Molecular Phylogenetics of the Neotropical Electric Knifefish Genus Gymnotus (Gymnotidae, Teleostei):

Biogeography and Signal Evolution of the Trans-Andean Species

by

Kristen Brochu

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Ecology and Evolutionary Biology University of Toronto

© Copyright by Kristen Brochu 2011

ii

Molecular Phylogenetics of the Neotropical Electric Knifefish

Genus Gymnotus (Gymnotidae, Teleostei): Biogeography and

Signal Evolution of the Trans-Andean Species

Kristen Brochu

Master of Science

Graduate Department of Ecology and Evolutionary Biology

University of Toronto

2011

Abstract

Gymnotus, the banded electric knifefish, is a diverse genus with a range that extends from

Argentina to southern Mexico and includes species distributed both east (cis-Andean) and west

(trans-Andean) of the Andes. Each Gymnotus species exhibits a distinctive electric organ

discharge (EOD), used for communication and navigation. Here, I present a new molecular

phylogenetic hypothesis for 35 Gymnotus species based on two mitochondrial (cyt b and 16S)

and two nuclear genes (RAG2 and Zic1). I found that the trans-Andean species are distributed in

four distinct lineages with varying amounts of divergence from their closest cis-Andean sister

taxa. I suggest that not all trans-Andean species evolved as a result of the orogeny of the Andes.

I evaluate EOD phase number evolution in Gymnotus and find a trend for reduced phase numbers

in both cis- and trans-Andean regions. Finally, I suggest hypotheses to account for the patterns of

EOD phase number diversification.

iii

Acknowledgments

This thesis would not have been possible without the advice and support that I received from

numerous sources. First, I would like to thank my supervisor, Nathan Lovejoy, for his helpful

insights and continued support. My time spent in his lab has helped me to develop important

skills as an independent researcher that I have no doubt will serve me well in the future. I would

like to thank my supervisory committee members, Hernan Lopez-Fernandez and Marc Cadotte,

for valuable feedback and advice. I am also very grateful to the members of the Lovejoy lab,

Megan McCusker, Eric Lewallen, and Devin Bloom for their guidance and encouragement. I

particularly appreciate their patience in answering my many questions and helping me to learn

new techniques. I would also like to thank all the wonderful people who made my time at the

University of Toronto a fun and rewarding experience.

My thesis was greatly enhanced through collaboration with William Crampton of the University

of Central Florida. I am extremely grateful for his help with equipment, recordings, and analysis

of EODs. His feedback and comments have always been prompt and constructive and his advice

is greatly appreciated.

My field work would not have been possible without a number of people who assisted me at

every step. Permit applications were facilitated in Panama by the Smithsonian Tropical Research

Institute (STRI), in Costa Rica by the Organization for Tropical Studies (OTS), and in Colombia

by the Instituto Alexander von Humboldt (IAvH). I would like to thank E. Bermingham and O.

Sanjur for supporting my trip to Panama. I am also indebted to the many people at STRI who

assisted me with specimens and field collections from both the Naos Island Laboratories and the

Bocas del Toro Research Station. I am also grateful to the many local fisherman and villagers

who assisted me in locating and catching electric fish. I would like to thank A. Summers, E.

Lewallen, and the students of the 2010 Marine Tropical Ecology Field Course for their role in

my first field collection experience. It will remain memorable for being both fun and successful.

I would like to extend a special thank you to Rigoberto Gonzalez at STRI who accompanied me

on many field collecting trips in Panama and who contributed greatly to making me feel at home

there. I would also like to thank Chielo for his resolute optimism and Kayla for her willingness to

accompany me on a collecting excursion on her vacation.

iv

I am very grateful to Francisco Campo and Ronald Vargas of OTS for their assistance in

organizing my trip to Costa Rica. Advice and collecting localities from William Bussing

(University of Costa Rica) proved invaluable and were very much appreciated. A special thank

you is owed to E. Lewallen for his field assistance on this trip and for helping to ensure that I

have fond memories of Costa Rica.

My trip to Colombia would not have been possible without the assistance of Javier Maldonado-

Ocampo. I am extremely grateful that he organized this trip and ensured that we were able to

collect the specimens that we needed. I would also like to thank D. Bloom, AO. Lara, and GC.

Rodriguez for their assistance in field collections. I would also like to thank them for helping to

make my time in Colombia very enjoyable.

This research was supported by a Natural Sciences and Engineering Research Council of Canada

(NSERC) Alexander Graham Bell Canada Graduate Scholarship (CGS) and Michael Smith

Foreign Study Supplement (MSFSS), a Fonds québécois de la recherche sur la nature et les

technologies (FQRNT) Bourse de maîtrise en recherche (B1), as well as a Sigma Xi (SX) Grant-

in-Aid of Research (GIAR) awarded to K. Brochu, in addition to an NSERC Discovery Grant to

NR. Lovejoy and an NSF Grant: NSF DEB-0614334 Evolution of Species and Signal Diversity

in the Neotropical Electric Fish Gymnotus (PI WGR. Crampton, CoPIS NR. Lovejoy, JS. Albert,

AA. Caputi). The University of Toronto also provided funds in the form of various Awards,

Grants, and TAships.

Above all, I would like to thank my family and friends for their support, encouragement, and

understanding while I continue to pursue my dream of studying amazing animals in

extraordinary places.

v

Table of Contents

Table of Contents

Abstract ........................................................................................................................................... ii

Acknowledgments .......................................................................................................................... iii

Table of Contents ............................................................................................................................ v

List of Tables ............................................................................................................................... viii

List of Figures ................................................................................................................................ ix

List of Abbreviations ..................................................................................................................... xi

List of Appendices ........................................................................................................................ xii

Chapter 1 Introduction .................................................................................................................... 1

1 Overview .................................................................................................................................... 1

2 Gymnotiform Biology and Phylogeny ....................................................................................... 2

3 Biology, phylogeny, and biogeography of Gymnotus ................................................................ 4

4 The Electrogenic and Electrosensory System (EES) ................................................................. 6

4.1 Electrogenesis ..................................................................................................................... 6

4.2 Pulse- vs. Wave-type Signals .............................................................................................. 8

4.3 Evolution of Multiphasic Signals...................................................................................... 10

5 Objectives, Hypotheses, and Predictions ................................................................................. 15

6 Significance .............................................................................................................................. 16

Chapter 2 Materials and Methods ................................................................................................. 17

1 Field Collection ........................................................................................................................ 17

2 Taxon Sampling ....................................................................................................................... 17

3 Molecular Phylogeny ............................................................................................................... 18

3.1 DNA Isolation, PCR, and Sequencing .............................................................................. 18

vi

3.2 Alignment ......................................................................................................................... 19

3.3 Phylogenetic Analysis ....................................................................................................... 20

4 Electric Waveform ................................................................................................................... 21

4.1 EOD Recordings ............................................................................................................... 21

4.2 EOD Character Evolution Analysis .................................................................................. 21

Chapter 3 Results .......................................................................................................................... 23

1 Molecular Dataset .................................................................................................................... 23

2 Phylogenetic Relationships ...................................................................................................... 23

2.1 Complete Dataset Analyses .............................................................................................. 23

2.2 Individual Gene Analyses ................................................................................................. 25

2.3 Molecular Sequence Divergence ...................................................................................... 26

3 EOD Evolution ......................................................................................................................... 27

3.1 Individual EOD Signals .................................................................................................... 27

3.2 Ancestral Character State Reconstruction ........................................................................ 27

Chapter 4 Discussion .................................................................................................................... 29

1 Gymnotus Phylogeny ............................................................................................................... 29

2 Biogeography of the trans-Andean species .............................................................................. 31

3 Electric Signal Evolution ......................................................................................................... 33

3.1 A Complicated History ..................................................................................................... 33

3.2 G. ardilai may be a recent introduction ............................................................................ 35

3.3 Mechanisms for a return to monophasy ............................................................................ 36

3.4 Adaptive significance of low-frequency energy ............................................................... 36

3.5 Cis-Andean Reductions in Phase Number ........................................................................ 41

3.6 Abiotic Selective Pressures ............................................................................................... 41

3.7 Corollaries ......................................................................................................................... 42

4 Future Directions ...................................................................................................................... 43

vii

5 Conclusions .............................................................................................................................. 44

References ..................................................................................................................................... 46

Appendices .................................................................................................................................... 81

viii

List of Tables

Table 1: List of specimens included in study.................................................................................54

Table 2: List of primers used for amplification and sequencing of the cyt b, 16S, RAG2, and Zic1

genes..............................................................................................................................................56

Table 3: Summary of EOD recordings..........................................................................................57

ix

List of Figures

Figure 1: Family level relationships of the order Gymnotiformes. Modified from Stoddard

(2002a)…………………………………………………………………………………………...59

Figure 2: Geographic distribution of gymnotiform species with delineation of biogeographic

regions. Modified from Albert et al. (2004)……………………………………………………...60

Figure 3: Type-locality map for 35 described Gymnotus species. Modified from Albert et al.

(2004)…………………………………………………………………………………………….61

Figure 4: Distribution Map for trans-Andean Gymnotus species………………………………..62

Figure 5: Morphological Hypothesis for Gymnotus after Albert et al. (2004)…………………..63

Figure 6: Molecular Hypothesis for Gymnotus after Lovejoy et al. (2010)...................................64

Figure 7: Electrostatic field of Gymnotiformes. Modified from Stoddard (2002a)……………...65

Figure 8: Spectral sensitivity of the two types of electroreceptor cells in Gymnotiformes.

