HARVARD UNIVERSITY Ernst Mayr Library of the Museum of Comparative Zoology MCZ LIBRARY OCCASIONAL PAPERS MAy j g ^ of the NATURAL HISTORY MUSEUM The University of Kansas Lawrence, Kansas NUMBER 171, PAGES 1-20 31 DECEMBER 1994 A Phylogenetic Analysis of the Lythrurus roseipinnis Species Group (Teleostei: Cyprinidae) with Comments on the Relationships of Other Lythrurus E. O. Wiley and D. Siegel-Causey1 Division of Ichthyology, Natural History Museum and Department of Systematica and Ecology, The University of Kansas, Lawrence, Kansas 66045-2454, USA ABSTRACT A phylogenetic analysis using morphological and allozyme charac- ters was performed on members of the genus Lythrurus and two outgroup species, Cyprinella lutrensis and Pteronotropis signipinnis. Of the 39 presumptive loci examined, 15 showed variation. Twenty-three morphological transformation series also were examined. The most parsimonious tree (68 steps), two trees one step longer, and five trees two steps longer have the following characteristics. ( 1 ) The L. roseipinnis species group is monophyletic; (2) L. roseipinnis is the sister species of L. atrapiculus; (3) L. umhratilis and L. ardens are sister species; and (4) Lythrurus species share several electrophoretic synapomorphies, as well as the morphological synapomorphies mentioned by earlier workers. The two trees of 69 steps are similar to the most parsimonious tree in recognizing the L. umhratilis — L. ardens group as the sister group to the L. roseipinnis group. Key words: Cyprinidae; Lythrurus; Systematics; Phylogenetics; Morphology; Elec- trophoresis. The Lythrurus roseipinnis species group is composed of three species of eastern North American freshwater minnows distributed along the northern Gulf Coastal Plain. Lythrurus roseipinnis (Hay in Jordan, 1885) is the 'Present address: Department of Biology, Nebraska Hall W436, University of Nebraska, Lincoln, Nebraska 68588-0514, USA. © Natural History Museum. The University of Kansas. Lawrence. ISSN:009 1-7958 2 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 western member of the group and is found in tributaries of the Mississippi River and Lake Pontchartrain, Louisiana, east to the lower reaches of the Alabama River (Mobile Bay Drainage). Lythrurus atrapiculus (Snelson, 1972) is found from the Escambia River Drainage, Florida and Alabama, east to the Apalachicola River Drainage of Georgia. Lythrurus bellus (Hay, 1 88 1 ) is found in the Mobile Bay Drainage of Mississippi and Alabama and in two small tributaries of the Tennessee River Drainage (Snelson, 1980a ). The Lythrurus roseipinnis species group is one of four species com- plexes recognized by Snelson (1972) and accorded generic status by Mayden (1989). Members of the genus share three synapomorphies (Snelson, 1972; Mayden, 1989): (1) the unique development of urogenital papillae in breeding females, (2) small scales, and (3) reduced anterodorsal squamation. The relationships of Lythrurus to other minnows and the composition of the species complexes are controversial. Snelson (1972) suggested that Lythrurus was closely related to the Notropis atherinoides group. Mayden (1989) presented evidence that Lythrurus is the sister group of a clade composed of Luxilus and Cyprinella, an hypothesis accepted in this paper for purposes of outgroup comparison. Within Lythrurus, Snelson recognized three species groups in addition to the L. roseipinnis group. The L.fumeus species group was composed of L.fumeus, a. species considered by Snelson (1972) to be the least special- ized of Lythrurus. Snelson (1973) reviewed the systematics of this species. Subsequently, Robison (1985) described Lythrurus snelsoni and added it to the L.fumeus complex. Snelson's (1972) L. umbratilis group consisted of L. umbratilis, the subspecific composition of which was refined by Snelson and Pflieger (1975). The