiu depending on which step matrix was used) and eight as the basal taxon. Overall, the most-parsimonious total- morphological character states (TS 24:1, TS 27:1, TS 29:1, evidence topology for the subgenus Ammocrypta is more TS 30:1, TS 34:1, TS 42:1, TS 44:1, and TS 50:0) that support similar to the allozyme-only trees than the morphology- Simons' (1992) hypothesis that £. vitreum is the sister taxon only trees. to the subgenus Ammocrypta.

The relationships of Etheostoma vitreiim differed among The subgenus Boleosoma was monophyletic in some the analyses. For the total-evidence trees (Figs. 5, 6) and analyses and not in others. The most parsimonious the morphology-only trees (Simons, 1989, 1992) £. vitrcum FREQPARS total-evidence tree (Fig. 5, 193.375 FREQPARS was always the sister taxon to the subgenus Amiiiocri/ptn. units long) provides the best-supported resolution of rela- Simons' (1992) analysis identified seven characters that tionships of the Boleosoma group. This tree has a mono- supported the monophyly of a clade composed of E. vit- phyletic Boleosoma with Etheostoma nigrum and E. reiim and the subgenus Ammocrypta, all of which were in- longimanum as sister taxa. This was also the result when a cluded in this study. Note that three allozymic character single, large step matrix directed the analysis of the states also support this grouping. However, the allozyme- allozyme data and one of the two trees resulting from the only trees had £. vitreum more closely related to members analysis of the allozyme data as directed by a series of step of the subgenus Boleosoma than to members of the subge- matrices (Fig. 3). An alternative, slightly longer FREQPARS nus Ammocrypta. Wood and Mayden (1997) found similar total-evidence tree (Fig. 6, 193.973 FREQPARS units long) results in an allozyme analysis. Simons (1992) also identi- has a paraphyletic Boleosoma with E. longimanum as the sis- fied two character states that potentially supported an E. ter group to Ammocrypta-loa and E. nigrum basal to these vitrciim-Boleosoiua clade—attachment of eggs on rocks and taxa. Additionally, the second of the two trees resulting darkened breeding coloration. Members of the subgenus from the analysis of the allozyme data as directed by a Boleosoma attach their eggs to the undersurfaces of rocks series of step matrices (Fig. 4) has a paraphyletic Boleosoma above the substrate (Page, 1985) and E. vitrcum attach their with E. nigrum sister to the species pair E. vitreum-E. eggs to the vertical surfaces of rocks (Winn and Picciolo, longimanum. This diversity of results suggests that the 1960). Other darters bury their eggs in the sand and gravel monophyly of the subgenus Boleosoma might be question- substrate, clump their eggs under large rocks where the able. Simons (1992) identified three character states that rock meets the gravel substrate, and deposit their eggs on supported the monophyly of the subgenus (presence of vegetation and submerged debris such as leaves, twigs, white knobs on the paired fins of breeding males, the ab- and roots (Page, 1985). The preferred spawning location sence of male breeding tubercles, and flattened, bilobed of most species of Ammocrypta is unknown, except for E. female genital papillae). Of these, he expressed doubt re- pellucidum that bury their eggs in a sand and gravel sub- garding the validity of the presence white knobs on the strate (Johnston, 1989). The preference for attaching eggs paired fins of breeding males, because similar knobs are to the undersurfaces of rocks is not unique to Boleosoma; present in other taxa of darters. He noted that removal of this behavior is also true of members of the subgenus this character from his