Modified from Stoddard (2002a)………………………………………………………………...66

Figure 9: Pulse and Wave-type signal discharges after Stoddard and Markham (2008)………...67

Figure 10: Voltage-time waveforms of monophasic and multiphasic signals. Modified from

Stoddard (2002b)………………………………………………………………………………...68

Figure 11: Electric organ discharge production after Stoddard (2002a)…………………………69

Figure 12: Voltage/time waveform of both the first phase and the full EOD of Brachyhypopomus

pinnicaudatus and their corresponding power spectrum plotted over the spectral sensitivity of

ampullary electroreceptors. Modified from Stoddard (2002b)…………………………………..70

Figure 13: Collecting localities in Panama and Costa Rica……………………………………...71

Figure 14: Collecting localities in Colombia…………………………………………………….72

x

Figure 15: Strict consensus phylogeny of 422 most parsimonious trees showing Gymnotus

relationships, based on the combined analysis of cyt b, 16S, RAG2, and Zic1 genes (3807

characters, 5037 steps, CI=0.47, RI=0.85).....................................................................................73

Figure 16: Maximum Likelihood phylogeny showing Gymnotus relationships, based on the

combined analysis of mitochondrial (cyt b and 16S) and nuclear (RAG2 and Zic1) genes..........74

Figure 17: Bayesian phylogeny showing Gymnotus relationships, based on the combined analysis

of mitochondrial (cyt b and 16S) and nuclear (RAG2 and Zic1) genes........................................75

Figure 18: Results of Maximum Parsimony analyses of individual mtDNA and RAG2

datasets...........................................................................................................................................76

Figure 19: Results of Maximum Likelihood analysis of individual mtDNA, RAG2, and Zic1

datasets...........................................................................................................................................77

Figure 20: Electric organ discharges of five species of trans-Andean Gymnotus visualized as

voltage over time waveforms…………………………………………………………………….78

Figure 21: Maximum Likelihood Optimization of electric organ discharge (EOD) phase number

of Gymnotus species over the total evidence Maximum Likelihood phylogeny...........................79

Figure 22: Parsimony Optimization of electric organ discharge (EOD) phase number of

Gymnotus species over the total evidence Maximum Likelihood phylogeny…………………...80

xi

List of Abbreviations

16S – 16S ribosomal gene

CI – Consistency Index

cyt b – cytochrome b gene

EOD – electric organ discharge

ML – Maximum Likelihood

MP – Maximum Parsimony

mtDNA – mitochondrial DNA

mya – million years ago

RAG2 – recombinase activating gene-2

RI – Retention Index

Zic1 – zic family member-1 gene

xii

List of Appendices

Appendix A: 16S Alignment.........................................................................................................81

1

Chapter 1 Introduction

1 Overview

Fishes are one of the most diverse groups of vertebrates. They exhibit a variety of strategies in

their morphology, behaviour, and ecology. While marine environments account for the vast

majority of the available aquatic habitat, these environments only harbour about half of the

diversity. In contrast, freshwater environments, which comprise only a small percentage of the

available habitat, also contain about half of the diversity of fishes (Cohen 1970). This notable

discrepancy prompts questions regarding how speciation and diversification occur within

freshwater environments.

Freshwater fish are particularly diverse within the Neotropical region, where there are over 6,000

described species (Albert et al. 2004). The dynamic environment found in these regions, along

with high species richness makes these regions of intense interest. Our understanding of the

immense diversity of freshwater fishes is still in its infancy. This gap in our knowledge makes

Neotropical fishes especially appealing for studies of evolution and diversification.

Investigations into how this variation is generated and maintained are exceptionally important to

our appreciation and preservation of not only the Neotropical fauna, but of all freshwater fishes.

This is especially important in light of the many land use changes that are currently underway in

the Neotropics.

In this introduction, I begin with a consideration of general Gymnotiform biology and an

overview of the phylogenetics and biogeography of the group. Next, I examine these topics in

more detail with respect to the electric knifefish genus Gymnotus (Gymnotidae: Gymnotiformes).

Finally, I evaluate some of the hypotheses relating to the evolution of signal complexity in the

group. In the remainder of my thesis, I investigate the evolutionary history of Gymnotus, which

constitutes an important part of the Neotropical freshwater fish fauna. I present the results for

phylogenetic analyses of four genes using three different methods. I also test some predictions

arising from one particular hypothesis about the evolution of signal diversity. I consider signal

evolution in both a phylogenetic and biogeographic context. Finally, I examine the implications

2

arising from the analysis of electric signal evolution and provide some hypotheses for the

patterns that emerge.

2 Gymnotiform Biology and Phylogeny

The teleost order Gymnotiformes, the electric knifefishes, is a highly diverse group that is widely

distributed in the Neotropics. There are over 170 species within approximately 30 genera

distributed among five families. They are found in drainage basins from southern Mexico

(15°N), to northern Argentina (36°S), and also on the Caribbean island of Trinidad.

Gymnotiforms are found in many habitats and constitute an important component of the

Neotropical freshwater fish fauna (Lundberg et al. 1987, Crampton 1996, Albert 2000, Albert et

al. 2004, Albert and Crampton 2005a, Lovejoy et al. 2010).

Gymnotiforms are especially interesting due to their active electrogenic-electroreceptive system,

which allows them to sense electric fields and to generate electric signals using specialized

electric organs. Gymnotiforms generate a species-specific electric organ discharge (EOD), which

they use for electrolocation and electrocommunication (Lissman 1958, Stoddard 2002a, 2002b,

Albert et al. 2004, Lovejoy et al. 2010). These signals are stereotypical and quantifiable, and as a

result can assist in the identification of morphologically cryptic species. These signals allow

these fish to be active at night and exploit niches in dark sediment-rich rivers in the Neotropics.

Studies on this group offer an interesting perspective on the role of communication in mating

systems and species recognition (Albert et al. 2004, Lovejoy et al. 2010).

Most Gymnotiforms are nocturnal, which means that visual cues are generally less important.

Both the visual and olfactory systems in gymnotiforms are less developed than in most other

teleost fish. This reduction in senses probably occurred due to the extreme reliance of

gymnotiforms on the electrosensory system. In fact, gymnotiforms tend to have increased brain

size compared to other fish, due to a large amount of brain tissue specialized for interpreting

electric signals (Lissman 1958, Albert 2000, Crampton and Albert 2006). Due to the extreme

importance of this sensory modality in the evolution of the Gymnotiformes, most, if not all,

aspects of Gymnotiform ecology and biogeography are in some way related to the electric sense.

This phenomenon will be discussed in more detail below.

3

In one striking example of how the electrosensory system influences the biology of

gymnotiforms, the characteristic culteriform or knife-like body shape of all known gymnotiforms

actually optimizes the detection of electric fields along the body. This body shape is

accompanied by a long anal fin, which is used for locomotion. Gymnotiform fishes undulate the

anal fin allowing them to keep their body relatively straight while they swim both forwards and

backwards detecting fine scale difference in electric field variation (Lissman 1958, Albert 2000,

Nanjappa et al. 2000, Albert and Crampton 2005b).

The order Gymnotiformes has undergone rapid taxonomic change in the past 25 years, with

many new species being discovered, and many species limits being re-evaluated or redescribed

(Albert and Miller 1995, Campos-da-Paz 1996, Albert et al. 1999, Albert and Crampton 2003,

Crampton and Albert 2003, Crampton et al. 2003, 2005, Maldonado-Ocampo and Albert 2004,

Cox Fernandes et al. 2004, Fernandes et al. 2005, Cognato et al. 2007, Richer-de-Forges et al.

2009). There are likely even more species awaiting discovery among museum specimens and in

field collections from new locations (Albert and Crampton 2005a). It has also been suggested

that examination of chromosomes could lead to the discovery of even more cryptic species

differing in karyotypes (Milhomem et al. 2008).

The monophyly of Gymnotiformes is well-supported (Alves-Gomes et al. 1995, Alves-Gomes

1999, Albert 2000). Gymnotiformes are thought to be most closely related to the catfish order

Siluriformes (Fink and Fink 1981, Alves-Gomes et al. 1995), with which they share a system for

passively sensing electric fields; however, there is some disagreement about the relationships

between the Gymnotiformes, Siluriformes, and Characiformes, with several studies suggesting

alternate relationships (Dimmick and Larson 1996, Ortí and Meyer 1996, 1997, Saitoh et al.

2003, Lavoué et al. 2005, Peng et al. 2006, Li et al. 2008, Poulsen et al. 2009, Nakatani et al.

2011). Synapomorphies for Gymnotiformes include possession of an electrogenic system, a

culteriform body plan, subdermal eyes, lack of pelvic, dorsal, and adipose fins, and the ability to

regenerate a large portion of the caudal segment of their bodies (Alves-Gomes 1999, Albert

2000, Albert and Crampton 2005b).

Within the Gymnotiformes, the relationships are somewhat contentious. It has been suggested

that both Sternopygidae and Hypopomidae are paraphyletic (Alves-Gomes et al. 1995). The

monophyly of Gymnotidae, including Gymnotus and the monotypic Electrophorus electricus is

4

well-supported. Currently recognized family-level relationships are summarized in Figure 1.

Under this scheme, the family Gymnotidae represents the basal group, although Sternopygus has

been suggested to be the sister group to all other Gymnotiforms (Alves-Gomes et al. 1995,

Alves-Gomes 1999, Albert 2000).

Gymnotiforms are believed to have originated in South America about 100 million years ago at

approximately the time of separation of Africa and South America. No extant or fossil

Gymnotiforms are known from Africa. There are 11 fossil fragments from Bolivia that can be

ascribed to the Gymnotiformes; however, their placement on the phylogeny is ambiguous (Albert

and Fink 2007). Gymnotiforms diversified widely during the Tertiary and dispersed into a

variety of habitats from their inferred original habitat of small upland streams (Albert 2000,

Albert and Crampton 2005a). Gymnotiform lineages occur on both the eastern side of the Andes

(cis-Andean) and the western side (trans-Andean) (Figure 2), suggesting that considerable

lineage diversity was present before the uplift of the Andes mountains.

3 Biology, phylogeny, and biogeography of Gymnotus

Gymnotus, the banded electric knifefish, is the most diverse genus in the Gymnotiformes, with

35 known species, and several additional species awaiting description (Albert et al. 2004, Albert

and Crampton 2005a, Richer-de-Forges et al. 2009). Gymnotus is in the family Gymnotidae with

the monotypic Electrophorus electricus. Gymnotus is relatively ancient and is found across the

range of the gymnotiforms, with species on both the eastern side of the Andes (cis-Andean) and

the western side (trans-Andean) (Figure 3). Seven species are exclusively trans-Andean in

distribution (Figure 4) (Albert et al. 2004, Albert and Crampton 2005a, Lovejoy et al. 2010).

