Left running head: Sullivan, Lavoue and Hopkins Right running head: Analysis of a riverine species flock

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1 John P. Sullivan Revision Date: November 16, pages 8972 Words 1 table, 7 figures File: brieno cytb formatted.doc Left running head: Sullivan, Lavoue and Hopkins Right running head: DISCOVERY AND PHYLOGENETIC ANALYSIS OF A RIVERINE SPECIES FLOCK OF AFRICAN ELECTRIC FISHES (MORMYRIDAE: TELEOSTEI) by John P. Sullivan 1,3, Sébastien Lavoué 2, Carl D. Hopkins 1 1 Department of Neurobiology and Behavior, Cornell University, Ithaca, NY, USA Museum National d Histoire Naturelle, Ichtyologie Générale et Appliquée; 43 rue Cuvier 75005, Paris, France. 3 Department of Ichthyology, American Museum of Natural History, Central Park West at 79 th St, New York, NY, USA Correspondence to: Carl D. Hopkins cdh8@cornell.edu phone: fax: Keywords: electric fish, species flock, speciation, cytochrome b, phylogeny

2 ABSTRACT We have discovered surprising undescribed diversity within a clade of mormyrid electric fish in our recent collections from the Ogooué and Ntem River basins of Gabon, Africa. We recognize 38 distinct forms within this group on the basis of unique morphologies and electric organ discharge (EOD) characteristics of which only four clearly correspond to described species. The remaining 34, treated here as operational taxonomic units (OTUs), may represent as many new species. We sequenced the complete cytochrome b gene from 86 specimens sampled from these OTUs and recovered 65 distinct cytochrome b haplotypes. Trees obtained by both maximum parsimony and maximum likelihood analyses, rooted with sequence data from outgroup taxa, provide strong support for the monophyly of all 38 of these OTUs. The small genetic distances between many distinct forms suggests an ongoing and rapid radiation. However, in many instances, the cytochrome b tree topology fails to support the monophyly of individual OTUs or close relationships between OTUs which are similar in morphology and EOD characteristics. In other cases, individuals from distinct 1

3 OTUs share identical, or nearly identical, haplotypes. We infer from these results a disjunction between the mitochondrial gene tree and the overall species phylogeny of these organisms. The presence of divergent haplotypes within single populations of some forms suggests incomplete mitochondrial lineage sorting, while the grouping of phenotypically distinct, but geographically proximate forms in other cases suggests local hybridization and introgression of mitochondrial genomes among forms. Both incomplete lineage sorting and introgression may contribute to the lack of congruence between the mitochondrial gene tree and the natural groups supported by morphological and EOD data. Diversification in this clade of riverine electric fishes and the problems associated with recovering a meaningful species-level phylogeny from mitochondrial data suggest a parallel to the well-known lacustrine fish species flocks. Social selection on EOD waveforms may be involved in the rapid radiation of these fishes. 2

4 INTRODUCTION The African weakly electric fish superfamily Mormyroidea (families Mormyridae + Gymnarchidae) represents a remarkable modern radiation from within the Superorder Osteoglossomorpha: one of the oldest and phylogenetically most basal groups of extant teleosts (Arratia 1997; Greenwood 1973; Lauder and Liem 1983; Patterson and Rosen 1977). This radiation in African freshwater riverine environments has occurred while, according to the fossil record, other osteoglossomorph lineages have undergone substantial reduction in their global diversity and distribution since the Early Tertiary (Li 1997). Over 200 species of mormyroids in 19 genera are recognized (Daget et al. 1984). In comparison, the remaining living osteoglossomorphs, although distributed on all continents except Europe and Antarctica, comprise only 18 species in 10 genera (Nelson 1994). Uniquely among the osteoglossomorphs and undoubtedly related to their evolutionary success, all mormyroids electrolocate and communicate by means of specialized electric organs and electroreceptors. In this paper we present evidence based on recent collections in Gabon in West Central Africa that this 3

5 radiation is ongoing explosively in at least one group of mormyroids. Within this group we have identified many taxonomically unrecognized forms possessing distinct electric organ discharge (EOD) waveforms and morphological characteristics. Based on the overall similarity of these characteristics we have sorted the specimens into operational taxonomic units (OTUs) and have investigated their phylogenetic relationships by analysis of sequence data from the mitochondrial cytochrome b gene. Our goals were to establish (1) whether morphological, EOD and molecular data would provide congruent definitions of OTUs and higher-level natural groups within this clade of fishes and (2), whether a molecular phylogeny of these entities would reveal patterns of EOD and electric organ evolution at the species level. Taxonomy and previous phylogenetic studies of the genera Brienomyrus and Paramormyrops The genus Brienomyrus was established by (Taverne 1971) as part of his taxonomic revision of the Mormyridae based upon osteology, but without explicit reference to any uniquely shared derived characters (synapomorphies). Within the genus, he recognized two subgenera: B.(Brienomyrus) and B.(Brevimyrus). Currently, there are 4

6 nine valid species within the first subgenus (B. brachyistius, B. longianalis, B. sphekodes, B. kingsleyae, B. curvifrons, B. longicaudatus, B. batesii, B. taverni, and B. hopkinsi) and a single species (B. niger) in the latter (Alves-Gomes and Hopkins 1997; Teugels and Hopkins 1998). The genus Paramormyrops was also established by Taverne (1977a) for the species Paramormyrops gabonensis from the Ivindo River of Gabon and to which he referred Marcusenius jacksoni Poll, from the Upper Zambesi basin of Angola. Taverne apparently did not include P. gabonensis within Brienomyrus due the presence of an ossified lateral ethmoid in this species, a character absent in the Brienomyrus that he examined. Recent studies have demonstrated the polyphyly of the genus Brienomyrus with several molecular datasets (Alves- Gomes and Hopkins 1997; Lavoué et al. 2000; Sullivan et al. 2000). Lavoué et al. (submitted) performed an unweighted parsimony analysis on the combined data used in these previous studies (from the mitochondrial 12S, 16S, cytochrome b and nuclear RAG2 genes) with new sequence data from two introns in the nuclear S7 gene for 38 species belonging to 17 nominal genera within the subfamily Mormyrinae. The single most parsimonius tree which results 5

7 (Fig. 1) indicates that Brienomyrus (Brienomyrus) brachyistius, the type species of the genus, is the sister species not to any other included Brienomyrus species, but to Isichthys henryi. These species together form the sister group to included species of the genus Mormyrus. Quite separate from this clade, Brienomyrus (Brevimyrus) niger is weakly supported as the sister species to Hyperopisus bebe at the base of a large clade containing species of Marcusenius, Hippopotamyrus, Gnathonemus and Campyolomormyrus. A third clade containing nominal Brienomyrus species (B.hopkinsi, B. longicaudatus) in addition to Paramormyrops gabonensis and an undescribed species (= VAD in this study) appears as the sister group to Marcusenius conicephalus. These species (including M. conicephalus) are endemic to a particular region of Lower Guinea: the Ogooué, Ntem, and Woleu/Mbini River basins of Gabon, southern Cameroon and Equatorial Guinea. The sister group of these taxa is a clade containing taxa endemic to the same region: Boulengeromyrus knoepffleri, Ivindomyrus opdenboschi and Pollimyrus marchei (Daget et al. 1984; Kamdem Toham 1998). In addition to high bootsrap and decay index values in this analysis, the monophyly of this third clade of Brienomyrus species plus Paramormyrops gabonensis is supported by a 6