analyses resulted in two additional Cato}wtus (Page, 1985). Moreover, Simons (1992) also ar- equally parsimonious trees, in one of which the node sup- gued that attaching eggs to the undersurfaces of rocks was porting the monophyly of Boleosoma collapsed resulting probably not the same character state as attaching eggs to in a paraphyletic Boleosoma. Our most parsimonious total- the vertical surfaces of rocks. Breeding males of Boleosoma evidence FREQPARS tree (Fig. 5) showed a monophyletic (Cole, 1967) and £. vitreum (Winn and Picciolo, 1960) Boleosoma supported by two allozymic character states (TS darken in the breeding season and lack any other bright 1: presence of c and loss of b, and TS 13: loss of d) and coloration. Although many breeding male darters exhibit three morphological character states (TS 33:1, TS 48:1, and in brilliant hues of red, orange, yellow, green, and blue, many TS 52:2), but the variation in trees suggests instability this of the tree. breeding males also have darkened bodies (e.g., members region of the Nothoiiotus, and subgenera Etheostoma, Vaillautia, Wiley and Mayden (1985) analyzed the pattern of spe- Catouotus) 1983). These two character states do not (Page, ciation in several groups of aquatic vertebrates distributed unite £. vitreum and Boleosoma because in each strongly along the northern Gulf Coastal Plain. They found that there are other taxa that share the derived state. The case, many groups had pairs of sister taxa with one species oc- differences the from the use between topologies resulting curring in the Mississippi River and drainages to the west that there is of different data sets, do however, indicate and with the other species occurring in drainages to the some homoplasy concerning the relationships of these taxa. east of the Mississippi River. They suggested that this pat- In spite of this, the most parsimonious hypothesis (Fig. 5) tern resulted from a "vicariance event loosely associated 12 Scientific Papers, Natural History Museum, The University of Kansas with the Mississippi River" (p. 605). This explanation re- distributions were not close enough to make this explana- quires that the taxa in question are sister taxa and it was tion acceptable. He suggested that the more northern dis- hypothesized to be true for Etheostoma clanim and the spe- tribution of £. vivax did not fit Wiley and Mayden's (1985) beaiiii. In results cies pair, £. bifascia-E. the present study, these taxa explanation. Our do not support these hypotheses, do not form a monophyletic group (Fig. 5), and therefore because sister group relationships among these taxa were this hypothesis is no longer vahd. Wiley and Mayden (1 985) not demonstrated (Fig. 5). However, the sister-group rela- also found that many groups had one sister species occur- tionship between £. bifascia and £. bcaiiii is strongly sup- ring in the Mobile Bay Basin and drainages to the west ported. Their distributions (£. bifascia in Gulf Slope drain- and the other sister species occurring in drainages to the ages east of the Mobile Bay Basin and £. beanii in the Mo- east of the Mobile Bay Basin. They suggested that the dis- bile Bay Basin and Gulf Slope drainages to the west) re- tribution of £. meridiammi and £. vivnx-E. pellucidiim was main an excellent example of a vicariant speciation event the result of a vicariance event in this region. Simons' (1992) associated with the origin of the Mobile Bay Basin. The results suggested that this explanation was possible (be- distributions of the rest of the subgenus Ammocn/pta are cause these were sister groups), but he thought that the best explained as resulting from dispersal and differentia- tion, not vicariant speciation.