Gymnotus species are found in all major river drainages of South America and occupy a variety

of habitat types. They form one of the major components of the fauna found in floating meadows

composed of aquatic macrophytes. Most Gymnotus species inhabit only one habitat, but two

species (G. carapo and G. arapaima) have been found to be more generalist and inhabit multiple

habitat types (i.e. blackwater rivers, terra firme streams, and whitewater floodplains) (Crampton

1996, Albert 2000, Albert et al. 2004). All species are active nocturnally and eat invertebrates or

small fish. Adult males are often territorial and exhibit nest guarding behaviour in several species

(Albert 2000, Crampton and Hopkins 2005). Most Gymnotus species are also capable of aerial

5

respiration, using the gas bladder as an accessory air-breathing organ when oxygen is scarce

(Crampton 1998, Albert and Crampton 2005a).

Gymnotus species are generally characterized as having alternating light and dark pigment bands,

hence the common name, banded electric knifefish. Since most Gymnotus are nocturnal, the

bands are likely not used for mating or social communication. Alternately, they have been

suggested to aid in avoidance of visual predation by disrupting visual recognition (Albert et al.

2004); however, there are some species that do not exhibit this colour pattern. In particular, all

the trans-Andean species are characterized by the breakdown of this trait to varying degrees.

Gymnotus cylindricus and Gymnotus maculosus have small spots of varying sizes on their body

(Albert and Miller 1995, Campos-da-Paz 1996). Gymnotus esmeraldas possesses a patchy

pattern of pale blotches along the majority of the anterior portion of the body with some banding

on the posterior portion of the body (Albert and Crampton 2003). Gymnotus henni and Gymnotus

choco show some breakdown of this banding pattern on the posterior portion of their bodies,

where the bands are irregular and wavy (Albert and Crampton 2003). Gymnotus ardilai exhibits

a loss of the banding pattern with increasing size (Maldonado-Ocampo and Albert 2004). If the

pigmentation bands do function as cryptic colouration, the trans-Andean species may have lost

this trait due to a reduction in predation pressure selecting for that character.

Due to the relatively high number of species, wide distribution, and great variation in character

traits, Gymnotus is a good group to answer questions about the evolution of diversity in the

Neotropics. However, in order to test hypotheses about diversification, it is necessary to have a

good phylogenetic framework so that this work can be placed in an evolutionary context.

Gymnotus species diversity has substantially increased in the past several years as the group has

been revised and new species have been added. The most comprehensive previous phylogenies

completed include Albert et al. (2004) which was based on morphology, as well as a molecular

analysis by Lovejoy et al. (2010) (Figure 5 and 6, respectively). Albert et al. (2004) proposed

three species groups, based on the major clades in their phylogenetic hypothesis: the G.

cylindricus group, the G. carapo group, and the G. pantherinus group. The G. cylindricus group,

composed of the two Middle American species G. cylindricus and G. maculosus, was suggested

to form a basal clade that is the sister clade to all the species of South America. Both their G.

pantherinus group and their G carapo group were monophyletic and contained species from both

6

sides of the Andes; however, support for the G. pantherinus group was weak. This phylogeny

suggested that the trans-Andean species are distributed in multiple clades; however, this has

never been tested rigorously with molecular evidence.

The phylogenetic hypothesis proposed by Lovejoy et al. (2010) was based on morphological

characters following Albert et al. (2004), as well as three genes, two mitochondrial (cyt b and

16S) and one nuclear (RAG2). Lovejoy et al. (2010) proposed five lineages based on the major

clades of their phylogeny: the G. carapo group (eight species), the G1 clade (five species), the

G2 clade (three species), G. pantherinus, and G. cylindricus. In all analyses except the

morphological one, the G2 clade was found to be the sister group to all other species, and G.

cylindricus was found to be the sister taxa to the G. carapo group. This phylogeny suggests that

the Middle American species are not found at the base of the phylogeny but are nested within

Gymnotus. This has important implications for reconstructions of both historical biogeography

and character evolution. This phylogenetic analysis did not test the hypothesis that the trans-

Andean species are distributed in multiple clades, as only one (G. cylindricus) of the seven

known trans-Andean species was included.

A major goal of this study is to clarify the phylogenetic position of trans-Andean lineages within

Gymnotus. This insight will provide valuable biogeographic information regarding the effects of

the Andes on Neotropical diversification. A new phylogenetic hypothesis will also be critical to

comparative studies of character evolution because it will allow the state changes to be viewed

within the context of evolutionary history, as well as provide some information regarding the

relative timing of the state changes (Alves-Gomes 1999). Finally, understanding the phylogeny

of trans-Andean lineages will allow a test of the Predator Avoidance Hypothesis for the

evolution of electric signals, as described below.

4 The Electrogenic and Electrosensory System (EES)

4.1 Electrogenesis

Electric signals are intimately linked with many aspects of Gymnotiform biology. They directly

impact fitness in this group and are influenced by several selective pressures related to the dual

functions of electrolocation and electrocommunication. Adaptations for one function can

seriously affect the other. Electric signals are also influenced by the physical constraints of the

7

environment on signal conduction and detection, as well as the physiological constraints of the

fish to produce and detect these signals. It is no surprise, therefore, that the evolution of electric

signals is a key factor contributing to diversification and habitat specialization (Stoddard 2002a,

Albert and Crampton 2005a).

While the capacity to sense electric signals has evolved in multiple groups of aquatic animals

(e.g. monotremes, elasmobranchs, and catfishes) (Hanika and Kramer 1999, 2000, Montgomery

and Bodznick 1999, Pettigrew 1999, Albert and Crampton 2005b), active electrogenesis has

evolved only a few independent times, including in the Neotropical order Gymnotiformes, the

African superfamily Mormyroidae, and some species within two families of African catfish in

the order Siluriformes (Stoddard 2002a).

All gymnotiforms except the electric eel emit only weakly electric discharges of generally less

than 100mV (Crampton and Albert 2006). The electric eel generally emits a discharge of

approximately 10mV for communication and navigation, but can emit a discharge of up to 600V

for prey capture or defence (Crampton and Albert 2006). While most gymnotiforms always emit

their signal, some species have been known to switch off their EOD when startled or exposed to

novel stimuli, which may have evolved as a mechanism to evade electroreceptive predators or to

avoid undesirable males (Curtis and Stoddard 2003, Crampton and Albert 2006); however, this

would allow only a brief respite.

Gymnotiformes possess a specialized electric organ that extends along the ventral portion of the

body from just behind the head to the tail. This specialized electric organ contains electrocytes,

which are cells specialized for generating an electric field around the body of the fish. When an

object enters the field, it distorts the wave in a stereotypical fashion depending on its resistance

and capacitance and allows the fish to discern a large amount of information about the object

(Figure 7). The fish are able to tell the size, shape, and material of an inanimate object, and can

also discern whether it‟s sensing a conspecific, potential mate, or potential predator. A 10-20cm

fish can communication within approximately 1 m from its body (Hopkins 1999, Stoddard

2002a). The distance for electrocommunication is greater than the distance for electrolocation

(Knudsen 1975, Albert and Crampton 2005b). The electroreceptor cells work in tandem with the

electric organ to detect changes in the fish‟s individual waveform, as well as the electric organ

discharges of other electric fish (Alves-Gomes 2001, Crampton and Albert 2006). Gymnotiforms

8

possess two types of electroreceptive cells: ampullary and tuberous (Figure 8). Ampullary

electroreceptors are used in passive electroreception and are tuned to low-frequency energy (~0-

100Hz). Tuberous electroreceptors are tuned to higher frequency energy (~100-3,000Hz)

(Hopkins 1974, Stoddard 1999, 2002a). Stoddard (2002a) suggested that tuberous

electroreceptors evolved for electrolocation. Tuberous electroreceptors tend to have a narrow

tuning range and greater sensitivity to the fish‟s own EOD than to those of conspecifics. If these

electroreceptors evolved for communication, they would be expected to be tuned to a much

broader range and be more sensitive to the signals of other fish.

4.2 Pulse- vs. Wave-type Signals

There are two main physiological types of electric signals (Figure 9). Pulse-type signals are

characterized by one to six phases (i.e. deviations from the 0V baseline) of alternating polarity

punctuated by brief periods of silence. In contrast, wave-type signals are characterized by a

pattern of one to four phases recurring in a continuous cycle (Albert 2000, Albert and Crampton

2005a). Pulse species tend to exhibit a broader range of physical and physiological states,

allowing them to navigate and communicate in a variety of conditions. They are also prone to

electrical interference and are especially vulnerable to predation by electroreceptive predators, as

discussed below. Wave species are restricted to a much narrower range of environmental

conditions due to constraints imposed on them by their consistent and continuous discharge.

They are, however, less affected by electrical interference and seem to sustain their EOD at such

frequencies as to be much less conspicuous to electroreceptive predators (Stoddard 2002a).

Both pulse-type and wave-type EOD signals can vary in phase number and polarity, as well as in

duration and amplitude for each phase (Stoddard et al. 2006). Both pulse-type and wave-type

species have evolved specialized physiology and habits to adapt to their environment (Stoddard

2002a). Hopkins and Heiligenberg (1978) proposed that wave-type species evolved due to the

need for greater temporal resolution. Fast flowing, well-oxygenated environments require a

higher and more stable EOD discharge rate in order to effectively navigate and capture prey in

such a dynamic environment. Wave-type species that are found in fast flowing habitats have

higher repetition rates of their EOD than wave-types species found in slower flowing habitats.

Even pulse-type gymnotiforms in fast flowing environments tend to have more regular EOD

rates and smaller changes between the day-night pulse rates. Increased rate of EOD discharge

9

provides a greater temporal acuity. Fast flowing environments tend to be highly dynamic and

change rapidly with time, demanding high temporal acuity from a fish foraging in that

environment. Prey items that are highly mobile might also demand a foraging fish to have high

temporal acuity (Crampton and Albert 2006).

The high dissolved oxygen content in fast flowing habitats is particularly important because of

the energetic considerations of EOD generation (Crampton 1998, Crampton and Albert 2006).