8 unique 22 base pair inversion in the first intron of the S7 gene (Lavoué et al. submitted). It is this third, taxonomically unrecognized clade with which we are concerned in this study. Despite their demonstrated remote relationship to the type species of Brienomyrus and the inclusion of Paramormyrops gabonensis, we henceforth refer to them as the Gabon-clade Brienomyrus. We have begun morphological studies to search for non-molecular synapomorphies of this group and species descriptions and a revision of their taxonomy are underway. The Gabon-clade Brienomyrus Gabon-clade Brienomyrus species range in adult size from about 100 to about 250 mm standard length. They have moderately compressed bodies which are relatively elongate compared to most other mormyrids, rounded non-tubular snouts with small terminal to somewhat subterminal mouths bearing 5-9 (upper jaw) and 6-9 (lower jaw) bicuspid pincer-like teeth. They have fleshy, somewhat bulbous chins, small but functional eyes and are light gray or light brown to near black in color with little patterning of body pigmentation. They are nocturnally active, and although their diet has not been studied, they are probably 7

9 benthic foragers of insect larvae as are other similarly sized mormyrids with similar dentition (Winemiller and Adite 1997). Within regions surrounded by forest, they are found in a wide variety of flowing water habitats: from the shallowest stream headwaters to rocky substrates in the deepest portion of large rivers. Species composition differs among these habitat types. Juveniles of some forms which as adults are collected exclusively in larger rivers can be found in small streams along with other streamrestricted forms. In streams they are frequently the numerically most abundant type of fish. Where they are most diverse, five or six species are commonly caught together along with species of other mormyrid genera (pers. obs.). Little is known regarding their life history, ecology and mating systems. The mormyrid electric organ and electric organ discharge (EOD) Mormyrids generate electric organ discharges using four parallel columns of electrocytes in the caudal peduncle (Bennett 1970; Bennett 1971; Bennett and Grundfest 1961; Lissmann 1951; Schlichter 1906). Each cell generates a pulse-like discharge waveform identical to the overall EOD waveform recorded from an entire fish. The pulse 8

10 duration varies between a fraction of a millisecond to 10 milliseconds. EODs are repeated at irregular intervals of 10 to 100 times per second. While the waveform of the discharge is controlled by the structure and physiology of the electrocytes making up the electric organ (Bass 1986b; Bennett 1971), the rhythm of the discharges is regulated by activity in the pacemaker in the medulla. Several studies have documented interspecific differences in EOD waveforms for several species assemblages of mormyrids (Bass 1986a; Bass 1986b; Bass et al. 1986; Hopkins 1980; Hopkins 1981; Hopkins 1986a). The species differ from one another by EOD duration, number and amplitude of peaks, polarity, and wave shape. For many, male and female EODs differ during the breeding season (Bass and Hopkins 1983; Hopkins 1980; Hopkins and Bass 1981; Kramer 1997). Adult male EODs are typically longer in duration than those of females. These EOD differences have behavioral relevance. For one Brienomyrus from Gabon, males discriminate between the EODs of conspecific and heterospecific females and between those of conspecific males and females (Hopkins 1986b; Hopkins and Bass 1981). Species and sex recognition is dependent upon temporal cues, e.g. the duration of the EOD waveform. 9

11 The relationship of certain species-specific EOD waveform characteristics to electrocyte anatomy is well understood. Electrocytes in mormyrids are flattened diskshaped cells that are electrically active on both anterior and posterior faces. A complex network of electricallyactive tube-shaped "stalks" which are part of each electrocyte, receive neural innervation and transmit electrical excitation from the synapse to the disk faces. Innervation is on the thickest portion of the stalks near the center of the electrocyte disk; the stalk system then branches repeatedly to form finer and finer stalklets which eventually merge with the posterior face of the electrocyte. In some species the stalks penetrate through the disk surface of the electrocyte before fusing with the non-innervated face on the opposite side of the cell. In other species, stalks are non-penetrating, and the stalklets fuse on the innervated face of the cell. Species with non-penetrating stalk electrocytes produce EODs with only two phases, or peaks, while species with penetrating stalk electrocytes have EODs with an additional initial phase (Fig. 3b). The complex anatomy of these electrocytes and the functional relationship between the stalks and the electrocyte faces in generating EOD waveforms have been 10

12 reviewed by (Bass 1986a; Bennett 1971) and recently in (Alves-Gomes and Hopkins 1997) and (Sullivan et al. 2000). In a previous study (Sullivan et al. 2000) we hypothesized that while non penetrating stalks was the primitive character state for mormyrid electric fishes, penetrating stalks evolved once, early in the modern history of mormyrids, and that multiple independent reversals to the ancestral non-penetrating condition had subsequently occurred within the family. This hypothesis was based on an unweighted parsimony reconstruction of electrocyte evolution on a phylogenetic hypothesis for momyroids derived from sequence data, in conjunction with observations of electric organ ontogeny. In that study, penetrating stalk electrocytes were reconstructed as the primitive condition for the clade of mormyrids which includes the Gabon-clade Brienomyrus OTUs and the outgroup taxa used in this study. MATERIALS AND METHODS Specimens Used in this Study We collected and recorded the electric signals of more than 1400 Gabon-clade Brienomyrus specimens (Table 1) during field trips to Gabon and the Central African 11

13 Republic between 1998 and Field sites are listed in Table 1 and shown in Fig. 2. Methods of capture included funnel traps baited with earthworms lowered to river and stream bottoms, hook and line, cast netting, rotenone exposure followed by resuscitation, and dip netting following localization of fishes EODs with handheld electrode/amplifier units. To record a fish s EOD, we placed each specimen individually into a plastic container with water from the field site. An electrode was positioned at least 10 cm from either end of the fish and oriented parallel to its body axis. Its EOD was amplified (CWE BMA-831/XR Bioamplifier, DC settings,0-50 khz bandwidth), captured by a DaqBook (IoTech, Inc.) analog to digital converter (16 bits at 100 khz) and recorded onto the hard disk of a laptop computer. We euthanized the fishes by overdose of the anesthetic MS222. Tissue samples were subsequently removed from the dorsal musculature with a clean scalpel blade and preserved in 90% ethanol or a saturated NaCl solution containing DMSO and EDTA (Seutin et al. 1991). All specimens were fixed in phosphate-buffered 10% formalin, later transferred to 75% ethanol, and catalogued at the Cornell University Museum of Vertebrates (CU) or the American Museum of Natural History 12

14 (AMNH). We attached permanent tags to all specimens bearing a unique number by which specimen, tissue sample and EOD recording are linked in our records. All animal procedures followed NIH guidelines under a protocol approved by the Cornell University Institutional Animal Care and Use Committee. Determination & naming of OTUs and choice of specimens for sequencing We assigned each specimen to an OTU upon visual examination of its external morphology and EOD waveform in the field. We created a new OTU whenever a specimen differed substantially in morphological and/or EOD characteristics from all those previously collected. After the field work, these OTU assignments were reevaluated (and in some cases amended) in the laboratory where more careful study of specimens and EOD records was possible. Our OTU determinations constitute a first pass partitioning of external morphological and EOD variation into provisional species units. We identified these OTUs by phenetic criteria (i.e., without consideration of the evolutionary polarity of the characters examined) and we have only indirect evidence of reproductive isolation between them (viz. the discontinuity of character states). 13

15 Therefore, it is possible that some of these OTUs may not correspond to single phylogenetic or biological species. Final determination of species status and taxonomic recognition for the new forms--which will to some degree hinge on the operational species concept employed (Harrison 1998; Turner 2000)--should await more detailed examination of morphological and molecular data beyond the scope of this study. We assigned a three-letter code to OTUs that do not correspond to described species. This code in some cases refers to the collection locality (e.g. NZO was collected near the village of Nzoundou, Gabon) and in other cases is an abbreviation of a manuscript name which is currently in preparation (e.g. CAB and LIS). For all others we used a descriptive three-character code containing either an S for sharp snout, a B for blunt snout, or an I for intermediate snout (Fig. 3) and either a P for penetrating stalk electrocyte or an N for non-penetrating stalk electrocyte, as indicated by the presence or absence of an initial head negativity to the EOD waveform and confirmed histologically in some specimens (see Bennett 1971). For one OTU with mixed penetrating and nonpenetrating stalked electrocytes we used a code "X." These 14