L. ardens complex was composed of L. ardens and L. Virus. Subsequently, Snelson (1980b) reviewed the systematics of L. lirus. Lythrurus matutinus (Cope, 1870), a species formerly composed of L. ardens populations from the Tar and Neuse river drainages of North Caro- lina, was added to the complex by Page and Burr (1991). Mayden (1989) hypothesized that L. ardens was the sister species of L. umbratilis and that this clade was the sister group of the L. roseipinnis group. He was not able to resolve a trichotomy among L.fumeus, L. lirus, and the L. ardens clade. Snelson (1972) suggested two evolutionary scenarios for evolution among species of the Lythrurus roseipinnis group. The first hypothesized that L. bellus gave rise to L. atrapiculus which, in turn, gave rise to L. roseipinnis as the complex moved westward. In phylogenetic terms, this suggests that L. bellus is the sister to a pair composed of L. roseipinnis and L. atrapiculus. The second scenario suggests that L. bellus gave rise independently to L. roseipinnis and L. atrapiculus. Stein et al. (1985) analyzed relationships among members of the Lythrurus roseipinnis group using electromorphs. Their analysis favored Snelson's (1972) second scenario because some populations of L. bellus (L. MAY 1 8 1995 FISHES OF THE LYTHRURUS ROSEIPINNIS SPECIES GROUP 3 b. alegnotus) were electrophoretically more similar to L. atrapiculus, oth- ers to L. roseipinnis, and still others to populations of all three species included in the analysis. Indeed, Stein et al. (1985) suggested the possibil- ity that L. atrapiculus was not a species distinct from L. bellus. The primary purpose of this study is to reassess the relationships within the Lythrurus roseipinnis species group using osteological, color-pattern, and electromorph characters. In the course of our study we examined at least one member of each species complex recognized by Snelson (1972), as well as outgroup species of Cyprinella and Pteronotropis. As a result, we were able to test the relationships recently proposed by Mayden (1989). MATERIALS AND METHODS Characters for analysis were obtained from alcohol-preserved, disar- ticulated, cleared-and-stained, and frozen specimens. Characters taken from Snelson (1972) and Mayden (1989) were checked against appropriate specimens. Material examined is listed in a separate section, below. Fishes used for electrophoresis were collected by seining, immediately frozen in liquid nitrogen, transported to the laboratory, and stored at -70°C. Voucher specimens are deposited in the Division of Ichthyology, Natural History Museum, The University of Kansas. Liver, skeletal muscle, and brain were dissected and homogenized sepa- rately in a 1:1 (v:v) mixture of tissue and 0.01 M Tris, 0.001 M EDTA, and 0.001 M mercaptoethanol, pH 6.8. Homogenates were centrifuged at 15,000xg for 10 min at 5°C. Within 72 h, the supernatant fractions were electrophoresed at 5°C on horizontal starch gels composed of 12% hydro- lyzed potato starch. Thirty-eight presumptive gene loci were visualized by histochemical staining following standard methods. Enzyme nomenclature follows the recommendations of the International Union of Biochemistry Nomenclature Committee (1984) and locus nomenclature follows the rec- ommendations of Buth (1983). Enzymes, loci, tissue sources, and electro- phoretic conditions are listed in Table 1. Electromorphs were coded a, b, c, etc., in order of increasing anodal mobility. Allelic designations are rel- evant to this study only. Specimens for which morphological data were taken were examined, and characters documented, using a Zeiss dissecting microscope coupled to a Pro Viz digitizing unit and a Macintosh II microcomputer. Drawings were traced from laser-printed digitized files and checked against the specimens in question. Data obtained from all sources were combined