Species with wave-type signals swim continuously, cannot modulate their EOD rate, and

generally emit signals at a higher rate than pulse-type species. These differences could indicate

that wave-type species have higher energetic requirements and therefore require well-oxygenated

waters. Oxygen consumption in both pulse- and wave-type species was found to be similar;

however, “scan swimming” (a foraging behaviour where the fish swims forwards and backwards

to scan the environment) has been shown to drastically increase the oxygen consumption

compared to resting. Scan swimming is not observed in pulse-type gymnotiforms; therefore it

could represent an additional energy cost to wave-type fish which may restrict them to well-

oxygenated waters (Julian et al. 2003, Albert and Crampton 2005b). In fact, wave-type species

have no physiological adaptations to deal with hypoxia and are generally restricted to well-

oxygenated waters. Pulse-types are dominant in hypoxic water, but are also dominant in some

well-oxygenated environments, such as terra firme streams (Crampton 1998, Julian et al. 2003,

Albert and Crampton 2005b, Crampton and Albert 2006). It has been suggested that they occupy

microhabitats with slower flow rates, where they are not subject to the fast flowing waters of the

main river channel (Albert and Crampton 2005b).

Changes in temperature are also important because they affect the speed of physiological

processes, which is reflected in longer or shorter EODs (Crampton and Albert 2006). Wave-type

species seem to require a temperature of 26°C ± 3-4°C, with most occurring in the exceptionally

thermostable environments of deep river channels. When temperatures change they induce

changes in the frequency of the EOD; however the electroreceptors do not become tuned to the

new frequency as rapidly as the temperature changes. When the environmental temperature

changes by more than 3 or 4°C the tuberous electroreceptors of wave-type species may no longer

be capable of sensing their own EOD due to the mismatch in the frequencies (Stoddard 2002b,

Albert and Crampton 2005b). Brachyhypopomus pinnicaudatus has been found to drastically

10

reduce the second phase of the EOD in response to high temperatures, possibly in relation to

cues that indicate the onset of the breeding season (Silva et al. 1999).

In contrast to wave-type species, pulse-type species have a higher spatial resolution due to their

wider range of frequencies that allows them to detect a wider range of capacitances from natural

objects that enter their electric field (Crampton and Albert 2006). This adaptation gives pulse-

type species an advantage in spatially complex environments, such as plant root masses;

however, pulse-type species are generally more restricted to high conductivity ecosystems

(Hopkins 1999, Crampton and Albert 2006).

Conductivity is important due to its correlation with primary productivity and pH. It is also

correlated to how easy it is to propagate the electric signal (Albert and Crampton 2005a,

Crampton and Albert 2006). The higher the conductivity, the less external resistance is

encountered by the electric current of the EOD. Aquatic ecosystems can be divided into low

conductivity (5-60µScm-1

) and high conductivity (60-700+µScm-1

) waters. In gymnotiforms,

some characteristic morphological adaptations are associated with changes in conductivity.

Species in low conductivity environments tend to have long, thin tails, representing more

electrocytes in series, whereas species in high conductivity environments tend to have short,

thick tails, representing more electrocytes in parallel. Electrocytes in series maximize the power

of an EOD where external resistance is high, i.e. low conductivity environments. Conversely,

electrocytes in parallel maximize the power of an EOD where external resistance is low, i.e. high

conductivity environments (Hopkins 1999).

4.3 Evolution of Multiphasic Signals

This thesis is primarily concerned with the evolution of the pulse-type signals in Gymnotus. One

of the most obvious differences among pulse-type signals of different species is the number of

phases. Monophasic signals are composed of one phase, while multiphasic signals are composed

of two to six phases (Figure 10). The arrangement of electrocytes and accessory electric organs

are important in determining phase number (Figure 11) (Hopkins 1988, Stoddard 2002a). The

plesiomorphic signal state in Gymnotiforms is thought to be the monophasic pulse-type

(Stoddard 1999, 2002a, 2002b, Alves-Gomes 2001, Albert and Crampton 2005a). The basal

family, Gymnotidae (comprised of Gymnotus and Electrophorus), exhibits pulse-type signals

(Crampton and Albert 2006) and E. electricus, the sister lineage to Gymnotus, has a monophasic

11

EOD. Additionally, all larval gymnotiforms examined exhibit monophasic or quasi-monophasic

signals with an ontogenetic development of the multiphasic signals (Stoddard 2002b, Lovejoy et

al. 2010, Crampton et al. 2011). The electric organ of all pulse-type gymnotiforms is derived

from hypaxial muscle. This electric organ remains the same throughout development in pulse-

type gymnotiforms, while the electric organ of wave-type species is replaced by a myogenic or

neurogenic adult electric organ early in their ontogeny (Albert and Crampton 2005b, Pereira et

al. 2007). In Gymnotus, the multiphasic signal usually develops when individuals reach a size

over 25mm, typically after a period of several weeks (Crampton and Hopkins 2005, Pereira et al.

2007, Kirschbaum and Schwassmann 2008). Based on these combined lines of evidence, the

monophasic pulse-type signal is considered to be plesiomorphic in Gymnotiformes (Stoddard

1999, 2002a, 2002b, Alves-Gomes 2001, Albert and Crampton 2005a).

Stoddard (1999, 2002a, 2002b) considered several hypotheses to explain why multiphasic signals

might have evolved from a monophasic ancestral condition. First, he considered that multiphasic

signals might provide an advantage in electrolocation. Most electric fish have a high density of

electroreceptors on their head, therefore this should be the area where these fish have the highest

sensory acuity, and indeed, some electric fish have been observed to explore new environments

and forage exclusively with their heads (Nanjappa et al. 2000). This evidence would suggest that

if multiphasic signals evolved to support electrolocation functions, the localized electric field

around the head of the electric fish should exhibit a multiphasic waveform. The electric organ

discharge can vary spatially and temporally in the localized area surrounding the fish. Due to the

physiology of the electric organ, several genera, Brachyhypopomus, Rhamphichthys, and

Gymnotus, are known to lack the second phase of the EOD at their heads. Increased signal

complexity at the tail end of the fish, where electrolocation is less critical, would suggest that

multiphasic signals did not evolve to enhance electrolocation. This hypothesis could be better

tested in a laboratory experiment with the foraging ability of both monophasic and multiphasic

individuals evaluated in complex vs. simple habitat types and with moving vs. stationary prey.

Multiphasic signals are sexually dimorphic in some groups of electric fish (Stoddard 2002b,

Albert and Crampton 2005a), which could indicate that they evolved to aid in mate attraction or

mate competition. Stoddard (2002b) suggests that if multiphasic signals evolved for sexual

selection then either only the displaying sex should have evolved additional phases, or if both

species evolved additional phases, they should have always been sexually dimorphic. In

12

multiphasic species, both sexes exhibit the same number of phases and do not always exhibit

sexual dimorphism (Curtis and Stoddard 2003, Crampton and Albert 2006). If sexual selection

was the main force driving evolution of multiphasic signals, then sexual dimorphism should be

present throughout multiphasic lineages. This pattern is not observed in any of the pulse-type

families (Stoddard 2002b). In light of this evidence, Stoddard (2002b) concluded that it is

unlikely that multiphasic signals evolved to enhance sexual signalling, although it does appear

that additional phases have secondarily been adapted for that purpose in several groups,

promoting further diversification.

EODs have also been suggested to play a role in species recognition (Hopkins and Bass 1981,

Hopkins 1999, Albert 2000, Crampton and Albert 2006). A larger number of phases increases

signal complexity, providing more parameters that can potentially be differentiated to increase

the distinction between species. If multiphasic signals evolved to facilitate reproductive isolation,

all phases would be expected to exhibit a high degree of differentiation and in particular, the

second phase would be expected to exhibit a large degree of interspecific differentiation. In the

gymnotiform Brachyhypopomus and the mormyrid Brienomyrus the first phase exhibits more

interspecific variation, while the other phases exhibit more intraspecific variation (Stoddard

2002a, 2002b). This evidence suggests that it is unlikely that multiphasic signals evolved to

improve species recognition; however, reproductive isolation does likely drive diversification

and complexity within the group.

Many species of gymnotiforms are territorial; therefore any adaptation that could increase

territory size would help to increase fitness (Knudsen 1975, Stoddard 2002b). In electric fish,

greater signal amplitude allows the signal to be broadcast further, effectively increasing signal

space. An extra phase in the electric signal would theoretically increase the amplitude without

any additional power output, so territorial species would be expected to be more likely to have

multiphasic signals than non-territorial species. Unfortunately, comparisons of territorial

behaviour are not available for most species, much less between closely related monophasic and

multiphasic species. Alternately, Stoddard (2002b) suggested that if multiphasic signals evolved

to improve territory defence by increasing the signal amplitude, then multiphasic signals should

exhibit greater amplitude than monophasic species. In the genus Brachyhypopomus, the

monophasic species has far greater signal amplitude than its multiphasic congeners (Stoddard

2002a, 2002b). Additionally, all strongly electric fish possess monophasic EODs with high

13

amplitude. One species of mormyrid in the genus Hyppopotamyrus has been found to have a

very low amplitude monophasic EOD (Hanika and Kramer 2000); however, the genus is

relatively nested in the mormyrid lineage and therefore its monophasy is likely a derived

condition and cannot be considered as homologous to the ancestral monophasic condition

(Stoddard 2002b). Therefore, Stoddard (2002b) concluded that he found no evidence to support

territory defence as the selective pressure driving the evolution of multiphasic signals although

he did concede that the evidence against this hypothesis is largely based on comparisons of EOD

amplitude instead of territorial behaviour and thus could be much stronger.