16 two letters are followed by a number indicating the order of discovery. From a total of 1450 voucher specimens (Table 1), we selected 86 individuals for sequencing representing all 38 OTUs. When possible, we sequenced more than one representative of each OTU from different populations. In general, we sequenced additional specimens from an OTU if the first two sequences failed to form a monophyletic group in a preliminary parsimony analysis. PCR and Sequencing DNA was extracted from tissue samples with the QIAamp tissue kit (Quiagen, Inc, Valencia, CA, USA). Sequences of the primers used to amplify the mitochondrial cytochrome b gene were taken from Palumbi (1996) and are: 5 -TGA TAT GAA AAA CCA TCG TTG-3 (L14724) and 5 -CTT CGA TCT TCG rtt TAC AAG-3 (H15930). PCR volumes of 50 µl consisted of approximately ng genomic DNA, 1X Perkin Elmer GeneAmp Gold PCR buffer, 1.25 units of Perkin Elmer AmpliTaq Gold, concentrations of 0.2µM of each primer, 200 µm of each dntp, and 3 mm of MgCl. Amplifications were carried out on a Hybaid TouchDown thermocycler (Hybaid Limited, Teddington, Middlesex, England) using an initial 95 C 15

17 denaturation step for 10 min followed by 35 repetitions of a three-step cycle consisting of 94 C for 1 min, annealing for 1 min at 42, and extension at 72 C for 1.5 min, followed by a final extension at 72 C for 7 min. We purified our PCR products with the Promega Wizard PCR Preps DNA purification kit (Promega, Madison, WI). We sequenced the double-stranded PCR products directly in both directions with the primers used for amplification by automated dye-terminator cycle chemistry on an Applied Biosystems 377 automated sequencer. We edited the sequences from the gel chromatograms with the Sequencher software package (Gene Codes Corp., Ann Arbor, MI). Choice of outgroups Outgroup cytochrome b sequences were available from our previous study (Sullivan et al. 2000) in which we had sequenced the same cytochrome b fragment from 41 species belonging to all 17 recognized genera of mormyrid fishes. This study and Lavoué et al. (submitted) together indicate that Marcusenius conicephalus is the sister group to the four included species of Gabon-clade Brienomyrus: Paramormyrops gabonensis, Brienomyrus hopkinsi, Brienomyrus longicaudatus and sp. 2 (= VAD in this study) and that sister to this group is a clade containing Boulengeromyrus 16

18 knoepffleri, Ivindomyrus opdenboschi and Pollimyrus marchei (Fig. 1). For this reason, we chose Boulengeromyrus knoepffleri and Ivindomyrus opdenboschi as outgroups and included Marcusenius conicephalus as an ingroup taxon to test the monophyly of the additional putative Gabon-clade Brienomyrus OTUs in this study. Phylogenetic analysis We reconstructed cytochrome b phylogeny using both Maximum Parsimony (MP) and Maximum Likelihood (ML) analyses in PAUP* version 4.0b3 (Sinauer, Inc., Sunderland, MA, USA) on an Apple PowerMac G4 computer. For the MP analysis we performed a heuristic search on the cytochrome b dataset in which all characters and classes of substitution were equally weighted. Starting trees for tree bisectionreconnection (TBR) branch swapping were obtained by 100 iterations of the random stepwise addition sequence. PAUP*'s default settings for a heuristic MP search were used in all other cases. Relative support for the internal nodes of the MP trees was estimated by bootstrap analysis (Felsenstein 1985) consisting of 1000 pseudoreplicates in PAUP* (starting trees obtained by single iteration of random stepwise addition; MAXTREES set to 2000; otherwise parameter settings were identical to MP heuristic search). 17

19 For the ML analysis, we performed an initial parameter-rich heuristic tree search using the generaltime-reversible model with rate heterogeneity (Yang 1994) in which the six-way substitution rate matrix was estimated from the dataset by ML as were site specific rates for each of the three codon positions. Assumed nucleotide frequencies were those determined empirically from the dataset, a molecular clock was not enforced, and starting branch lengths were obtained using the Rogers-Swofford approximation method. TBR branch swapping began from a starting tree obtained by neighbor-joining. The option to collapse branches of essentially equal length was used. Because of the impractical amount of computation time required to complete branch swapping in a parameter-rich analysis of a dataset this large, we halted the analysis after it had found a best tree which had remained unchanged through 2000 subsequent branch swappings. The substitution rate matrix parameters and codon-specific substitution rates estimated for this tree were entered as fixed parameters in a subsequent heuristic ML search in which branch swapping was allowed to continue to completion. Mutational saturation in the dataset was estimated by looking for non-linearity in a plot of pairwise observed raw distance, (PAUP* s adjusted character distance ) 18

20 against tree-corrected distance (PAUP* s patristic distance ) for each codon position and each class of nucleotide substitution on one of the most parsimonious trees resulting from the MP analysis as described by Hassanin (1998) RESULTS OTU diagnosis By visual comparison of morphologies and EOD characteristics we sorted these specimens into 38 OTUs (Fig. 4). Of these, four could be assigned unequivocally to the described species Brienomyrus curvifrons (Taverne et al. 1977b), B. longicaudatus (Taverne et al. 1977b), B. hopkinsi (Taverne and Thys Van Den Audenaerde 1985), and Paramormyrops gabonensis (Taverne et al. 1977a) based on comparison to type material and the original descriptions. We could not confidently associate three other previously described Gabon-clade Brienomyrus species from this region, B. sphekodes (Sauvage 1880), B. batesii (Boulenger 1906), B. kingsleyae (Günther 1896) to any of our remaining 36 OTUs. However, we cannot rule out the possibility that these species are represented among them. Table 1 lists 19

21 these OTUs and data for the voucher specimens and their sequences. Dataset characteristics Sixty-five unique 1143 base pair cytochrome b haplotypes were recovered from the 85 Gabon-clade Brienomyrus specimens sequenced. The ingroup dataset for the parsimony analysis included the sequences of 70 putative Gabon-clade Brienomyrus specimens (a haplotype was included more than once if it was obtained from specimens assigned to different OTUs) and sequences of two Marcusenius conicephalus. We observed no indels, nonesense mutations, or ambiguous sites in any of the ABI chromatograms which might indicate ampification of nonmitochondrial copies of the gene. Uncorrected P-distance between the outgroup sequences of Boulengeromyrus knoepffleri and Ivindomyrus opdenboschi and the ingroup taxa is 9-10%, between M. conicephalus and the other ingroup taxa is 8-9% and between other ingroup taxa, 0 to 8%. We observed no evidence of mutational saturation for transitions or transversion for any codon position in our plots of pairwise "adjusted" character distance against patristic distance as calculated in PAUP*. 20

22 Out of 287 variable sites in the sequences, 232 were parsimony-informative characters: 188 (81%) in the third codon position, 33 (14%) in the first codon position and 11 (5%) in the second codon position. Average base frequencies across all sites are C=32%, A=29%, T=25%, G=14%, but the informative sites of the third codon position show increased high C, low G bias (C=43.5%, A=29.5%, T=22%, G=5%) as reported previously for mormyroid fishes (Lavoué et al. 2000; Sullivan et al. 2000), for other fishes (Lydeard and Roe 1997; Meyer 1993), and for other vertebrate groups (Irwin et al. 1991; Kornegay et al. 1993). A Chi-square test failed to reveal any significant heterogeneity in base frequencies among the sequences. Phylogenetic analysis results An unweighted maximum parsimony analysis in PAUP* yielded 680 MP trees, each of 636 steps (CI=0.49. RI=0.84, RC=0.41, uninformative sites excluded). A strict consensus of these trees, shown as a phylogram (using ACCTRAN character optimization) is shown in Fig.5. A consensus tree of 1000 parsimony bootstrap pseudoreplicates in which nodes receiving less than 50% bootstrap level support are collapsed is shown in Fig 6. Well-supported nodes are labelled A-J in Figs 5 & 6. 21