into a single data matrix. For electrophoretic data, the locus was considered the transformation series (column, "character") and the allele was considered the character (matrix cell, "character state"). For morphological data, each transformation series UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 Table 1 . Enzymes, loci. International Union of Biochemistry Nomenclature Com- mittee numbers, tissue sources, and electrophoretic conditions for examination of the Lythrurus roseipinnis species group and related species. Tissue 1 Electrophoretic Enzyme IUBNC No. Locus source conditions* Aconitate hydratase 4.2.1.3 M-Acon-A Muscle A Adenosine deaminase 3.5.4.4 Ada-A Muscle B Adenylate kinase 2.7.4.3 Ak-A Muscle E Alkaline phosphtase 3.1.3.1 Akp-A Liver A Aspartate amino- 2.6.1.1 M-Aat-A Muscle E transferase Calcium binding Nonspecific Cbp-1 Muscle E proteins Cbp-2 Muscle E Creatine kinase 2.7.3.2 Ck-A Muscle E Ck-B Brain E Fructose bisphosphate 4.1.2.13 Fba-C Brain A aldolase Fumarate hydratase 4.2.1.2 Fum-A Muscle B General protein Nonspecific Gp-1 Muscle E Guanine deaminase 3.5.4.3 Gda-A Muscle E Glucose-6-phosphate 1.1.1.49 G6pdh-B Liver C dehydrogenase Glucose-6-phosphate 5.3.1.9 Gpi-A Muscle A isomerase Gpi-B Muscle A Glutathione reductase 1.6.4.2 Gsr-A Muscle B Glyceraldehyde-3-phos- 1.2.1.12 Gapdh-A Muscle C phate dehydrogenase Gapdh-B Brain C Glycerol-3-phosphate 1.1.1.8 G3pdh-A Muscle B dehydrogenase Isocitrate dehydrogenase 1.1.1.42 M-Icdh-A Muscle B S-Icdh-A Muscle B L-Lactate dehydrogenase 1.1.1.27 Ldh-A Muscle A Ldh-B Muscle A Ldh-C Liver A Malate dehydrogenase 1.1.1.37 M-Mdh-A Liver B (NAD+-dependent) S-Mdh-A Liver B S-Mdh-B Muscle B Malate dehydrogenase 1.1.1.40 S-Me-A Muscle B (NADP+-dependent) Mannose-6-phosphate 5.3.1.8 M6pi-A Muscle H isomerase Peptidases 3.4.13.11 Pep-A Muscle F Pep-B Muscle F Pep-C Brain F Pep-E Muscle F Pep-S Muscle F FISHES OF THE LYTHRURUS ROSEIPINNIS SPECIES GROUP Table 1. Continued. Tissue Electrophoretic Enzyme IUBNC No. Locus source conditions* Phosphoglucomutase 5.4.2.2 Pgm-A Brain G Phosphogluconate 1.1.1.44 Pgdh-A Brain A dehydrogenase - Purine nucleoside 2.4.2.1 Pnp-A Muscle B phosphorylase Pyruvate kinase 2.7.1.40 Pk-A Muscle E Superoxide dismutase 1.15.1.1 Sod-A Liver G Triose phosphate 5.3.1.1 Tpi-A Brain B isomerase Tpi-B 1 Brain B Tpi-B2 Brain B Xanthine dehydrogenase 1.1.1.204 Xdh-A Muscle G *A: Tris-citrate-NADP pH 7.0. 8 V/cm 10 h; B: Tris-citrate pH 8.0, 6.5 V/cm 10 h; C: Tris-borate-EDTA-NAD-NADP pH 9. 1 , 1 1 V/cm 14 h; D: Borate-NAD pH 8.8, 7 V/cm 1 1 h; E: Histidine-citrate-NADPpH 7.0, 5 V/cm 1 1 h; F: LiOH 14 v/cm 10 h; G: Poulik-NADP, 10 V/cm 10 h.; H: phosphate-citrate pH 7.0, 3 V/cm 18 h. was treated as an independent statement of homologous character evolu- tion. Phylogenetic analyses were performed using PA UP 3.0. Pteronotropis signipinnis was the designated outgroup and characters provided by May den (1989) were included to insure that Cyprinella lutrensis would perform as the sister (and functional outgroup) of a monophyletic Lythrurus. Transformation series containing more than two characters were analyzed as unordered. Taxa having more than one character in a transformation series were treated as polymorphic. Both DELTRAN and ACCTRAN optimizations were utilized. Because of the small number of taxa involved, the exhaustive search option was used to evaluate all possible tree topolo- gies. Material Examined Material examined is listed in four categories: alcohol preserved, disar- ticulated, cleared-and-stained, and electrophoretic. Specimens from the same lot and with the same catalogue number may appear in each category. State, drainage, catalogue number and number of specimens are given for each lot where they first appear and the number of specimens (in parenthe- ses) refers only to the number of specimens for that category. Only