LITERATURE CITED

Bailey, R. M., and D. A. Etnier. 1988. Comments on the subgenera of dart- Hay, O. P. 1883. On a collection of fishes from the lower Mississippi val- ers (Percidae) with descriptions of two new species of Etlwostomn ley. Bulletin of the U.S. Fisheries Commission 1882. 2:57-75. iUlocentra) from southeastern United States. Miscellaneous Publi- Hennig, W. 1966. Ph\/logenetic Systematics. Translated by Davis, D. D. and cations of the Museum of Zoology, University of Michigan 175;1- R. Zangerl. Chicago: University of Illinois Press. 48. Howell, W. M. 1968. Taxonomy and distribution of the percid fish, Bailey, R. M., and W. A. Gosline. 1955. Variation and systematic signifi- Etheostoma stigmaeum (Jordan), with validation and redescription of cance of vertebral counts in the American fishes of the family Etheostoma dai'isoni Hay. Ph.D. dissertation, University of Alabama, Percidae. Miscellaneous Publications of the Museum of Zoology, Tuscaloosa, Alabama. University of Michigan 93:1^14. International Union of Biochemistry Nomenclature Committee. 1984. Brooks, D. R., and E. O. Wiley. 1985. Theories and methods in different Enzyme Nomenclature. Orlando: Academic Press. 606 p. approaches to phylogenetic systematics. Cladistics 1:1-12. Jenkins, R. E. 1971. Nuptial tuberculation and its systematic significance Buth, D. G. 1983. Duplicate isozyme loci in fishes: Origins, distribution, in the percid fish Etheostoma Oon) vitreum. Copeia 1971:735-738. phyletic consequences and locus nomenclature. Pp. 381^00 in M. Jenkins, R. E., and N. M. Burkhead. 1993. Freshwater Fishes of Virginia. C. Rattazzi, J. G. Scandalios, and G. S. Whitt (eds.). Isozymes: Ci(r- Bethesda: American Fisheries Society. 1079 p. rent Topics in Biological nnd Medical Research. Vol. 10. New York: Alan Johnston, C. E. 1989. Spawning in the Eastern sand darter, Ammocrypta R. Liss. pelhicida (Pisces: Percidae), with comments on the phylogeny of Cavalli-Sforza, L. L., and A. W. F. Edwards. 1967. Phylogenetic analysis: Ammocn/pta and related taxa. Transactions of the Illinois State Acad- Models and estimation procedures. Evolution 21:550-570. emy of Science 82:163-168. Churchill, S. P., E. O. Wiley, and L. A. Hauser 1985. Biological realities Jordan, D. S. 1877. Contributions to North American ichthyology based and the proper methodology: A reply to Duncan. Taxon 34:124-130. primarily on the collections of the United States National Museum. Cole, C. F. 1967. A study of the eastern johnny darter, Etlwostonia olmstedi 2. A—Notes on Cottidae, Etheostomatidae, Percidae, Centrarchidae, Storer (Teleostei, Percidae). Chesapeake Science 8:28-51. Aphrododeridae, Dorysomatidae, and Cyprinidae, with revisions Collette, B. B. 1 965. Systematic significance of breeding tubercles in fishes of the genera and descriptions of new or little known species. Bulle- of the family Percidae. Proceedings of the U.S. National Museum tin of the U.S. National Museum 10:5-68. 117:567-614. Jordan, D. S., and S. E. Meek. 1885. List of fishes collected in Iowa and Edwards, A. W. F. 1971. Distances between populations on the basis of Missouri in August, 1884, with descriptions of three new species. gene frequencies. Biometrics 27:873-881. Proceedings of the U.S. National Museum 8:1-17. Edwards, A. W. P. 1974. Distance measures for phylogenetic trees. Pp. Kluge, A. G. 1984. The relevance of parsimony to phylogenetic inference. in 41^3 J. F. Crow and C. Denniston (eds.). Genetic Distance. New Pp. 24-38 11! T. Duncan and T Stuessy (eds.), Cladistics: Perspectives York: Plenum Press. on the Reconstruction of Evoliitionari/ History. New York: Columbia Etnier, D. A., and W. C. Starnes. 1993. The Fishes of Tennessee. Knoxville: University Press. University of Tennessee Press. Kluge, A. G. 1985. Ontogeny and phylogenetic systematics. Cladistics Farris, J. S. 1972. Estimating phylogenetic trees from distance matrices. 1:13-28. American Naturalist 106:645-668. Kluge, A. G. 1989. A concern for evidence and a phylogenetic hypothesis S. 1982. Farris, J. Outgroups and parsimony. Systematic Zoology 31:328- of relationships among Epncrates (Boidae, Serpentes). Systematic 334. Zoology 38:7-25. S. 1983. basis of Farris. the evolu- Farris, J. The logical phylogenetic systematics. Pp. 7-36 Kluge, A. G., and J. S. 1969. Quantitative phyletics and in N. I. Platnick and V. A. Funk (eds.). Advances in Cladistics. Vol. 2. tion of anurans. Systematic Zoology 18:1-32. York: Columbia Press. P. 1993. data: Ex- New University Mabee, M., and J. Humphries. Coding polymorphic and A. G. 1985. 42:166- Farris, J. S., Kluge. Parsimony, synapomorphy, and examples from allozvmes and ontogeny. Systematic Biology planatory power: A reply to Duncan. Taxon 34:130-135. 181. and A. G. 1986. Fishes and Farris, ]. S., Kluge. Synapomorphy, parsimony, and evi- Mettee, M. F, P. E. O'Neil, and J. M. Pierson. 1996. of Alabama dence. Taxon 35:298-306. the Mobile Basin. Birmingham: Oxmoor House, Inc. 820 p.