23 The ML analysis yielded a tree with an Ln-likelihood score of Estimated relative substitution rates were for position one, 0.08 for position two, and 2.58 for position three. Because branches of essentially equal length were collapsed during the ML search, some nodes are not resolved. The topology of this ML tree (not shown) is extremely similar to that of the MP consensus tree: the only topological incongruence between them concerns the pattern of interrelationships of clades H, I and J (Figs 5 & 6), none of which have significant character support. Clades H and I are weakly linked under parsimony (Fig. 5), while clades I and J are weakly linked under maximum likelihood. Both analyses indicate that Marcusenius conicephalus is the sister group to all other ingroup specimens sequenced (node A, Figs 5 & 6), supporting the monophyly of the additional, putative Gabon-clade Brienomyrus with those included in previous studies. Mean raw cytochrome b P- distance between M. conicephalus and the Gabon-clade Brienomyrus taxa is 8.3%. Within the Gabon-clade Brienomyrus, nodes B, C, D, E and F (Figs 5 & 6) are well supported by long branch lengths and high parsimony bootstrap values. Nodes B and E define the most basal division within the Gabon-clade 22

24 Brienomyrus. The OTUs in these two clades are separated from each other by seven percent cytochrome b distance. Clade B includes the OTUs VAD and SZA both of which belong to separate, well-defined clades (D & C, respectively, separated from each other by an average 6.5 percent cytochrome b distance). The two VAD specimens sequenced, one from the Woleu Basin and the other from the Ivindo, appear as a monophyletic group which is sister to OTU BP from the Okano basin. Sister to these taxa is clade C in which two distinctly different OTUs, IP1 and LIS, both known from only single populations, are nested within the SZA OTU, individuals of which are known from a number of localities in the Ivindo, Ntem, Woleu and Okano Rivers in northern Gabon. All members of clade B have penetrating stalk type electric organs (Fig. 7,d). The most basal division in the remaining taxa separates the OTU ROB, an OTU only known from the Louétsi River in southern Gabon, from clade F. Clade F, which is supported by an exceedingly long branch relative to others on the tree, is in turn divided into a large clade (G) whose sister group consists of two individuals of the OTU BP1: one from the Okano River, the other from the Woleu River Basin. 23

25 Outside of clade G, all ingroup OTUs have penetrating stalk type electric organs. Within clade G, OTUs have a mix of penetrating and non-penetrating stalk-type electric organ (Fig. 7d). Because penetrating stalks are found in the sister group to clade G (BP1 Okano + BP1 Woleu) and in the following three successive outgroups( ROB, clade B, and the two M. conicephalus) the penetrating stalk character state is reconstructed by unweighted local parsimony optimization (Maddison et al. 1984) as primitive for G. Primitive retention and derived loss of penetrating stalks within the Gabon-clade Brienomyrus is consistent with the more global reconstruction of this character in (Sullivan et al. 2000) which suggests a single origin of the penetrating stalked electrocyte from the nonpenetrating stalk electrocyte at the base of the subfamily Mormyrinae, followed by reversals to the non-penetrating condition within several genera. The reconstruction in (Sullivan et al. 2000) further suggests that penetrating stalk electrocytes were present in the most recent common ancestor of all the ingroup and outgroup taxa considered in this study and that a reversal to the non-penetrating character state took place in the common ancestor of Boulengeromyrus knoepffleri and Ivindomyrus opdenboschi. 24

26 Above node G the remainder of the Gabon-clade Brienomyrus (32 OTUs) segregate into three relatively wellsupported clades--h, I, and J--that are of uncertain relationship to each other and that are poorly resolved internally. Within these clades specimens of different OTUs are often very closely allied genetically (i.e. often separated by cytochrome b P-distances of less than 0.5%), while in some cases specimens assigned to distinct OTUs share identical cytochrome b haplotypes (e.g. BP6 3547/BN2 3415; BP1 3016, 2530, 2704/SN3 2619; SZA 3788/IP1 1692). Furthermore, only two OTUs represented by sequences from multiple populations in this large clade appear monophyletic on the tree (OFF in Figs 5 & 6, and Paramormyrops gabonensis in Fig. 5 only). Other OTUs (SP2, SP4, MAG, TEN, SN2, SN3, CAB, NZO, and BP1) represented by multiple sequences, either from the same or from different populations, appear non-monophyletic on the tree. The remaining OTUs above node G are represented by single individuals, therefore their monophyly cannot be tested. While lack of OTU monophyly may in some cases result merely from lack of resolution of the data, i.e. few characters supporting any particular topology, conflict with OTU monophyly is found in other parts of the tree where support is strong for haplotype relationships (e.g. the non- 25

27 monophyly of OTU TEN within clade H or of BP1 within clade F). As with OTU monophyly, expectations of higher level relationships between OTUs based on certain morphological, electric organ and EOD characteristics are not reflected in the topology obtained above node G. While these characters were too few to warrant coding them into a separate data matrix for an MP phylogenetic analysis, we consider them below and in Fig. 7. Accepting the monophyly of clade G, the tree topology below node G and the outgroup relationships shown in Fig. 1, we identified five unambiguous characters states each of which appears uniquely within several OTUs of clade G (those shown in black in Fig. 7a-d). These are: (1) the very sharp snout combined with jutting lower jaw morphology of 9 OTUs (Fig 7a), (2) 16 circumpeduncular scales found in four OTUs versus 14 or fewer in all others (Fig. 7d), (3) EOD waveforms reversed in their polarity found in four OTUs (Fig. 7b), (4) monophasic EOD waveforms (Fig. 7c), and (5) non-penetrating stalk electrocytes (Fig. 7d and mentioned above). The absence of these character states is clearly primitive for the Gabon-clade Brienomyrus as a whole. However, in no case do OTUs which share these character 26

28 states correspond to a monophyletic group of cytochrome b haplotypes. DISCUSSION The departure of the tree topology from expectations of OTU monophyly and of close relationships between similar OTUs may result from a failure of OTUs as we defined them to represent natural (monophyletic) groups, or from incongruence between the mitochondrial haplotype tree and overall organismal phylogeny. OTU non-monophyly would result from use of primitive or convergent similarities instead of shared uniquely derived similarities (synapomophies) in their determination. Gene tree/species tree incongruence has been suggested in studies of other fish radiations and attributed to one of two phenomena. The first of these, incomplete gene lineage sorting, has been implicated in studies of mbuna cichlids of Lake Malawi (Moran and Kornfield 1993; Parker and Kornfield 1997) and in Indo-Pacific butterflyfishes (McMillan and Palumbi 1995). This results when the rate of speciation exceeds the rate at which genetic polymorphisms can coalesce within the parental species such that these polymorphisms are passed on to daughter species. The phylogeny of alleles sampled in such daughter species will then be decoupled 27

29 from larger organismal phylogeny (Harrison 1991; Pamilo and Nei 1988). In this case, the most recent common haplotype ancestor for two sequenced individuals of different species will be older than the most recent common ancestor of the species themselves. The second explanation for gene tree/species tree incongruence is introgression by hybridization (Smith 1992). This is possible among interfertile species in which reproductive isolation is mediated by extrinsic barriers or by behaviors which can break down under some conditions. In a case of introgression, the most recent common haplotype ancestor for two sequenced individuals of different species will be younger than the most recent common ancestor of the species themselves. Arnold (1997) and Dowling (1997) have recently reviewed cases of introgression by hybridization in fishes and other animals. To some extent, we can evaluate these alternative hypotheses. The OTU SZA is a probable example of the use of primitive characteristics in OTU definition since it appears paraphyletic with respect to OTUs IP1 and LIS (Figs. 5 & 6). The morphological and EOD characteristics by which SZA was recognized likely evolved in the common ancestor of clade C, but are primitive with respect to alternative states found in IP1 and LIS. SZA is known from 28