cata- logue number and number of specimens are given in subsequent references to the same lot. Complete locality data are available on request. Alcohol-preserved specimens. — Cyprinella lutrensis: KANSAS: Neosho River Dr.: KU 8146 (100). Lythrurus ardens: ALABAMA: Ala- bama River Dr.: KU 22161 (14). Lythrurus atrapiculus: ALABAMA: 6 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 Yellow River Dr.: KU 18887 (15): KU 18976 (5), KU 21896 (2). Lythrurus bellus: ALABAMA: Alabama River Dr.: KU 8789 (29), KU 20352 (12), KU 20471 (12), KU 22147 (7). Tombigbee River Dr.: KU 8762 (21), KU 14511 (38), KU 14538 (18), KU 17377 (18), KU 22147 (7), KU 21871 (15), KU 21876 (1). MISSISSIPPI: Tombigbee River Dr.: KU 20302 (96). Lythrurus fumeus: TENNESSEE: Big Sandy River Dr.; KU 9794 (7). Lythrurus lirus: GEORGIA: Alabama River Dr.; KU 18986 (15). TEN- NESSEE: Tennessee River Dr.; KU 18933 (20). Lythrurus roseipinnis: ALABAMA: Alabama River Dr.; KU 22893 (2). Escatawpa River Dr.; KU 22895 (41). LOUISIANA: Pearl River Dr.; KU 19621 (9). Lake Pontchartrain Dr.; KU 19471 (9). MISSISSIPPI: Pascagoula River Dr.; KU 15513 (50), KU 16909 (50). Pearl River Dr.; KU 15122 (15), KU 16857 (75), KU 16872 (22). Wolf River Dr.; KU 17832 (40). Lythrurus umbratilis: ARKANSAS: Ouachita River Dr.; KU 6179 (9). KANSAS: Marais des Cygnes River Dr.; KU 22500 (27). Neosho River Dr.; KU 21958 (3). KENTUCKY: Tradewater River Dr.; KU 15142 (5). Pteronotropis signipinnis: FLORIDA: Yellow River Dr.: KU 14475 (30). Disarticulated skeletons. — Cyprinella lutrensis: KU 8146 (5). Lythrurus atrapiculus: KU 18887 ( 1 ), KU 18976 (2). Lythrurus bellus: KU 14511 (2), KU 20471 (1), KU 21884 (2). Lythrurus fumeus: KU 9794 (5). Lythrurus lirus: KU 18986 (4). Lythrurus roseipinnis: KU 16857 (5). Lythrurus umbratilis: KU 6179 (1), KU 15142 (1), KU 21958 (3). Pteronotropis signipinnis: KU 14475 (5). Cleared-and-stained skeletons. — Cyprinella lutrensis: KANSAS: Ver- digris River Dr.; KU 15783 (6); KU 16386 (2). Lythrurus ardens: KEN- TUCKY: Ohio River Dr.; KU 4136 (5). Lythrurus atrapiculus: KU 18887 (10); KU 18976 (3). Lythrurus bellus: KU 14511 (15). Lythrurus fumeus: Sabine River Dr.; KU 6244 (8). Lythrurus lirus: KU 18933 (15). Lythrurus roseipinnis: KU 16857 (14). Lythrurus umbratilis: KU 15521 (13). Pteronotropis signipinnis: KU 16858 (13). Electrophoretic specimens. — Cyprinella lutrensis: KANSAS: Marais des Cygnes River Dr.; KU 23018 (15). Lythrurus ardens: KU 22161 (15). Lythrurus atrapiculus: ALABAMA: Chactawhatchee River Dr.; KU 23019 (10), Escambia River Dr.; KU 23020 (10). Yellow River Dr.; KU 21896 (10); KU 23021 (10). Lythrurus bellus: ALABAMA: Alabama River Dr.; KU 22147 (15). Tombigbee River Dr.; KU 21871 (15); KU 23022 (10). Lythrurus fumeus: TENNESSEE: Tennessee River Dr.; KU 23023 (9). Lythrurus lirus: TENNESSEE: Tennessee River Dr.; KU 23024 (9). Lythrurus roseipinnis: ALABAMA: Alabama River Dr.; KU 23025 (15), LOUISIANA: Lake Pontchartrain Dr.; KU 23026 (10), MISSISSIPPI: Biloxi River Dr.; KU 23027 (10), Pearl River Dr.; KU 22873 (15). Lythrurus umbratilis: KU 22500 (20). Pteronotropis signipinnis: FLORIDA: Escambia River Dr.: KU 23028 (15). FISHES OF THE LYTHRURUS ROSEIP1NNIS SPECIES GROUP RESULTS Presumptive loci and alleles. — Thirty-nine presumptive loci were ex- amined. Twenty-four were fixed for all species and 15 showed variation among and/or within species. The following loci were fixed: Aat- A, S-Acd- A, Acon-A, Ak-A, Akp-A, Cbp-A, Cbp-B, Ck-B, Fum-A, Gda-A, Gapdh- A, G6pdh-A, G6pdh-B, Gpt-A, M-Icdh-A, S-Icdh-A, Ldh-A, S-Me-A, Pep-D, Pgam-A, Pgm-A, Pk-A, Pnp-A, Xdh-A. Allelic frequencies for all variable presumptive loci are listed in Table 2. Alleles within transformation series are designated by letters in Table 3 and appear by transformation series and letter (i.e., 24a, 26b, etc.) in Figure 10. The presumptive locus Gpi-B was dropped from phylogenetic analysis. Morphological characters. — Twenty-three morphological transforma- tion series were examined and found to vary. These are listed below. In each case, characters are coded with their transformation series number (1, 2, etc.) "a" or "b" and this coding is indicated in figures where illustrated. Thus "1-b" denotes the forked condition of the pterotic bone and is shown in Figure 1, left. A similar notation is used for the phylogenetic trees (e.g., la, lb, etc.). 