The Predator Avoidance Hypothesis proposes that multiphasic signals evolved to provide crypsis

from electroreceptive predators. Gymnotiforms have probably always had electroreceptive

predators, considering that their sister group, the Siluriformes (catfishes), possess a system for

sensing electric fields. Many important predators that occupy the same habitats as gymnotiforms

(including freshwater rays, catfishes, and other gymnotiforms) possess ampullary

electroreceptors that allow them to detect electric signals. If multiphasic signals evolved to be

cryptic to electroreceptive predators, then multiphasic signals should be less detectable to

electroreceptive predators. Ampullary electroreceptors are sensitive to low-frequency energy in a

range of approximately 0-100Hz, with a maximum sensitivity at approximately 30Hz for

gymnotiforms and 8Hz for catfish (Hanika and Kramer 1999, 2000, Stoddard 1999, 2002a). The

amount of low-frequency energy in an EOD is a function of the amount of asymmetry in the

voltage-time waveform. Signals that have equal amounts of energy above and below 0V cancel

out the low-frequency DC component of their signal and therefore have a reduced amount of

energy in the low-frequency range. Signals that have an asymmetric distribution of energy above

and below 0V tend to have an increased low-frequency component to their EOD. Monophasic

signals possess no energy below 0V, representing the asymmetrical extreme. Monophasic signals

have a large low-frequency component to their signal, which falls well within the range of

ampullary electroreceptors. Multiphasic signals tend to emit much higher frequency energy,

which can be detected by tuberous electroreceptors possessed by gymnotiforms but not catfish,

while being relatively cryptic to ampullary electroreceptors (Figure 12). This evidence supports

the proposal of the Predator Avoidance Hypothesis for the evolution of multiphasic signals,

which suggests that multiphasic waveforms evolved as a mechanism to avoid electroreceptive

predators (Stoddard 1999, 2002a, 2002b).

14

Stoddard (1999) tested whether multiphasic signals provide crypsis by training an electric eel to

respond to electric playback signals and then presenting it randomly with either a biphasic signal

or a monophasic signal (the same biphasic signal with the second phase digitally deleted). The

eel was much more likely to approach the monophasic signal than the biphasic signal. These

results imply that a monophasic signal may be more likely to elicit a predation attempt.

Multiphasic signals appear to be less detectable by the electric eel, suggesting that a multiphasic

species would be more adept at evading predation attempts. Hanika and Kramer (1999, 2000)

also found similar results studying the catfish Clarias gariepinus and the mormyrid Marcusenius

macrolepidotus. C. gariepinus was found to prey almost exclusively on males of M.

macrolepidotus. Playback experiments determined that catfish could always detect the signals of

males, but never of females. Males of M. macrolepidotus increase the duration of their EODs

upon maturation, increasing the low-frequency energy content of those signals. This change in

EOD parameter tends to attract more females, but also more predators. They also tested several

other species of mormyrids and found that species with a monophasic, or quasi-monophasic

discharge were also easily detected by C. gariepinus. These results provide additional support for

the idea that low-frequency energy is easily detectable by ampullary electroreceptors and that

this predation pressure is a strong force selecting for crypsis in electric fish.

Stoddard (1999, 2002a, 2002b) also proposed biogeographic support for the Predator Avoidance

Hypothesis. Electric eels are not present in the trans-Andean region, and both freshwater rays

and pimelodid catfish, which represent key electroreceptive predator groups, exhibit reduced

abundance and diversity in this region (Miller 1966). Species that are exclusively trans-Andean

in distribution may therefore experience reduced predation pressure. The Predator Avoidance

Hypothesis predicts that in areas of low predation, species would retain the ancestral monophasic

condition. The only trans-Andean Gymnotus species to have their EOD examined (G. cylindricus

and G. maculosus) show monophasic EODs. In contrast all adult pulse-type gymnotiform species

examined to date in cis-Andean habitats, where predation by electroreceptive predators is

expected to be high, exhibit multiphasic waveforms, except for one Brachyhypopomus species,

which may be a Batesian mimic of the electric eel, and the electric eel itself (Stoddard 1999,

Lovejoy et al. 2010). This evidence is in line with the Predator Avoidance Hypothesis and

supports the idea that predation pressure drove the evolution of multiphasic signals. These

patterns indicate that the monophasic Gymnotus species retained the ancestral monophasic

15

condition due to a lack of predation pressure selecting for crypsis, while cis-Andean groups

evolved multiphasic signals to evade electroreceptive predators.

The obvious geographic patterns predicted by the Predator Avoidance Hypothesis in Gymnotus,

make it an ideal group in which to test these ideas. Following this line of evidence, the

phylogeny of Lovejoy et al. (2010) examined the evolution of EOD phase number, including the

evidence that all adult cis-Andean Gymnotus species with a known EOD exhibit multiphasic

signals, while two trans-Andean species show monophasic discharges (G. cylindricus and G.

maculosus). This study was only able to include one trans-Andean species (G. cylindricus),

which did not contribute findings on the signal evolution of trans-Andean species. The available

evidence provides some support for the Predator Avoidance Hypothesis; however, this theory

would have more robust support in Gymnotus if more trans-Andean species are examined.

Although multiple hypotheses have been proposed to explain the evolution of multiphasic

signals, an evaluation of all of these is outside the scope of this thesis. Since the Predator

Avoidance Hypothesis has traditionally been considered to be the best explanation of multiphasic

signal evolution, with multiple lines of supporting evidence, and testable predictions, my thesis

will focus on the evaluation of this hypothesis within the genus Gymnotus. In order to evaluate

the Predator Avoidance Hypothesis, I will collect EOD signal recordings from trans-Andean

Gymnotus species and explore EOD evolution in a phylogenetic context.

5 Objectives, Hypotheses, and Predictions

The first objective of this thesis is to propose a new phylogenetic hypothesis for Gymnotus, using

additional genes and species that were not included in previous datasets. This phylogeny will be

used to evaluate previous phylogenies proposed by Albert et al. (2004) and Lovejoy et al. (2010).

It will also be used to determine the phylogenetic position of the trans-Andean Gymnotus

species. I will explicitly test the hypotheses that (1) trans-Andean species do not constitute a

monophyletic group, and are distributed in multiple clades, as proposed by Albert et al. (2004),

and (2) that the trans-Andean G. cylindricus lineage is not the sister group to all other Gymnotus

species, as proposed by Lovejoy et al. (2010).

The second objective of this thesis is to test the hypothesis that trans-Andean Gymnotus species

exhibit monophasic EODs, as predicted by the Predator Avoidance Hypothesis. To do this,

16

EODs from trans-Andean Gymnotus species will be recorded in the field and used to determine

EOD phase number. The evolution of EOD phase number will also be considered in a

phylogenetic context, to test the prediction that trans-Andean lineages should show evolution of

monophasic EODs. If multiple independent lineages of trans-Andean fauna are recovered in the

phylogeny, it will provide more independent tests for this prediction.

6 Significance

Understanding the phylogeny of Gymnotus will provide valuable information concerning

Neotropical speciation and biogeography. This study will be the first to include six of the seven

trans-Andean species of Gymnotus in a molecular phylogenetic analysis and will represent the

most complete phylogenetic hypothesis generated to date for the genus. My work will provide

new information on EOD waveforms for the trans-Andean fauna, as well as an analysis of the

evolution of phase number. My results will help to clarify the evolution of electric fish

communication signals and evaluate the relevance of the Predator Avoidance Hypothesis for

Gymnotus. Results from my thesis will provide the groundwork for further studies on Gymnotus,

such as the variation and evolution of other aspects of EOD parameters, and corresponding

implications for species recognition and mate choice.

17

Chapter 2 Materials and Methods

1 Field Collection

Field collection expeditions to three countries were undertaken to collect various species of

trans-Andean Gymnotus. The first trip was to Panama from February-May 2010, with the

assistance of the Smithsonian Tropical Research Institute (STRI). An additional trip to Costa

Rica was carried out for two weeks in April 2010 with the assistance of the Organization for

Tropical Studies. A map of Panamanian and Costa Rican collecting localities is shown in Figure

13. A collecting trip was organized to Colombia in June 2010. A map of Colombian collecting

localities is shown in Figure 14.

Specimens were located in the field by the authors and colleagues using an electric fish detector,

which consists of a differential amplifier and speaker connected to electrodes that are placed on

the end of a pole and submerged in the water (Wells and Crampton 2006, Crampton et al. 2007).

Specimens were then collected using dipnets with 3-4 mm mesh size. Fish were kept individually

in aerated buckets in the field for EOD recording procedures. Subsequently, they were

euthanized according to animal care protocols. Muscle tissue was then sampled and stored in 95-

100% ethanol. Specimens were preserved using 10% formaldehyde and then transferred into

70% ethanol for permanent storage. Vouchers have been deposited in museum collections (See

Table 1 for voucher numbers).

2 Taxon Sampling

A total of 35 Gymnotus species were included in this analysis, comprising 22 cis-Andean species

and six trans-Andean species out of the 35 described species, along with an additional seven cis-

Andean species that have not yet been formally identified. Two individuals were sequenced for

each species whenever possible. More individuals were sequenced across the range of those

species with an extensive distribution (G. coropinae and G. carapo) to consider any geographic

variation. Eight additional taxa were selected from among the gymnotiform families, including

Gymnotidae (Electrophorus), Rhamphichthyidae (Rhamphichthys), Hypopomidae (Hypopomus,

Brachyhypopomus), and Sternopygidae (Sternopygus). Sternopygus macrurus and S. astrabes

were used as the outgroup taxa. A total of 97 individuals were analyzed, including 95 ingroup

18

individuals and 2 outgroup individuals. Table 1 provides a complete list of specimens included in

this study.

3 Molecular Phylogeny

3.1 DNA Isolation, PCR, and Sequencing

Existing sequences were available for cyt b, 16S, and RAG2 for 27 cis-Andean and one trans-

Andean Gymnotus species, as well as the eight outgroup species (Lovejoy et al. 2010). Here I

added two cis-Andean species and five trans-Andean species, as well as the new gene Zic1.