30 a number of localities whereas IP1 and LIS are known from only single populations. IP1 and LIS may thus represent examples of local speciation within a widespread homogeneous ancestral species. However, because as in this case, OTU paraphyly should often be interpretable as such (since some OTU coherence on the tree will be maintained), it is unlikely that use of primitive character states in OTU definition explains all the cases of OTU non-monophyly and incongruence. Geographic signal within the tree Other patterns in the data seem more consistent with unnatural OTU definition as a result of character convergence among geographically separated lineages, or with genetic introgression. This is reflected in the many instances of tree-implied relationships above node G which reflect geographic proximity of the individuals rather than OTU membership. For instance, within clade H, Ivindo and Ntem forms of TEN belong to separate clades (TEN 2011/2091 and TEN 3850); in both cases the TEN haplotypes appear more closely related to haplotypes of different OTUs from the same localities. Implied relationships on the tree again reflect geographic proximity of populations instead of OTU similarity in the case of SP & 2672 and SP & 29

31 2673 in clade H, all from a single tributary of the Louétsi River, and four specimens each of SP2 and SP4 in clade J from Mouvanga Creek, an affluent of the Ngounié River, only about 40 km distant. (Another SP4 haplotype in clade J specimen SP is discussed below). Neither within clade H nor within clade J is genetic differentiation found between these two OTUs which are distinguished only by substantially different EODs (see Fig. 3c). These two forms may in fact be a single species with an EOD polymorphism. Nevertheless, in both clades H and J these SP2 and SP4 individuals appear more closely related to specimens of other, dissimilar OTUs with which they are sympatric than to allopatric individuals of their own OTU. Likewise, three BP1s from Mouvanga Creek in clade J appear more closely related to some sympatric SN3s than to other BP1s from nearby localities in the Louétsi River (also in J). Geographic signal is also seen at higher phylogenetic levels in the tree: for instance all the specimens appearing in the large clade J are from southern Gabon, although other specimens of these same OTUs from the same localities are appear again in clades H and I. To explain this geographical signal in the data one of two phenomena (or a combination of the two) needs to be invoked: either 30

32 rampant convergent evolution in the characters we used to recognize OTUs in different areas, or extensive local hybridization and mitochondrial introgression among forms. We accept that convergence in morphology and EOD characteristics is possible and perhaps likely in some cases. The higher-level phylogenetic hypothesis for the family Mormyridae (Sullivan et al. 2000) indicates that tubular snouts have evolved independently in three separate lineages: Mormyrops, Campylomormyrus and Mormyrus. It is not hard to accept, therefore, that on a smaller scale of differentiation, "blunt snouts" and/or "sharp snouts," as well as other morphological characteristics, may have evolved more than once in the Gabon-clade Brienomyrus. Evolutionary constraints on electrocyte structure may make EOD convergence likely in different areas. One possible case of EOD convergence in Gabon-clade Brienomyrus can be found in the allopatrically distributed OTUs TEN and BN2 (Fig. 7c). These two OTUs are morphologically quite dissimilar despite their very similar, unique monophasic EOD waveforms. However, for convergent evolution to explain the cases outlined above which illustrate the geographical signal within the clade G topology, both morphology and EOD characteristics would have had to co-evolve identically in 31

33 different localities. There is no reason to believe that selection should favor the association of a particular EOD waveform with a particular head/body morphology. In these cases, introgression seems the more acceptable explanation. Further evidence for introgression is provided by a number of cases in which individuals of different OTUs share identical or nearly identical cytochrome b haplotypes (In clade I: BP6 3547/BN2 3415; SN7 3666/SN2 3415; in clade J: BP1 3016, 2530, 2704/SN3 2619). In all cases these are sympatric and quite distinct forms. It seems impossible that such significant morphological and EOD evolution could occur with zero concomitant genetic divergence and much more likely that mitochondrial introgression is obscuring their overall degree of genetic relatedness. If introgression is the most likely explanation of this geographic pattern in which dissimilar but sympatric OTUs appear more closely related to each other than to seemingly identical individuals from other populations, this introgression must have crossed multiple species boundaries in more than one instance. Hybridization would have to be frequent enough to achieve introgression of some genes (in this case the mitochondrial genome) and yet not be so extensive as to break down OTU-specific characteristics. We consider introgression a more likely 32

34 explanation for the data than the alternative of multiple independent origins of OTUs. Retention of ancestral polymorphisms Other patterns of incongruence between the cytochrome b tree and OTU monophyly appear to be more consistent with incomplete genetic lineage sorting than with introgression or convergence. For instance, in the cases of OTUs SN3 and SP4, individuals from the same populations are found to have divergent cytochrome b haplotypes which place them into different, well supported subclades of clade G (Figs 5 & 6). This observation suggests that the retention of ancestral polymorphisms within populations may be at least partly responsible for the lack of intuitive groupings above node G and that it may be futile to interpret the pattern of haplotype relationships above this level in terms of OTU relationships. Phylogenetic studies on recently formed species flocks in fishes (Kornfield and Parker 1997; Moran and Kornfield 1993; Parker and Kornfield 1997; Strecker et al. 1996) and in Darwin's finches (Sato et al. 1999) have attributed similar findings to incomplete mitochondrial lineage sorting. Supplemental sampling of individuals within and among populations of OTUs would help to clarify the degree of 33

35 such polymorphism but may not improve phylogenetic resolution within clade G. All mitochondrial genes being linked, addition of another mitochondrial marker to this dataset would likely only strengthen those relationships already well supported by cytochrome b. Only an independent dataset could help resolve the relative roles of OTU definition and patterns of introgression and incomplete lineage sorting in this clade. Whether such a dataset could be obtained from the nuclear genome in which evolutionary rates and lineage sorting processes are generally slower than for mitochondrial genes (Avise 1994; Palumbi and Cipriano 1998) is questionable. Estimating the age of the Gabon-clade Brienomyrus radiation Similar terminal branch lengths in the phylograms resulting from the MP analysis (Fig 5) and the ML analysis (not shown) suggest that base substitutions are accumulating at relatively equal rates in different lineages of Gabon-clade Brienomyrus. Alves-Gomes (1999) calculated a substitutional clock rate of 0.23%/MY for mitochondrial 12S and 16S rrna genes in mormyroid fishes. Data from (Sullivan et al. 2000), indicate that cytochrome b distances are on average 3.0 times greater than 12S and 16S distances in corresponding pairwise comparisons of 34

36 mormyrid taxa, consistent with the relative evolutionary rates of these genes in other organisms (Avise 1994). Accepting Alves-Gomes s suggested 12S/16S clock rate thus implies a clock rate for Gabon-clade Brienomyrus cytochrome b in the neighborhood of 0.7%/MY. To calculate this clock, Alves-Gomes (1998) used mean uncorrected P-distance between the mormyroids and their sister group, the notopteroids, and used the 65 MY old fossil Ostariostoma, putatively the sister taxon to these two groups (Li and Wilson 1996), as a calibration point. Recently, however, a 100 MY old fossil notopterid, Paleonotopterus greenwoodi has been described and phylogenetically placed as the sister group to the extant notopteroids (Forey 1997; Taverne and Maisey 1999), pushing back the minimum age of divergence of the mormyroids from the notopteroids to 100 MYA. By itself, this finding would mean that this clock rate is overestimated by at least a factor of 1.5. However, pairwise mormyroid and notopteroid 12S and 16S P-distances plotted against the corresponding pairwise P-distances from the slowly-evolving RAG2 nuclear gene (in Fig. 1 of Sullivan et al. 2000) demonstrate that due to mutational saturation, P-distance underestimates actual genetic divergence in this case by at least a factor of three. Applying these corrections doubles the implied 35