1 . Pterotic (Fig. 1 ). — The pterotic is broad and unforked in the outgroups and all ingroup taxa (1-a) except Lythrurus roseipinnis, in which it is forked (1-b). 2. Frontal (Fig. 2). — In the outgroups and most ingroup taxa the frontal is short relative to its width (2-a). Lythrurus roseipinnis and L. atrapiculus have elongate frontals (2-b). 3. Palatine shaft (Fig. 3). — The shaft of the palatine varies from short and relatively thick in Cyprinella lutrensis, Lythrurus ardens and L.fumeus (3-a) to relatively long inL. umbratilis, L. lirus, and the L. roseipinnis group (3-b). The palatine shaft of Pteranotropis signipinnis is short but constricted. It was coded as "short" (i.e., 3-a). 4. Posterodorsal metapterygoid flange (Fig. 4). — A posterodorsal metapterygoid flange is present above the hyomandibular- metapterygoid articulation in the outgroups Lythrurus fumeus, L. umbratilis and L. ardens (4-a), whereas it is absent or greatly reduced in L. lirus and the L. roseipinnis group (4-b). 5. Spacing of the articulation points of the metapterygoid with the hyomandibular and symplectic (Fig. 4). — The posterior border of the metapterygoid articulates via cartilage with both the hyomandibular and the symplectic. In the outgroups as well as Lythrurus lirus, L. fumeus, L. umbratilis, and L. ardens these points of articulation are relatively widely spaced (5-a), whereas in the L. roseipinnis group they are closely spaced (5-b). 8 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 Table 2. Allele frequencies among samples of Lythrurus and two outgroups examined in this study. Number in parentheses after the locus name refers to the transformation series number in Table 3. ] ^ocus/Allele Ada- A (24) Ck-A(25) Gpdh-A (26) Gpi-A (27) Taxon a b a b a h a b c d P. signipinnis 1.00 1.0C I 1.00 1.00 C. lutrensis 1.00 1.0C 1 1 .00 1.00 L. lirus 1.00 1.00 1.00 1.00 L. fumeus 1 .00 1.00 1.00 0.90 0.05 0.05 L. umbratilis 1.00 1.00 1.00 0.10 0.05 0.85 L. aniens 1.00 1.00 1.00 0.05 0.95 L. be 1 1 us 1.00 1.00 1.00 0.95 0.05 L. roseipinnis 1.00 1.00 1.00 0.05 0.90 0.05 L. atrapiculus 1.00 1.00 1.00 0.95 0.05 Locus/Allele Gsr-A(28) Ldh-A (29) M-Mdh-A(30) S-Mdh-A(31) Taxon a b a b a b a b P. signipinnis 1.00 1.00 1.00 1.00 C. lutrensis 1.00 0.90 0.10 1.00 1.00 L. lirus 1.00 1.00 1.00 1.00 L. fumeus 1.00 1.00 1.00 1.00 L. umbratilis 1.00 0.95 0.05 1.00 1.00 L. ardens 1.00 1.00 1.00 1.00 L. bellus 1.00 1.00 i.00 1.00 L. roseipinnis 1.00 1.00 1.00 0.90 0.10 L. atrapiculus 1.00 1.00 1.00 1.00 Anterodorsal metapterygoid flange (Fig. 4). — The anterodorsal metapterygoid flange articulates with the endopterygoid. In the outgroups, Lythrurus lirus, L. fumeus, L. umbratilis and L. ardens the flange is robust (6-a), whereas in the L. roseipinnis group it is gracile (6-b). Posteroventral flange of the endopterygoid (Fig. 5). — The posterior border of the endopterygoid is relatively straight and shallow in the outgroup taxa, Lythrurus lirus, L. fumeus and the L. roseipinnis group (7-a). Lythrurus ardens and L. umbratilis have a deep flange (7-b). FISHES OF THE LYTHRURUS ROSEIPINNIS SPECIES GROUP Table 2. Continued. Locus/Allele S-Mdh-B (32) M6pi-A (33) 1 3ep-A (34) Pep-B (35) Taxon a b a b a b a b c P. signipinni 1.00 1.00 1.00 1.00 C. lutrensis 1.00 1.00 1.00 1.00 L. lirus 1.00 1.00 1.00 1.00 L. fumeus 1.00 1.00 1.00 1.00 L. umbratilis 