DNA was extracted from muscle tissue using DNeasy Blood and Tissue Kits (QIAGEN). The

polymerase chain reaction (PCR) was used to amplify fragments of two nuclear genes and two

mitochondrial (mtDNA) genes using various combinations of primers (Primer sequences are

listed in Table 2). The nuclear gene fragments include 840 bp of the zic family member 1 gene

(Zic1), and 1295 bp of the recombinase activating gene-2 (RAG2). The mtDNA genes include

1126 bp of cytochrome b gene (cyt b), and 546 bp of the 16S ribosomal gene (16S). These genes

were selected based on their successful use in several taxa, particularly within the

Gymnotiformes, and represent a selection of both faster- and slower-evolving genes from the

mitochondrial and nuclear genomes (Palumbi et al. 1991, Meyer 1993, Palumbi 1996, Lovejoy

and Collette 2001, Li et al. 2007, Lovejoy et al. 2010, Maldonado-Ocampo 2011). Cytochrome b

exhibits variable rates of evolution across sites, with some regions being highly conserved, most

likely due to their importance in the function of the protein in the electron transport chain

(Palumbi 1996). 16S seems to be relatively conserved in some regions and hypervariable in

others, although it has been suggested to evolve more slowly compared to other genes in the

mitochondrial genome (Palumbi 1996). RAG2 evolves more slowly than mitochondrial genes

and is known to exhibit less homoplasy (Lovejoy and Collette 2001). The three genes where

existing sequences were available have been shown to provide good phylogenetic resolution in

gymnotiforms (Lovejoy et al. 2010). Zic1 was found to be a useful nuclear gene marker in a

study of 36 taxa of ray-finned fishes because it possesses a long, uninterrupted exon that is

relatively well-conserved (Li et al. 2007). It was also used with success in Sternopygidae,

another family of Gymnotiformes (Maldonado-Ocampo 2011). Using a diverse set of genes

assists in avoiding problems of systematic bias, such as nucleotide compositional bias (where

lineages with greater similarity in their nucleotide composition are grouped together, regardless

19

of their evolutionary history), long-branch attraction (when rapidly evolving lineages are

grouped together due to the accumulation of changes, regardless of their evolutionary history),

and heterotachy (changes in rates of substitution over time) (Li et al. 2007).

PCR reactions for cyt b, 16S, and RAG2 were carried out in 25µl amounts using 2.5µl 10x Taq

Buffer with (NH4)2SO4, 2µl of a 10µM mixture of each dNTP, 1.5µl of 25µM MgCl2, 1µl of 10

µM of each primer, 0.2µl of 1 U of Taq DNA Polymerase, and 2 µl of DNA. PCR reactions for

Zic1 were carried out in 25µl amounts using 2.5µl 10x Taq Buffer with (NH4)2SO4, 2µl of a

10µM mixture of each dNTP, 3µl of 25µM MgCl2, 1µl of 10 µM of each primer, 0.5µl of 1 U of

Taq DNA Polymerase, and 2µl of DNA.

Thermocycler conditions for cyt b, 16S, and Zic1 were: 95°C for 30s denaturation; 48-50°C (cyt

b), 54-58°C (16S), or 52°C (Zic1) for 60s annealing; and 72°C for 60s (16S), or 90s (cyt b, Zic1)

extension. This protocol was repeated for 35-38 (cyt b, 16S), or 40 (Zic1) cycles with hold steps

of 95°C for 30s (cyt b), 60s (16S), or 150s (Zic1) before the first cycle and 72°C for 300s after

the final cycle. Thermocycler conditions for RAG2 followed a touchdown protocol: 95°C for 30s

denaturation, 58°C, 56°C, 54°C, 52°C, (or 56°C, 54°C, 52°C, 50°C) for two cycles each, then

50°C (or 48°C) for 32 cycles annealing for 60s, and 72°C for 90s extension.

PCR products were purified using QIAQuick PCR Purification Kits (QIAGEN). Sequencing was

conducted at the DNA Sequencing Facility at the Toronto Sick Kids Hospital. Sequencing was

conducted using external primers, with one additional set of internal primers, Zic1_intF and

Zic1_intR, designed for this study using a preliminary Zic1 alignment.

3.2 Alignment

Sequences were edited and visually aligned using Sequencher 4.6 (Gene Codes Corp.). No

insertions or deletions were observed in cyt b or Zic1. A one codon indel was observed in one

RAG2 sequence. The final alignment for 16S followed Lovejoy et al. (2010). Regions of the

alignment surrounding indels where homology could not be determined were considered to be

ambiguous. Ambiguous regions were then excluded from the analyses. Ambiguity in RAG2

consisted only of four base positions surrounding the one indel. Ambiguity in 16S consisted of

several regions. The alignment for 16S can be viewed in Appendix A.

20

3.3 Phylogenetic Analysis

Nuclear and mitochondrial data were combined into a total evidence dataset using Geneious

(Drummond et al. 2011) consisting of 3807 characters for 97 OTUs. Data partitions were defined

as nuclear (RAG2 and Zic1) and mtDNA (cyt b and 16S). Analyses were conducted separately

for each nuclear gene, the mtDNA dataset, and the total evidence dataset.

Parsimony analyses were conducted in PAUP* (Swofford 2002). Sternopygus macrurus and S.

astrabes were used as the outgroup taxa. Gaps were treated as missing data. I used the following

parameters for all parsimony analyses: heuristic search algorithm, 1000 random-addition

sequence replicates, and TBR branch swapping. Bootstrap values (Felsenstein 1985) were also

calculated using the heuristic search algorithm with 1000 bootstrap replicates and 10 random-

addition sequence replicates.

Maximum Likelihood analysis (Guindon and Gascuel 2003) was performed using RAxML 7.2.8

(Stamatakis 2006, Stamatakis et al. 2008) with default parameters under the GTR+G model. I

used jModeltest (Guindon and Gascuel 2003, Posada 2008) to select models based on the

Corrected Akaike Information Criterion (AICc) and Bayesian Information Criterion (BIC). The

best fitting model for the total evidence dataset, according to both AICc and BIC, was TIM2+G;

however, the differences in AICc and BIC scores between this model and the GTR+G model

were small and the GTR+G model was calculated to have a higher likelihood, therefore, I opted

to use the GTR+G model.

Bayesian analysis was implemented in MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). All

partitions were unlinked. Two runs were performed, composed of four Markov Chains, with a

heating value of 0.065. Markov Chains were run for 20 million generations, sampling every 1000

generations. I assessed convergence by several methods. The average standard deviation of split

frequencies was 0.012 in MrBayes. The potential scale reduction factor (PSRF), which is a

convergence diagnostic for branch length posterior probabilities, approached one as the runs

converged (Gelman and Rubin 1992). Log-likelihood scores and other parameters were also

plotted in Tracer 1.5 (Rambaut and Drummond 2007) to assess convergence. It was determined

that convergence was achieved within the first 25% of sampled trees, which were then discarded

as burn-in, and the remaining trees were taken to represent the posterior probability distribution.

21

Uncorrected pairwise sequence divergences were calculated for each gene using PAUP*

(Swofford 2002).

4 Electric Waveform

4.1 EOD Recordings

Five trans-Andean species were examined for their EOD, including Gymnotus panamensis from

Panama (eight individuals), G. cylindricus and G. maculosus from Costa Rica (51 individuals),

and Gymnotus henni and Gymnotus ardilai from Colombia (17 individuals), with a total of 76

individuals recorded. A minimum of five adults were measured for each species (See Table 3 for

a summary of EOD recordings). EOD waveforms were digitized and recorded for individual fish

in the field no more than 48 hours after capture, using the head-to-tail standard and procedures

described by (Crampton et al. 2008). Fish were held in a mesh “sock" and placed in a tank of

water. Electrodes were placed directly in front of and behind the stationary fish. Water was kept

within 27°C ± 0.2°C during the day and 27°C ± 1°C at night. Conductivity was also kept

between 30-70µScm-1

, generally as close to 50µScm-1

as possible. Signals were amplified using

a custom-designed AC-coupled differential amplifier and digitized with an E-MU 0202 USB 2.0

Audio/MIDI Interface to a computer at a sampling rate of 192 kHz with 32-bit resolution. The

resultant EODs were saved in the ASCII format. The digitized signals can be visualized as a

voltage-time waveform which allows characterization of the number of phases. Phase number

was considered to be a positive or negative deviation from the 0 volt baseline greater than 1.5%

of the positive phase with highest amplitude.

4.2 EOD Character Evolution Analysis

The number of phases in adult Gymnotus range from one (monophasic) to six phases

(multiphasic). An EOD phase is characterized as a substantial deviation from the 0V baseline

(Figure 10) (Crampton and Albert 2006). While EOD phase number is generally a stereotypical

trait within species, some species with higher phase number (4+) are known to exhibit some

intraspecific variation, with phase number ranging from four to six. Due to this variability, EOD

phase number was coded as 1, 2, 3, or 4+ phases. This character was optimized as an unordered

multistate character using Maximum Likelihood and Parsimony methods in Mesquite 2.73

(Maddison and Maddison 2010). For the ML optimization the Markhov k-state 1 parameter

22

model (Mk1) of evolution was used, which considers each character change equally probable. In

order to resolve equivocal nodes in the MP optimization, ancestral states were examined in

further detail by viewing all Maximum Parsimony Reconstructions, using the MPR Mode in

Mesquite. The Maximum Likelihood consensus phylogeny was used for this analysis because it

did not include polytomies. The tree was also pruned to include only one individual per species,

except in the case of G. carapo, where it was necessary to include more than one individual to

maintain branching patterns.

23

Chapter 3 Results

1 Molecular Dataset

The cyt b dataset consisted of 1126 characters, with 545 parsimony informative characters. The

16S dataset consisted of 546 characters with 152 parsimony informative characters. The RAG2

dataset consisted of 1295 characters, with 289 parsimony informative characters. The Zic1

dataset consisted of 840 characters with 121 parsimony informative characters. The total

evidence dataset consisted of 3807 characters for 97 individuals comprising 35 Gymnotus species

and eight outgroup taxa. This dataset represents the most complete phylogenetic analysis of

Gymnotus to date. Compared to the most recent phylogenetic hypothesis (Lovejoy et al. 2010),

my dataset includes an additional nuclear gene, as well as an additional 17 species, almost

doubling the previous number of included species (Table 1). These additions include seven

species that have not yet been formally identified, as well as key trans-Andean taxa. This dataset

includes six out of the seven trans-Andean species, representing the most complete geographic

sampling of the genus.

2 Phylogenetic Relationships

2.1 Complete Dataset Analyses

Figure 15 shows the strict consensus tree of the 422 most parsimonious trees showing Gymnotus

relationships, based on two nuclear (RAG2 and Zic1) and two mitochondrial (cyt b and 16S)

genes. This phylogeny consists of 5037 steps, with a Consistency Index (CI) of 0.47, and a

Retention Index (RI) of 0.85. Figure 16 shows the results of the Maximum Likelihood analysis

using the GTR+G model (Final ML Optimization Likelihood Score=-26085.84) and Figure 17

shows the results of the Bayesian analysis, both based on two nuclear (RAG2 and Zic1) and two

mitochondrial (cyt b and 16S) genes.