37 (maximum) clock rates to 0.46%/MY for 12S/16S and about 1.5%/MY for cytochrome b. This proposed (maximum) cytochrome b clock rate is roughly in the middle of the range for mitochondrial coding gene clock rates (0.9% - 2.5%) estimated for or applied to fishes in other studies (McCune 1997). While the confidence interval on this estimate for a cytochrome b clock rate in mormyroids must be thought to be exceedingly wide, applying this rate to the data implies an age for the Gabon-clade Brienomyrus of four to five million years (based on an average pairwise uncorrected distance of 7.0% between Marcusenius conicephalus versus taxa in clade E in Fig. 6). Likewise, this rate implies an age of about two million years for clade F (based on average pairwise uncorrected distance of 3.0% between ROB and members of clade G) and thus that the bulk of the Gabon-clade Brienomyrus diversity originated within the Quaternary, much of it probably within the past half million years. Endemism and biogeographic patterns The precise distributional boundaries of the Gabonclade Brienomyrus in African freshwaters remain poorly defined, but available data suggest that they may be restricted to the river basins of Lower Guinea and the 36

38 Congo (Teugels and Hopkins 1998). Within the region they are known to occur, the center of diversity of the Gabonclade Brienomyrus appears to be within the Ogooué and Ntem Basins of Gabon and Cameroon. While most of our collection effort to date has been concentrated in this region, this conclusion is supported by study of the existing taxonomic literature and all available museum collections, as well as by our collections in a single Congo River tributary, the Sangha River of the Central African Republic. There, only a single species from this clade was found (SAN in our analysis) in habitats similar to those in Gabon where five or six species are typically found together. In addition to being their center of diversity, phylogenetic and distributional data suggest that the Ogooué and Ntem Rivers are as well the probable center of origin of this clade, since the two sequential outgroups to the Gabon-Brienomyrus (Marcusenius conicephalus and the clade containing Boulengeromyrus knoepffleri, Ivindomyrus opdenboschi and Pollimyrus marchei) are themselves Ogooué/Ntem endemics. Faunal exchange between these two basins is probably maintained during periods of high water in swamp-forest regions where both rivers have headwaters. One of these, the Ayina River, is thought to have been captured by the Ogooué system from the Ntem at some time in 37

39 the past (Olivry 1986; Thys van den Audenaerde 1966). Assuming an origin in the Ogooué/Ntem region, members of this clade would have dispersed at some point into the Congo Basin and into other Lower Guinea drainages. The importance of the Ogooué/Ntem region from the standpoint of diversity and endemism of Gabon-clade Brienomyrus species is paralleled in many other forestassociated groups of plants and animals (the "Cameroon/Gabon Core Area" of Hamilton 1982) as well as in the forest-dwelling cyprinodontiform fish genus Aphyosemion for which Wildekamp (1993) lists 39 species and subspecies from the Ogooué, Ntem, and associated coastal drainages alone. These contemporary patterns of organismal distribution in conjunction with pollen-based reconstructions of floral change indicate the persistence of several forest refugia in portions of the Ogooué and Ntem Basins throughout the most arid periods of the Pleistocene (Maley 1987; Maley 1991; Maley 1996). Thus it seems possible that the forest-dependent Gabon-clade Brienomyrus could have diversified in situ in this region during the Quaternary time frame suggested by the molecular clock considerations. 38

40 The Gabon-clade Brienomyrus as a riverine species flock Within fishes, the term species flock has largely been applied to intralacustrine radiations: most famously the haplochromine cichlids of the Rift Valley lakes of East Africa (Brooks 1950; Echelle and Kornfield 1984; Goldschmidt 1996; Greenwood 1984; Kornfield and Smith 2000; Meyer et al. 1990), but also to tilapiine cichlids of the crater lakes of Cameroon (Schliewen et al. 1994), sculpins of Lake Baikal (Berg 1965; Taliev 1955), cyprinids in both Lake Lanao, Philippines (Kornfield and Carpenter 1984) and in Lake Tana, Ethiopia (Nagelkerke et al. 1994), killifishes in Lake Titicaca (Parenti 1984; Parker and Kornfield 1995), pupfishes in Lake Chichancanab, Mexico (Humphries 1984), and to a Mesozoic radiation of semionotid fishes in North American lakes (McCune 1996; McCune et al. 1984), among others. Arguing that the term need not be restricted to lacustrine fishes, Johns & Avise (1998) have applied it to marine radiations of northeastern Pacific Sebastes rockfishes and to Antarctic nototheniod icefishes (Ritchie et al. 1996) for which there is evidence of rapid speciation in the past. While definitions of a fish species flock can differ (see Greenwood 1984; Ribbink 1984), we find it necessary and sufficient that it be a monophyletic assemblage of species at least largely 39

41 restricted to the geographical area of their origin (i.e. autochthonous), exhibiting a high level of sympatry and rapid, or explosive, speciation relative to their nearest relatives in neighboring regions. We have shown how all of these criteria likely apply to the Gabon-clade Brienomyrus. River basins are similar to lakes in that they are habitats circumscribed by a boundary inside of which gene flow is possible, but across which dispersal and invasion are rare events. Thus, at least in this respect, there is no a priori reason to believe that the criterion of autochthony for a fish species flock could not as easily be met in a river basin as in a lake. However, unlike many of the lakes hosting fish species flocks which were biologically depauperate environments after a discrete geological origin or last re-filling subsequent to a drydown, the rivers of West Central Africa have probably always harbored fish communities, although their courses and interconnections may have changed dynamically through time. Perhaps because available ecological niches in these riverine environments have always been occupied, the Gabonclade Brienomyrus flock does not appear to represent an adaptive radiation in which species have diversified greatly in ecology and morphology. 40

42 Speciation, sympatry and electric organ & EOD diversification within the Gabon-clade Brienomyrus The diversity of EOD waveforms in natural communities of mormyrid electric fishes has long been recognized (Bass 1986b; Hopkins 1979; Hopkins 1981), but the genealogical relationships of the species concerned was unknown. This study provides evidence for the existence of a great diversity of EOD waveforms among a monophyletic group of numerous, very closely related species of mormyrid fishes. To what can all this EOD and species diversity be attributed? Given the exceedingly close genetic relationships of many of these forms as well as their morphological and apparent ecological similarity, it seems improbable that EOD diversification could be largely the result of either genetic drift or adaptation to different physical environments. Far more likely, EOD diversification is the result of some form of direct social selection (West-Eberhard 1983) and is associated in some way with the process of speciation. Patterns which emerge from the cytochrome b tree may be instructive. Within the Gabon-clade Brienomyrus, only in clade G has there been significant EOD/electrocyte morphology evolution in the in the form of one or more apparent reversals to non-penetrating stalk morphology and 41

43 hence to EODs which lack an initial head-negative phase (see Fig 7d). (OTU BX1 may in fact represent such a reversal from penetrating to non-penetrating stalks in progress. EODs in some populations show an extremely weak initial head-negativity, presumably too weak to be of significance in communication, associated with poorly developed penetrating stalks. These features are absent, or nearly so, in other populations of the species.) If rapid speciation and selection for novel EOD types/electrocyte morphologies are causally linked processes (regardless of the directionality), the association should be clearest in clade G given the tremendous flush of speciation it has undergone and is probably still undergoing. The 32 OTUs in clade G, among which one finds significant sympatry and overlap of distributions, are all within only three percent cytochrome b distance of each other. Sullivan et al. (2000) noted that species with the putatively derived non-penetrating stalk electrocytes usually have longer duration EODs than do their counterparts with penetrating stalk electrocytes. We hypothesized that selection may favor reversal to nonpenetrating stalks within those primitively penetrating stalk lineages in which contact between many recently 42