Fildes, R. A., and H. Harris. 1966. Genetically determined variation of Miller, R. J., and H. W. Robison. 1973. The Fishes of Oklahoma. Stillwater: adenylate kinase in man. Nature (London) 209:261-263. Oklahoma State University Press. Phylogenetic Relationships of Sand Darters 13

Fishes. in Moore, G. A. 1968. Pp. 21-65 W. F Blair, A. P Blair, P Brodkorp, Systenuitics. Htstoncnt Ecologx/, & North American Freshwater Fishes. F. R. Cagle, and G. A. Moore (eds.), Vcrtcbmtcs of the United States. Stanford: Stanford University Press. New York: McGraw-Hill. Stevens, P. F 1980. Evolutionary polarity of character states. Annual Re- R. W. D. G. Murphy, W., J. Sites, Jr., Buth, and C. H. Haufler. 1990. Pro- view of Ecology and Systematics 11:333-358. teins I: Isozyme electrophoresis. Pp. 45-126 in D. M. Hillis and C. Swofford, D. L. 1981. On the utility of the distance Wagner procedure. Moritz (eds.). Molecular Systeinatics. Sunderland: SLnauer Associates. Pp. 25-43 III V. A. Funk and D. R. Brooks (eds.), Advances in Cladis- Page, L. M. 1983. Handbook of Darters. Neptune City, Horida: TFH Publi- tics: Proceedings of the First Willi Hennig Society. New York: New York cations. Botanical Garden. Page, L. M. 1985. Evolution of reproductive behaviors in percid fishes. Swofford, D. L. 1988. FREQPARS: Frequency Analysis Using Parsimony. lUinois Natural History Survey Bulletin 33:275-295. Version 1 .0. Computer program distributed by the Illinois Natural Page, L. M., and G. S. Whitt. 1973a. Lactate dehydrogenase isozymes of History Survey, Champaign, Illinois. darters and the inclusiveness of the genus Percina. Illinois Natural Swofford, D. L. 1993. PAUP: Phylogenetic Analysis Using Parsimony, History Survey, Biological Notes 82:1-7. Version 3.1.1. Computer program distributed by the Illinois Natu- Page, L. M., and G. S. Whitt. 1973b. Lactate dehydrogenase enzymes, ral History Survey, Champaign, Illinois. malate dehydrogenase isozymes and tetrazolium oxidase mobili- Swofford, D. L., and S. H. Berlocher. 1987. Inferring evolutionary trees ties of darters (Ftheostomatini). Comparative Biochemistry and from gene frequency data under the principle of maximum parsi- Physiology 44B:61 1-623. mony. Systematic Zoology 36:293-325. Pflieger, W. L. 1997. Ttic Fishes of Missouri, revised edition. Jefferson City: Swofford, D. L., and R. B. Selander. 1981. BlOSYS-1: A FORTRAN pro- Missouri Department of Conservation. gram for the comprehensive analysis of electrophoretic data in popu- Putnam, F. W. 1863. List of the fishes sent by the museum to different lation genetics and systematics. Journal of Heredity 72:281-283.

institutions, in exchange for other specimens, with annotations. Wiley, E. 0. 1981 . Phylogenetics: The Theon/ and Practice of Phylogenetic Sys- Bulletin of the Museum of Comparative Zoology, Harvard Univer- tematics. New York: John Wiley and Sons. in sity 1(1):2-16. Wiley, E. O., and R. L. Mayden. 1985. Species and speciation phyloge- 1972. distance. netic the fish Rogers, J. S. Measures of genetic similarity and genetic systematics, with examples from North American Studies in Genetics 7:145-153. fauna. Annals of the Missouri Botanical Garden 72:596-635. E. R. 1975. of the fishes of Selander, R. K., M. H. Smith, S. Y. Yang, W. Johnson, and J. Gentry Williams, J. D. Systematics percid the subgenus