1.00 1.00 1.00 1.00 L. arden 1.00 1.00 1.00 1.00 L. bellus 1.00 1.00 1.00 1.00 L. roseipinnis 1.00 1.00 1.00 1.00 L. at rapi cuius 1.00 0.95 0.05 1.00 1.00 Locus/Allele Pep-C (36) Pep-E (37) Pep-A (38) Taxon a b c a b c a b c P. signipinnis 1.00 1.00 1.00 C. lutrensis 1.00 1.00 1.00 L. lirus 1.00 1.00 1.00 L. fumeus 1.00 1.00 1.00 L. umbratilis 1.00 1.00 1.00 L. ardens 1.00 1.00 1.00 L. bellus 1.00 1.00 1.00 L. roseipinnis 1.00 1.00 1.00 L. at rapi cuius 1.00 1.00 1.00 Endopterygoid length (Fig. 5). — The endopterygoid is relatively long in the outgroup taxa and in Ly thrums lirus, L. fumeus, and the L. roseipinnis group (8-a). The bone is relatively short in L. umbratilis and L. ardens (8-b). Symphysial process of lower jaw. — Pteronotropis signipinnis and all members of Lythrurus, except L. lirus and L. fumeus, have a process on the anterior end of the lower jaw (Fig. 6). Cyprinella lutrensis, L. lirus and L. fumeus lack this process. 10 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 -o O T3 3 & " T3 en 8^ Q VH tS en X *■' — 2 « H E o S 03 03 'o -a o o 03 3 ail I csj ja E? — -Ci *£, 3 <3 -Q £ C3 CCU-J>-J>-j>-J'-J>-4>-j •S ? ex. c •~- JU cr, cu SO IT) rn m o en Os CJO (N fN SO fN fN fN fN fN fN 03 CDXXXXXXX 03 03X OX OXXX 03 03X O X X X X X cj 03XXXXXXX 03 03XXXXXXX 03 SL 03 03 03 o3 03 o3 o3Xo3o3Xo3o3o3X o3XX 03X 03X-X 03 03XXXXXXXX X X 03 *- 03 03 r03o3o3o3 03 03 03 03XXXXXXX 03 03 X O, O. £ £ J £ 03 03XXXXXX C 03 03XXXXXXX P3Xo3o3Xo3o3o3o3 03XXXXXXXX 03 03 03 03XXXXX >" ^ a -jS w • ? 5 ~ "P .a .«* ■§ 3 a: U »j ^ >-i kj kj kj >~j FISHES OF THE LYTHRURUS ROSEIPINN1S SPECIES GROUP 1 1 10. Articulatory head of the maxilla (Fig. 7). — The posterior part of the articulatory process of Lythrurus roseipinnis, L. atrapiculus and Cyprinella lutrensis is distinctly wide (10-b) compared to being nar- row in Pteronotropis signipinnis and other Lythrurus (10-a). 11. Dorsal flange of anguloarticular (Fig. 8). — Cyprinella lutrensis, Lythrurus umbratilis, and L. aniens have a high flange (11 -a). Pteronotropis signipinnis and all other Lythrurus have a low flange (lib). 12. Dorsal ridge of anterior arm of cleithrum (Fig. 9). — Lythrurus atrapiculus has a high ridge ( 12-b), whereas all other taxa have a low ridge (12-a). 13. Posterior flange of the ascending arm of the cleithrum (Fig. 9). — The outgroups, Lythrurus umbratilis, L. Urns, and L.fumeus have distinctly narrow flanges (13-a), whereas L. aniens and the L. roseipinnis group have wide flanges (13-b). 14. Ventral edge of posterior flange of ascending arm of the cleithrum (Fig. 9). — The edge is nearly horizontal relative to the anterior arm in the outgroups and most Lythrurus ( 14-a), whereas it is inclined upward in members of the L. roseipinnis group (14-b). 15. Anterodorsal flange of the ascending arm of the cleithrum (Fig. 9). — This flange is rounded in the outgroups, Lythrurus umbratilis, L. Urns, and L. fumeus (15-a), whereas it is triangular in the L. roseipinnis group and L. aniens (15-b). 16. Dorsohorizontal shelf of anterior arm of cleithrum (Fig. 9). — This shelf is narrow in the outgroups and most Lythrurus (16-a), but is wide in L. ardens and L. umbratilis (16-b). 17. Posterior flange of scapula. — The outgroups, Lythrurus fumeus and L. lirus lack this flange (17-a), whereas other Lythrurus have it (17-b: Mayden, 1989; pers. obs.). 18. Size of scales. — The outgroups have relatively large scales (18-a), whereas Lythrurus has relatively small scales (18-b: Snelson, 1972; Mayden, 1989). 19. Predorsal squamation. — The outgroups have uncrowded scales ( 19-a), whereas Lythrurus has crowded scales (19-b: Snelson, 1972; Mayden 1989). 20. Urogenital papillae. — The outgroup females lack enlarged urogenital papillae (20-a) whereas Lythrurus females have them (20-b: Snelson, 1972; Mayden 1989). 