44 speciated forms necessitates novel EOD characteristics, such as increased duration, which can enhance species recognition (Hopkins and Bass 1981). This proposed scenario assumes a reinforcement model in which speciation is initiated in allopatry or parapatry and reduced fitness of hybrids upon secondary contact selects for the elaboration of pre-zygotic reproductive barriers such as signals used for mate recognition. Another possibility, which does not exclude a role for reinforcement, is that disruptive sexual selection on EOD variation within populations is itself producing the high rate of speciation in these fishes. Sympatric speciation driven by sexual selection has been proposed for a number of fish species flocks e.g. (Berrebi and Valiushok 1998; Schliewen et al. 1994; Seehausen 2000; Seehausen et al. 1999; Turner 1994) and recently has received increased theoretical support (Kondrashov and Kondrashov 1999; Turner and Burrows 1995) Sexual dimorphism in EODs appears to be ubiquitous among the Gabon-clade Brienomyrus: EOD waveforms in reproductive males are longer in duration than those of juveniles and females and often markedly different in waveform characteristics (see superimposed male and female EODs of CAB, VAD, and IN1 in Fig.4) indicating the probable 43

45 importance of female choice in EOD evolution (Bass et al. 1986). A parallel may exist in South American gymnotiform electric fishes of the genus Brachyhypopomus in which reproductive males have longer duration EODs (Franchina and Stoddard 1998; Hopkins 1991; Hopkins et al. 1990). Hopkins et al. (1990) and Hopkins (1999) argued that the sexual dimorphism in Brachyhypopomus may have evolved as the result of female choice, since long duration EODs, being more costly to produce than shorter EODs of the same amplitude, may serve as reliable and honest indicators of male quality. The direct current (DC) component found in the EODs of some male gymnotiforms and mormyrids may also be evidence of female choice. Catfishes, which are major predators of electric fishes on both continents (Merron 1993; Reid 1983; Zuanon 1990), are insensitive to the high frequencies of most EODs but sensitive to direct current (DC). Hopkins (1990) pointed out that the extra DC component often associated with these longer EODs may impose a handicap on males (Zahavi 1975) as do male dimorphic characters in other organisms. While male discrimination of conspecific female EODs from those of conspecific males and of heterospecifics has 44

46 been observed in the field in a Gabon-clade Brienomyrus (=VAD in this study, Hopkins 1986b; Hopkins and Bass 1981), EOD-mediated female choice and assortative mating remain to be demonstrated. Sympatric speciation in Gabon-clade Brienomyrus remains to be proven. However, the evident rapidity of speciation within this group of fishes, particularly within clade G, is at least consistent with the theoretical prediction of much a shorter time for sympatric speciation versus allopatric speciation (Kondrashov 1998; Kondrashov and Mina 1986) and also with empirical comparisons of time to speciation for fishes of allopatric origin versus those of presumed sympatric origin (McCune 1997; McCune and Lovejoy 1998). Surveying a number of studies using cytochrome b in fishes, McCune and Lovejoy (1998) estimated that divergence between sister species of presumed allopatric origin was between 2.0 and 5.6% (post-speciation divergence subtracted) relative to 0 to 1.25% for presumed sympatrically derived sister species. In clade G of the Gabon-clade Brienomyrus, there are 32 OTUs, all potentially good species, within no greater than three percent cytochrome b divergence. While for reasons stated above, sister group relationships cannot be confidently postulated within clade G directly from the cytochrome b tree (which 45

47 if accepted uncritically as a species phylogeny would indicate frequent sympatry of sister forms), genetic distance between many taxa within clade G would necessarily fall into this range for presumed sympatrically speciated forms. Intriguing as these hypotheses are, much more needs to be learned about the mating system, behavior, ecology, life history, and population structure of Gabon-clade Brienomyrus species before we can make more specific conclusions regarding the nature of speciation and EOD diversification within this group. CONCLUSIONS We have identified a species flock of mormyrid fishes in West Central Africa. This is the first freshwater fish species flock phenomenon described within a group of weakly electric fishes and also the first described wholly within a riverine, as opposed to a lacustrine, environment. Electric communication in these fishes is likely to be causally related to their rapid diversification: social selection on species-specific EOD waveforms that serve as prezygotic isolating mechanisms among closely related 46

48 sympatric species may help complete the speciation process, or may initiate it. While analysis of cytochrome b sequences provides evidence for the monophyly of this flock and suggests its recent origin, our study underscores the limitations of using mitochondrial data, or any single molecular marker, to resolve phylogeny within a rapidly speciating clade of organisms. The implications are serious, since population genetic studies of speciation in such groups require a phylogenetic framework to guide the choice of populations to be sampled. If fish species flock studies are to greatly contribute to our understanding of the speciation process, new approaches for estimating their phylogenies-- in particular those that overcome the problems associated with single molecular markers--need to be pursued. ACKNOWLEDGEMENTS For help in Gabon we wish to thank Mr. J.D. Mbega and Mr. J.H. Hervé Mve of IRAF; Drs. Paul Posso and B. Bouroubou from IRET; Drs. O. Langrand and A. Kamdem Toham of WWF-CARPO; the Sisters of the Catholic Mission in Lambaréné; the Christian and Missionary Alliance of Bongolo; CIRMF, Franceville; Jim Beck and Christian Ella 47

49 from the U.S. Peace Corps, and Conrad Aveling of ECOFAC. For help in the Central African Republic we thank Jean Bernard Kindi-Moungo and the WWF office in Bangui. From Cornell, M. Arnegard helped collect fishes, EODs and tissues in Gabon as did J. Friel who additionally oversaw the curation of specimens at the Cornell Museum of Vertebrates and G. Harned prepared histological slides of electric organs. We thank Dr. M.L.J. Stiassny and Joel Cracraft of the American Museum of Natural History for making possible JPS s collection trip to the Central African Republic. M. Arnegard, J. Lundberg, A. McCune, G. Teugeuls, B. Turner, and K. Zamudio provided helpful comments on an early draft of the manuscript. Funding for this work came from the following grants to CDH: NSF International Program Grant # INT , National Geographic Society , and the National Institute of Mental Health MH

50 TABLE AND FIGURE LEGENDS Table 1. List of all 89 specimens sequenced in this study, organized by operational taxonomic unit (OTU). Table includes brief explanation of the meaning of the OTU abbreviation, the total number of voucher specimens in the field samples, the number of individuals sequenced, collection locality and GenBank, museum, and individual specimen numbers. NPp = electrocyte with non-penetrating stalk, posterior innervation; Pa = electrocyte with penetrating stalk, anterior innervation. Figure 1. Phylogenetic relationships among 38 species belonging to 17 nominal genera of the Mormyrinae as reflected by the most parsimonious tree recovered from an unweighted analysis of the combined molecular sequence datasets from 12S, 16S, cytochrome b, RAG2 and S7 intron 1 and 2 (4256 aligned base pairs, 939 informative characters, CI=0.51, RI= 0.62). The Genus Brienomyrus is shown to be polyphyletic. The "Gabon-clade Brienomyrus is distinct from Brienomyrus (Brienomyrus) brachyistius, the type species for Brienomyrus Taverne, Both are distinct from Brienomyrus (Brevimyrus) niger Taverne, as indicated. The numbers above nodes are bootstrap values (shown if 49