Monophyly of Gymnotus and Gymnotidae is well supported across analyses. I define six major

clades, five of which were recovered by Lovejoy et al. (2010): the G1 clade (five species), G.

pantherinus, the G2 clade (four species), the G3 clade (two species), the G. cylindricus group

(three species), and the G. carapo group (20 species). These clades were consistently recovered

24

across analyses. In all analyses, there are four independent lineages that include trans-Andean

species, one of which consists of all the Middle American species. This result is consistent with

the prediction of the morphological hypothesis of Albert et al. (2004).

The three analyses differed primarily in their placement of G. pantherinus. In the MP analysis G.

pantherinus appears at the base of a clade corresponding to the G2 clade of Lovejoy et al.

(2010), consisting of G. aff. anguillaris, G. pedanopterus, G. n. sp. FRIT, and G. cataniapo. In

the ML and Bayesian analyses G. pantherinus is found to be a separate lineage representing the

sister taxa to all Gymnotus, except the G1 clade. Within the G2 clade, the positions of G.

pedanopterus and G. aff. anguillaris are not well supported in any analysis.

A new clade composed of G. tigre and G. henni was found to be the sister group to the clade

consisting of the G. cylindricus group and the G. carapo group. I define this new clade as the G3

clade. The G. cylindricus clade contains all of the Middle American species. G. panamensis was

found to form the sister group to G. cylindricus + G. maculosus. The analyses also suggested that

some G. cylindricus individuals may be more closely related to G. maculosus than to other G.

cylindricus.

Within the G. carapo group, there are five major lineages with relatively stable positions across

analyses (Figures 15, 16, 17). Clade A consists of seven species and Clade B consists of three

species. Clade C consists of G. bahianus and the „G. carapo complex‟ (three species). Clade D

consists of three species, of which two are trans-Andean in distribution. Clade E consists of the

„G. sylvius complex‟ (four species). The species composition of these clades remains the same,

except in the Parsimony analysis where G. omarorum appears as the sister taxon to clade C +

(D+E) instead of within clade B.

Some species relationships vary across analyses, including the closely related G. carapo, G.

arapaima, and G. ucumara, as well as G. sylvius and the newly discovered species G. n sp.

CORU, G. n sp. MAMA, and G. n sp. RS1. G. chaviro is sometimes nested within G. varzea and

G. mamiraua is sometimes nested within G. n. sp. ITAP. Both G. choco and G. ardilai are found

in one clade within the G. carapo group; however, some G. carapo from the Orinoco appear to

be more closely related to G. ardilai than to other G. carapo from the same region.

25

2.2 Individual Gene Analyses

Individual gene analyses were performed using MP and ML methods (Figure 18 and 19,

respectively). For these analyses, cyt b and 16S were combined into one mtDNA dataset. All

individual MP gene analyses recovered the same major clades but differed slightly in their

placement of individual taxa, particularly within the G. carapo group. The mtDNA MP analysis

could not resolve the positions of the G2 and G3 clades or G. pantherinus. The mtDNA MP

analysis also differed in its placement of G. omarorum. Both RAG2 MP and Zic1 MP analyses

exhibit a significant loss of resolution compared to the mtDNA MP dataset, particularly within

the G. carapo group. The Zic1 MP analysis did not resolve any major clades. This result is

expected based on the smaller amount of parsimony-informative characters in these datasets and

the slower rate of evolution in nuclear genes. The RAG2 MP analysis places G. pantherinus as

the sister taxon to the clade consisting of the G3 clade + (G. cylindricus group + G. carapo

group).

Individual ML gene trees also recover all major clades with slight variations in their positions

across genes. The mtDNA ML dataset places the major clades in the same positions as the total

dataset analysis. It varied from the total evidence analysis in the placement of Clade D within the

G. carapo group. The RAG2 ML analysis differed from the total evidence and mtDNA MP

datasets in the placement of G. stenoleucas and G. pantherinus. It also differed in the positions

of some clades within the G. carapo group. The Zic1 MP analysis was the most different from

the total evidence dataset with a basal G2 clade, and G. pantherinus as the sister taxon to the rest

of the species. The G1 clade was then the sister clade to the G3 clade + (G. cylindricus group +

G. carapo group). All individual genes varied somewhat in their placement of some individuals

within the G. carapo group, particularly within Clades C and D.

The topology of the individual gene phylogenies was largely consistent across analyses, with the

major clades consisting of the same species. There were some differences in arrangement of the

clades and individual species relationships, mostly within the G. carapo group. The individual

gene phylogenies generally exhibited improved resolution in the ML analyses.

26

2.3 Molecular Sequence Divergence

Uncorrected pairwise distances were compared for key species and clades. These distances were

calculated for multiple genes, but cyt b distances are presented here, because of the widespread

use of the cyt b gene in phylogenetic studies of fishes. Hereafter, divergences refer to pairwise

cyt b distances.

E. electricus was approximately 25% divergent from Gymnotus species. Interspecific

divergences within Gymnotus were generally greater than 1%. Intraspecific divergences were

generally less than 1%, with a few exceptions. The G. carapo group was found to have more

closely related species groups, while more basal clades were found to exhibit greater divergences

between species.

Trans-Andean clades were found to have varying degrees of divergence from their cis-Andean

sister clades. G. maculosus, G. cylindricus, and G. panamensis are on average 11.4% divergent

from members of their cis-Andean sister clade, the G. carapo group. G. henni is on average

11.3% divergent from its cis-Andean sister taxa G. tigre. G. choco is on average 1.5% divergent

from its cis-Andean sister taxon, G. carapo (OR). In contrast, G. carapo from the Orinoco was

only found to be between 0-0.2% diverged from G. ardilai.

Divergences between regions for the widespread G. coropinae and G. carapo were noticeably

larger than within regions. Within G. coropinae, Central Amazon individuals were 0% divergent

from each other, and individuals from the Guyanas were on average 1% divergent from each

other. Divergences between the two regions for G. coropinae were an average of 4.3%. Within

G. carapo, individuals from the Orinoco were 0.2% divergent, individuals from the Central

Amazon were 0% divergent, and individuals from the Western Amazon were 0.6% divergent.

Orinoco individuals were on average 3.9% divergent from the other regions. G. carapo from the

Central and Western Amazon constitute members of the „G. carapo complex,‟ with G. arapaima

and G. ucamara. Members of the „G. carapo complex‟ were on average 0.4% divergent. G.

bahianus was found to be approximately 1.5% diverged from the „G. carapo complex.‟

Several other closely related species groups were also found within the G. carapo group. G. n.

sp. CORU, G. n. sp. MAMA, G. n. sp. RS1 and G. sylvius were all closely related. G. n. sp.

MAMA individuals were on average 0.1% divergent; however, no individuals within this

27

complex were more than 0.36% divergent. Divergence among individuals of G. n. sp. ITAP was

approximately 1%. It was very closely related to G. mamiraua with only 1.7% divergence

between them. G. chaviro was only 0.08% divergent from G. varzea, compared to 0%

divergence among G. varzea individuals. G. n. sp. XING was found to be on average 2.7%

divergent from both G. n. sp. and G. pantanal, compared to the average of 1.3% divergence

between these two species. G. curupira and the specimens considered to be G. tigre (2019 and

2024) by Lovejoy et al. (2010) were 0.5% divergent. Finally, within the G2 clade, G. n sp. FRIT

was 11% divergent from G. cataniapo and 11.5% divergent from G. pedanopterus.

3 EOD Evolution

3.1 Individual EOD Signals

EODs of five trans-Andean species were recorded and digitized, including Gymnotus

panamensis from Panama (eight individuals), G. cylindricus and G. maculosus from Costa Rica

(51 individuals), and Gymnotus henni and Gymnotus ardilai from Colombia (17 individuals),

with a total of 76 individuals recorded. A minimum of five individuals was recorded per species

(See Table 3 for a summary of EOD recordings). Some intraspecific variation was observed

when comparing individuals of different size; however only adult EODs were included in the

analysis of EOD phase number. Phase number was considered to be a positive or negative

deviation from the 0 volt baseline greater than 1.5% of the positive phase with the highest

amplitude.

Figure 20 shows representative EOD signals of five trans-Andean species. G. cylindricus and G.

maculosus (from Costa Rica) were both confirmed to be monophasic. G. henni (from Colombia)

was also found to be monophasic. G. ardilai (from Colombia) and G. panamensis (from Panama)

were both found to be multiphasic, with tetraphasic and triphasic EODs, respectively.

3.2 Ancestral Character State Reconstruction

I used character reconstructions to trace evolutionary changes in EOD phase number across

phylogenetic reconstructions. Results were found to be consistent across tree topologies, thus I

report here the optimization of EOD phase number using Maximum Likelihood and Maximum

Parsimony methods for reconstruction ( Figure 21 and 22, respectively) using the Maximum

Likelihood consensus phylogeny. Maximum Parsimony Reconstructions of the ancestral state

28

recovered eight most parsimonious reconstructions. Ancestral states are reported as proportions

of their occurrence within the eight trees.

In both reconstruction methods, the ancestral state in the family Gymnotidae is inferred to be

monophasic and the ancestral state in the genus Gymnotus is inferred to be 4+ multiphasic. There

is a general trend of phase number reduction in most clades. The evolution of a triphasic state is

inferred in the ancestor of the clade consisting of the G3 clade, the G. cylindricus group, and the

G. carapo group. Further, a reversal to a multiphasic state is observed in at least one clade within

the G. carapo group. The optimizations suggest two independent phase number reductions in cis-

Andean clades within the G. carapo group: G. omarorum evolved a triphasic signal from a

tetraphasic ancestor, while G. obscurus evolved a biphasic signal from a triphasic ancestor. Two

independent reversals to a monophasic state are also observed, both within trans-Andean

lineages. Both G. henni and the G. cylindricus + G. maculosus clade evolved a monophasic

signal from a triphasic ancestor. For each trans-Andean clade, except G. ardilai, a reduction in

phase number compared to the cis-Andean sister group was observed.