12 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 Fig. 1. Left pterotics of Lythrurus in ventral view. Left: L. roseipinnis (KU 16857-5). Right: L. umbratilis (KU 21958-2). Labels refer to characters discussed in text. Scale = 5mm. 21. Vertical bars on body. — Breeding male Lythrurus umbratilis and L. ardens have darkened vertical bars on the body (21-b), whereas other Lythrurus and the outgroups lack them (21 -a). 22. Dorsal fin spot. — Lythrurus umbralilis, L. ardens, and species of the L. roseipinnis group have a dark spot at the origin of the dorsal fin (22-b). Pteronotropis signipinnis , Cyprinella lutrensis, L.fumeus, and L. lirus lack this spot (22-a). 23. Character Suite 7 of Mayden (1989). — Lythrurus, Cyprinella, Luxilus, and Notropis share three synapomorphies (23-b) missing in Pteronotropis (23-a): (1) large cephalic breeding tubercles, (2) sub- marginal breeding tubercles on body scales, and (3) a triangular pa- latine. These characters are included in the analysis as a single trans- formation series. Because weights of one step and three steps result in the same outgroup topology, we used a weight of 1 step to minimize overall tree length. Data Analysis. — Electrophoretic and morphological characters were combined to produce the data matrix shown in Table 3. PAUP analysis evaluated 135 tree topologies. One tree of length 68 steps (CI [Consistency Index] = 0.794, RI [Redundancy Index] = 0.731, RC [Rescaled Consis- tency Index] = 0.580) was found. Two trees of 69 steps (CI = 0.783, RI = 0.712, RC = 0.557) and five trees of 70 steps (CI = 0.771, RI = 0.692, RC = 0.534) were also considered. All eight trees had the following character- istics: (1) the Lythrurus roseipinnis species group is monophyletic. (2) Lythrurus roseipinnis is the sister species of L. atrapiculus. (3) Lythrurus umbratilis and L. ardens are sister species. (4) Lythrurus species share several electrophoretic synapomorphies, as well as the morphological synapomorphies mentioned by Snelson (1972) and Mayden (1989). FISHES OF THE LYTHRURUS ROSEIPINNIS SPECIES GROUP 13 Fig. 2. Left frontals of Lythrurus in dorsal view. Left: L. bellus (KU 20471-1). Right: L. atrapiculus (KU 18976-3). Scale = 5mm. Fig. 3. Left palatines of Lythrurus in medial view. Left: L. ardens (KU 4136- 2). Right: L. bellus (KU 14511-2). Scales = 5mm. The two trees of 69 steps are similar to the most parsimonious tree (Fig. 10) in recognizing the Lythrurus umbratilis — L. ardens group as the sister group to the L. roseipinnis group. Relationships and Character Evolution. — We will use the most parsi- monious tree, shown in Figure 10, as the basis for discussing the relation- ships among the members of the L. roseipinnis species group and the relationships of the group to other Lythrurus. Monophyly of the group: Lythrurus roseipinnis, L. atrapiculus, and L. bellus share four (ACCTRAN optimization) or six (DELTRAN optimiza- tion) morphological synapomorphies concentrated on two bones. On the metapterygoid, the posterodorsal flange is absent or greatly reduced (TS4- b; also found in L. lirus); the articulatory points for the hyomandibular and symplectic are closely spaced (TS5-b), and the anterodorsal flange is gracile (TS6-b). On the cleithrum, the posterior flange of the ascending arm 14 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 Fig. 4. Left metapterygoids of Lythrurus in medial view. Left: L. atrapiculus (KU 18976-2). Right: L. aniens (KU 1436-s). Numbers refer to characters dis- cussed in the text. Scale = 5mm. 8-a 8-b Fig. 5. Left pterygoid series and quadrate of Lythrurus in medial view. Left: L. ardens (KU 4136-2). Right: L. roseipinnis. KU 16857-2). Numbers refer to charac- ters discussed in the text. Scale = 5 mm. is wide (TS13-b, shared with L. ardens); the ventral edge of the ascending arm is inclined upward (TS14-b), and the anterodorsal flange is triangular (TS16-b, shared with