51 above 50%), those below nodes are Bremer decay indices. (Adapted from Lavoué et al., submitted) The monophyly of the Mormyrinae and sister group relationship between Myomyrus macrops and remaining Mormyrinae was established by Lavoué et al. (2000) and by Sullivan et al. (2000). Figure 2. Map of Central West Africa with the collection localities of the specimens of the Gabon-clade Brienomyrus indicated as closed circles. All field sites are in the either Gabon or the Central African Republic. Locality names indicated here identify the collection basin for each specimen listed in Table I. Figure 3. Operational taxonomic units (OTUs) are diagnosed using both external morphology, and electric organ discharge characteristics. A.) Tracings of the dorsal profiles of the heads of four Gabon-clade Brienomyrus OTUs illustrate the difference between blunt snouts (top) and sharp snouts (bottom). The electrocytes in the electric organ have non-penetrating stalks with posterior innervation (Type NPp, left) or penetrating stalks with anterior innervation (Type Pa), as illustrated by a schematic of a single electrocyte in sagittal view. B.) Four types of EODs recorded from each of the OTUs in A. 50

52 Individuals with Type NPp electrocytes produce EODs with only two phases: peak P1, and peak P2. OTUs with penetrating stalks (Type Pa) have EODs with an initial head-negative phase (Po) in addition to P1 and P2. For each OTU, multiple EODs have been normalized to the same peak-peak amplitude, and superimposed with head positivity upward. C.) Three OTUs illustrate subtle differences in the duration of the EOD waveform. The first, BEN, from the Ivindo, has a relatively short EOD compared to the others. SP4 from the Louétsi River has a similar morphology but slightly longer EOD duration; SP7 differs in head shape and in EOD duration. Figure 4. Photographs of live or preserved specimens from each OTU recognized in this study. All EODs are plotted on the same time base with head positivity upward. For OTU's with pronounced sex differences in EODs, both male and female waveforms are shown. Figure 5. A strict consensus tree of 680 equally parsimonius trees, each of 636 steps, shown as a phylogram using ACCTRAN character optimization, produced from an unweighted parsimony analysis of the complete cytochrome b gene from Gabon-clade Brienomyrus and outgroup taxa. For 51

53 all trees, CI=0.49. RI=0.84, RC=0.41 with uniformative sites excluded. Table in inset lists the different OTUs, according to the origin of the names. OTU's NGO, NZO, OFF, and SAN all are abbreviations for one of the collecting localities where these fish were captured. Some of the OTU names refer to manuscript names of two manuscripts currently in preparation, others refer to the shape of the snout (B=blunt; I=intermediate; S=sharp) and to the type of electrocytes in the electric organ (N= NPp; P= Pa). The remaining OTUs are described species. The letters in this and the following figure refer to nodes that are discussed in the text. Figure 6. Consensus MP bootstrap tree based on 1000 pseudoreplicates on the same dataset used for Fig. 5. Nodes supported by bootstrap proportions greater than or equal to 50% are shown with bootstrap values indicated. Figure 7. The cytochrome b tree fails to recover intuitive higher level groups of Gabon-clade Brienomyrus suggested by morphological and electric organ characteristics, all presumably derived characteristics within the group based on outgroup comparison. The phylogenetic tree from Fig. 5 52

54 is reproduced showing the presence (in black) or absence (gray) of each character state for each OTU. A.) OTUs MAG, BEN, SP2, SP4, SP6, SP7, SP8, B.curvifrons and B.hopkinsi all share very sharp snouts with terminal mouths and a jutting lower jaw. Yet these taxa do not form a monophyletic group on the cytochrome b haplotype tree. B.) OTUs with 16 circumpeduncular scales (inset) do not form a monophyletic group and in particular OFF and B. longicaudatus which additionally share an elongate caudal peduncle, a unique sloping head shape and large adult size do not appear as sister taxa. C.) OTUs TEN and BN2 that share monophasic EOD waveforms (inset) similarly do not form a monophyletic group. D.) OTUs SP6, SP8, MAG and SP2 all possess reversed polarity EOD waveforms (inset) in which the initial head negative P 0,derived from current flowing through penetrating stalks, becomes a major phase of the EOD and P2 is reduced. These OTUs do not form a monophyletic group on the tree. E.) OTUs possessing nonpenetrating stalk electrocytes with posterior innervation (type NPp) do not form a monophyletic group. 53

55 BIBLIOGRAPHY Alves-Gomes, J., and C. D. Hopkins Molecular insights into the phylogeny of mormyriform fishes and the evolution of their electric organs. Brain Behav. Evol. 49: Alves-Gomes, J. A Systematic biology of gymnotiform and mormyriform electric fishes: Phylogenetic relationships, molecular clocks and rates of evolution in the mitochondrial rrna genes. J. Exp. Biol. 202: Arnold, M. L Natural Hybridization and Evolution. Oxford University Press, Oxford. Arratia, G Basal teleosts and teleostean phylogeny. F. Pfeil, München. Avise, J. C Molecular markers, natural history and evolution. Chapman & Hall, New York. Bass, A. H. 1986a. Electric organs revisited: evolution of a vertebrate communication and orientation organ. Pp in T. H. Bullock and W. Heiligenberg, eds. Electroreception. John Wiley & Sons, New York. Bass, A. H. 1986b. Species differences in electric organs of mormyrids: substrates for species-typical electric 54

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72 Gabon-clade Brienomyrus Brienomyrus (B.) hopkinsi Paramormyrops gabonensis Brienomyrus (B.) longicaudatus Brienomyrus (B.) sp. VAD Marcusenius conicephalus Pollimyrus marchei Ivindomyrus opdenboschi Boulengeromyrus knoepffleri Stomatorhinus (3 spp.) Pollimyrus (3 spp.) Gnathonemus petersii Campylomormyrus (3 spp.) Marcusenius sp. Genyomyrus donnyi Marcusenius moorii Marcusenius greshoffi Hippopotamyus (2 spp.) Marcusenius senegalensis Hippopotamyus pictus Hyperopisus bebe Brienomyrus (Brevimyrus) niger Isichthys henryi Brienomyrus (B.) brachyistius Mormyrus (2 spp.) Mormyrops (3 spp.) Myomyrus macrops Fig. 1

73 Sullivan, Lavoué, & Hopkins C.A.R. EQUATORIAL GUINEA CAMEROON Ntem River Sangha River Coastal Pointe Mbini Woleu River 0 Coastal Libreville Lower Ogooué Ngounié River Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano Okano River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River River Ogooué River GABON Ivindo River Upper Ogooué CONGO Mouvanga / Ngounié Upper Ogooué Louétsi / Ngounié Kilometers Coastal/Mayumba Fig. 2

74 A NPp Pa blunt B. sp. TEN B. sp. VAD sharp B. sp. OFF B. sp. MAG B P 1 P 1 P 0 P 2 P 1 P 2 P 1 1 ms P 2 P 0 P 2 C n= 9 B. sp. BEN (Ivindo) n= 10 B. sp. SP4 (Louétsi) n= 10 B. sp. SP7 (Ntem) 1 msec Fig. 3

75 Gabon-clade Brienomyrus Blunt snout, nonpenetrating stalk Blunt snout, penetrating stalk Sharp snout, nonpenetrating stalk BN TEN 1689 BP CAB 1031 f m SN SN BN BP SN PAR 3459 BP OFF 3662 BX BP B. longicaudatus 2176 NZO 2551 SZA 3930 B. curvifrons 2085 Paramormyrops gabonensis 2040 Sharp snout, penetrating stalk ROB 3004 P LIS 3372 VAD 1486, 1491 f m B. hopkinsi 2067 SN SP BP SN MAG 1022 SP LIB SAN ms B. kingsleyae EOD unknown NGO ms B. sphecodes EOD unknown Intermediate snout, penetrating stalk BEN 1021 SP B. batesii EOD unknown IP Intermediate snout, non-penetrating stalk f SP IN m SP ms Fig. 4