1971. Biochemical polymorphism and systematics in the genus Ammocri/pita , Genus Animocryptn, with descriptions of two new spe- Perotnyscus. I. Variation in the old-field mouse (Peromyscus cies. Bulletin of the Alabama Museum of Natural History 1:1-56. R. polionotus). Studies in Genetics 6:49-90. Winn, H. D., and A. Picciolo. 1960. Communal spawning of the Glassy Simons, A. M. 1989. Phylogenetic relationships of the sand darters Darter Etheostoma vitreum (Cope). Copeia 1960:186-192. (Teleostei: Percidae). M.A. thesis. University of Kansas, Lawrence, Wood, R. M., and R. L. Mayden. 1997. Phylogenetic relationships among Kansas, 66 p. selected darter subgenera (Teleostei: Percidae) as inferred from Simons, A. M. 1991. The phylogenetic relationships of the crystal darter, analysis of allozymes. Copeia 1997:265-274. S. and the Vol. 4: Crystallarm asprella. Copeia 1991:927-936. Wright, 1978. Ei'olution Genetics of Populations. Varmbility Simons, A. M. 1992. Phylogenetic relationships of the Boleosonia species in and among Natural Populations. Chicago: University of Chicago group (Percidae: Etheostoma). Pp. 268-292 m R. L. Mayden (ed.). Press.

APPENDIX 1

Specimens Examined.

All material examined is deposited in the ichthyological collection of the Natural History Museum of The Univer- the of actual of sity of Kansas (KU). Vouchers for the electrophoretic analysis include remains specimens (number specimens indicated in parentheses).

Etheostoma meridianum.— Mississippi: Noxubee Etheostoma beanii.—Pearl River Drainage: Missis.sippi: Leake Co.: Pearl Tombigbee Drainage: Co.: Creek at 5.6 mi. S of Mashulaville. KU 23148 River, 2 miles S of Carthage on Mississippi Highway 35. KU 22868 (15). Hashuqua highway 490, (1). Winston Co.: Noxubee River, 5 mi. S of KU 23149 (7). Etheostoma bifascia. —Escambia River Drainage: Florida: Escambia Sturgis. Etheostoma —Kansas River Kansas: Wabaunsee Co.: Pine Barren Creek at Florida highway 29, just S of Pine Barren. KU nigrutn. Drainage: Creek. 23143 22146 (15). Co.: East branch of Mill Creek at mouth of Nehring KU Etheostoma clarum.—White River Drainage: Arkansas: Lawrence Co.: (15). Etheostoma —Wabash River Indl\n a: Fulton Co.: Strawberry River at Arkansas highway 25, 2.5 miles NE of Strawberry. pellucidum. Drainage: at KU 23145 (15). Tippecanoe River Talma. KU 23150 (10). Etheostoma vitreum.—Roanoke River Virginia: Franklin Etheostoma davisoni. —Choctawhatchee River Drainage: Alabama: Drainage; Co.: Blackwater River at Route 220 5.8 airmiles SE of Boones Mill. Bullock Co.: Sec. 26, T12N, R24E, Spring Creek at Bullock County Road bridge, KU 23144 (15). 14, 11 miles SE of Union Springs. KU 23141 (15). Etheostoma vivax. —St. Francis River Arkansas: Co.: Etheostoma longunanum.—James River Drainage: Virginia: Roanoke Drainage: Clay St. Francis River at the Arkansas-Missouri border on Arkansas Co.: Catawba Creek at Route 311 bridge, 0.5 miles SE Catawba. KU 23142 highway miles 1.5 miles of Rector. 23146 (15). (15). 90, 8.75 E and S KU 14 Scientific Papers, Natural History Museum, The University of Kansas

APPENDIX 2

Enzymes, International Union of Biochemistry Nomenclature Committee numbers, loci, tissue sources, and electrophoretic conditions for examination of the subgenus Ammociypta.

Tissue Enzyme lUBNC No. Locus Phylogenetic Relationships of Sand Darters 15

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