L. ardens). It is equally parsimonious to consider characters 13b and 16b as synapomorphies of the L. roseipinnis species group plus the L. umbratilis — L. ardens pair with each character reversed in L. umbratilis (ACCTRAN optimization). Relationships within the L. roseipinnis group: Lythrurus roseipinnis and L. atrapiculus share two morphological synapomorphies not shared with L. bellus. Both have elongate frontals (TS2-b), a character also seen in L. lirus. Both have a wide posterior articulatory process of the maxilla (TSlO-b), a character also seen in the outgroup species Cyprinella lutrensis. L. roseipinnis and L. atrapiculus differ from one another in three FISHES OF THE LYTHRURUS ROSEIPINNIS SPECIES GROUP 15 Fig. 6. Left dentary of Lythrurus atrapiculus (KU 18976-1) in lateral view. Scale = 5 mm. 10-b Fig. 7. Left maxillae of Lythrurus in dorsal view. Left: L. atrapiculus (KU 18976-2). Right: L. umbratilis (KU 21953-1). Numbers refer to characters dis- cussed in the text. Scale = 5mm. 16 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 Fig. 8. Left angular-articular and retroarticular in lateral view. Upper: Lythrurus lints (KU 18933-3). Lower: Cyprinella lutrensis (KU 16386-2). Num- bers refer to characters discussed in text. Scale = 5 mm. autapomorphies. Uniquely, L. roseipinnis has a narrow, forked posterior pterotic process (TSl-b). Uniquely, L. atrapiculus has a high ridge on the anterior arm of the cleithrum (TS12-b). These species also differ in one fixed electromorph of S-Mdh-A (TS32-b). Relationship of the L. roseipinnis species group to other Lythrurus: The Lythrurus roseipinnis group shares three morphological synapomorphies with the L. umbratilis group. The lower jaw has a distinct symphysial process (TS9-b; also found in Pternotropis signipinnis); there is a posterior flange in the scapula (TS17-b; Mayden, 1989), and there is a fin spot at the origin of the dorsal fin (TS22-b; Mayden, 1989). The most parsimonious tree suggests that Lythrurus lirus is the sister species of the L. roseipinnis group + L. umbratilis group. All share a unique electromorph at the Pep-C locus (TS36-b) and a synapomorphy for the Pep-E locus (TS37-b). {Lythrurus ardens has an autapomorphic electromorph, TS37-C.) All species except L. ardens also have a long and relatively thin palatine shaft (TS3-b). This grouping is not supported in the trees of 69 steps in which L. lirus and L.fumeus were grouped as sisters or as part of a trichotomy with the clade composed of the L. umbratilis and L. roseipinnis groups. FISHES OF THE LYTHRURUS ROSEIP1NNIS SPECIES GROUP 17 12-b 13-b 16-a 16-b Fig. 9. Left cleithra of Lythrurus in lateral (upper, slightly rotated) and medial (lower) views. Upper left: Anterior ami of Lythrurus umbratilis (KU 21958-2). Upper right: Anterior arm of L. atrapiculus (KU 18976-1). Lower left: L. atrapiculus (KU 18976-2). Lower right: L. umbratilis (KU 21958-3). Numbers refer to characters discussed in the text. Scale = 5 mm. Monophyly of Lythrurus: The monophyly of Lythrurus is supported both by characters previously reported by Snelson (1972; TS18-b, 19-b, 20-b) and by a suite of electromorphs from the following loci: CK-A (TS25-b), G3pdh-A (TS26-b), Gpi-A (TS27-b), Gsr (TS28-b), Pep-A (TS34-b), Pep- B (TS35-b), Pep-S (TS38-b). DISCUSSION The relationships suggested by our analysis generally support Snelson's (1972) hypothesis that Lythrurus fumeus is a relatively old member of Lythrurus, even when Mayden's (1989) outgroup structure is used. Snelson (1972) and Mayden (1989) presented different hypotheses regarding the relationships of L. aniens, L. Urns, and L. umbratilis. Our study generally supports Mayden's (1989) hypothesis that the vertical bars of breeding males of L. umbratilis and L. ardens (TS21-b) represent a synapomorphy 18 UNIV. KANSAS NAT. HIST. MUS. OCC. PAP. No. 171 *•* tf if * \