The parsimony reconstruction method also optimizes the most parsimonious state for the

unknown EODs of G. n. sp. XING, G. bahianus, G. choco, and G. n. sp. RSI. G. bahianus, G.

choco, and G. n. sp. RSI are inferred to have tetraphasic signals and G. n. sp. XING is inferred to

have a triphasic signal.

29

Chapter 4 Discussion

1 Gymnotus Phylogeny

The work presented here represents the most complete phylogenetic analysis for Gymnotus to

date. The phylogenetic relationships proposed here support aspects of both Albert et al. (2004)

and Lovejoy et al.'s (2010) hypotheses and also clarifies some of the ambiguities in their

analyses.

In all analyses, the monophyly of Gymnotus and its position as sister group to E. electricus is

well-supported. The present analyses also find support for all five of the clades proposed by

Lovejoy et al. (2010), and suggest the addition of a new clade, called the G3 clade, composed of

G. tigre and G. henni (Figures 15, 16, 17). The G. cylindricus group was found to be the sister

clade to the G. carapo group in all analyses. All analyses also supported the G3 clade as the

sister group to these two clades. The G1 clade was always found to be the basal clade.

The position of G. pantherinus was found to be variable across analyses. It was alternately the

sister group to the G2 clade, which was then sister group to the G3 + (G. cylindricus group + G.

carapo group) clade, or the sister taxon to all clades except G1. Lovejoy et al. (2010) also

encountered this difficulty and noted that the position of G. pantherinus was not well-supported

in any analysis, including that of Albert et al. (2004). This ambiguity appears to be a

phylogenetic artefact. It is possible that the inclusion of additional molecular data or additional

species in the G1 and G2 clades will help to resolve the relationships of this species.

The trans-Andean species were found to be distributed in four independent lineages within three

separate species groups: the G. carapo group (G. choco and G. ardilai), the G. cylindricus group

(G. cylindricus, G. maculosus, and G. panamensis), and the G3 clade (G. henni). This result is

consistent with the prediction of Albert et al. (2004), although the species relationships found in

this study differ from that hypothesis.

My results confirm the monophyly of the G. cylindricus group, with the addition of G.

panamensis, which Albert et al. (2004) suggested was part of the G. pantherinus group. Albert et

al. (2004) suggested that G. panamensis was sister to G. pantanal and G. anguillaris within their

30

G. pantherinus group. This grouping was based mostly on external morphology as they had been

unable to collect internal anatomy data for G. panamensis. External morphological characters are

more susceptible to convergent evolution, which may have caused an erroneous placement of G.

panamensis. With the inclusion of G. panamensis in the G. cylindricus group, my analyses

indicate that this lineage includes all the Central American species, which appears more

parsimonious from a biogeographical perspective. My analyses support Lovejoy et al.'s (2010)

finding that the G. cylindricus group is the sister clade to the G. carapo group and not the sister

clade to the rest of the Gymnotus. The position of this clade was well-supported and stable across

analyses, which is significant for understanding patterns in Central American biogeography,

which will be discussed below.

G. henni was not included in Lovejoy et al. (2010)‟s study due to lack of available tissues, while

G. tigre was suggested to be the sister group to G. curupira. Albert et al. (2004) suggested that

G. tigre was the sister group to G. henni + G. esmeraldas. The results of the present analyses

seem to agree with the relationships proposed by Albert et al. (2004), which suggests that

although G. esmeraldas was not included in the present study, it may also belong to the G3

clade. The relationship found by Lovejoy et al. (2010) may have been the result of

misidentification, as the samples of G. tigre were obtained from juvenile fish (N. R. Lovejoy and

J. S. Albert, pers. comm.). Considering that there is only 0.5% divergence between those

individuals and G. curupira, it would suggest that they are in fact G. curupira, or else a new

closely related species, therefore they were treated as G. curupira in this study and new samples

of adult G. tigre were used.

My results confirmed the existence of the „G. carapo complex‟, consisting of G. carapo (CA), G.

carapo (WA), G. ucamara, and G. arapaima (Figure 15, 16, 17). Individuals of these three

species were consistently unresolved across analyses and often did not form monophyletic

species groups, suggesting that these species have only recently diverged. Incomplete lineage

sorting in this group is supported by the low divergence (~0.4%). Albert et al. (2004) originally

included G. carapo, G. ucamara, G. arapaima, and G. choco in the „G. carapo complex.‟

Lovejoy et al. (2010) confirmed the existence of this complex, but were not able to include G.

choco in their analysis due to a lack of tissue samples. In my analysis, G. choco was not included

in what I refer to as the „G. carapo complex,‟ but instead, groups with G. carapo (OR) + G.

ardilai. G. carapo (OR) was found to be on average 3.9% divergent from G. carapo of other

31

regions. This evidence suggests that the specimens of G. carapo from the Orinoco may represent

a distinct species. In the past, many specimens of Gymnotus were automatically considered to be

G. carapo based on the limited taxonomic work that had been done on Gymnotus (Albert et al.

1999). This practice resulted in specimens of diverse phenotypes being labelled G. carapo in

museum collections, which gave rise to the idea that this species has an extremely broad

geographic distribution. It is likely that many of these specimens represent distinct species.

These results further support the suggestion of Albert et al. (1999, 2004) that G. carapo is a

paraphyletic species and that its taxonomy and population genetics warrant further investigation.

In a similar vein, the specimens of G. coropinae from the Central Amazon and the Guyanas

exhibit an average divergence of 4.3%. These populations may be in the process of becoming

reproductively isolated and investigations into the morphology and population genetics of this

species could be interesting.

This study included several taxa that have been suggested to represent new species. Within the

G2 clade, G. n sp. FRIT was 11% divergent from G. cataniapo and 11.5% divergent from G.

pedanopterus, suggesting that it is in fact a distinct species. G. n. sp. XING was also found to be

relatively divergent (2.7%) from both G. n. sp. and G. pantanal, compared to the 1.3%

divergence between these two species. Additional evidence will be required to corroborate the

status of G. n. sp. as a distinct species. Divergence among individuals of G. n. sp. ITAP was

approximately 1%. It was very closely related to G. mamiraua with only 1.7% divergence

between them. These two species were not always found to be reciprocally monophyletic,

suggesting that G. n. sp. ITAP may represent a variation of G. mamiraua. Finally, G. n. sp.

CORU, G. n. sp. MAMA, G. n. sp. RS1, and G. sylvius were all observed to be very closely

related, forming a species complex that I refer to as the „G. sylvius complex‟ The analyses show

that these species do not always form species groups. G. n. sp. MAMA individuals exhibited on

average 0.1% intraspecific divergence; however, no individuals within this complex were more

than 0.36% divergent from each other. Additional evidence will be required to confirm the status

and relationships of this group.

2 Biogeography of the trans-Andean species

The trans-Andean species were found to be distributed in four independent lineages within three

separate species groups: the G. carapo group (G. choco and G. ardilai), the G. cylindricus group

32

(G. cylindricus, G. maculosus, and G. panamensis), and the G3 clade (G. henni). This result is

consistent with the prediction of Albert et al. (2004). Albert et al. (2004) suggested some

biogeographic scenarios for Gymnotus based on the relationships in their phylogeny. The

multiple trans-Andean lineages are inferred to have diverged as a result of vicariance due to the

rise of the Andes approximately 12 mya. Assuming that the trans-Andean lineages diverged

approximately 12 mya, much of the diversity in Gymnotus can be inferred to predate this time

period as there was already considerable lineage diversity within the genus.

If all trans-Andean lineages diverged as a result of Andean uplift, they should exhibit similar

amounts of diversification from their closest cis-Andean sister group (assuming relatively equal

rates of molecular evolution). In fact, when comparing uncorrected pairwise divergences, the

trans-Andean clades were found to have varying degrees of divergence from their cis-Andean

sister clades. G. maculosus, G. cylindricus, and G. panamensis are on average 11.4% divergent

from members of their cis-Andean sister clade, the G. carapo group. G. henni is on average

11.3% divergent from its cis-Andean sister species, G. tigre. In contrast, G. choco is on average

1.5% divergent from its cis-Andean sister taxon, G. carapo (OR) and G. ardilai was only found

to be between 0-0.2% diverged from cis-Andean G. carapo (OR). Thus, amounts of cis/trans

divergence are relatively similar for the G. cylindricus group and G. henni lineages, while much

less cis/trans divergence is evident for the G. choco and G. ardilai lineages. If G. henni and the

G. cylindricus group are inferred to have diverged as a result of the uplift of the Andes, then G.

choco and G. ardilai may have diverged more recently, via alternative biogeographic pathways.

This pattern could possibly be explained by stream capture events or human-facilitated dispersal.

The small amount of divergence between G. ardilai and G. carapo (OR), as well as the restricted

distribution of the former (Maldonado-Ocampo and Albert 2004), could indicate that G. ardilai

represents a human introduction of G. carapo (OR) into the trans-Andean region.

My results clarify the biogeography of the Central American Gymnotus species. While Albert et

al. (2004) proposed that two lineages of Gymnotus (a G. panamensis lineage and a G. cylindricus

+ G. maculosus lineage) were present in Central America; my analyses show that these lineages

represent a single monophyletic clade. The timing of the dispersal event of this lineage into

Central America is somewhat contentious. Myers (1966) proposed that due to the relatively

species-poor fauna of primary freshwater fish species in Central America, it was likely that those

groups, including Gymnotus, were relatively recent invaders, most probably making use of the

33

Panamanian landbridge approximately 3mya. Conversely, Bussing (1976, 1985) suggested that

some fishes, including Gymnotus, reached Central America much earlier, by the late Cretaceous

or Paleocene, approximately 65mya. At this time, an ancient landbridge between North and

South America is hypothesized to have existed, which would have allowed dispersal into Central

America (Briggs 1994). This hypothesis is partly based on the fact that Gymnotus is the only

gymnotiform that is distributed farther North than Costa Rica. Considering the long time spans

involved in freshwater dispersal across land-masses, it follows that Gymnotus could only have

achieved such a Northerly distribution over extremely long time periods.

The phylogeny of Albert et al. (2004) provided some support for the hypothesis that the G.

cylindricus group represents a relatively ancient invasion into Central America. The trans-

Andean lineages were distributed in multiple lineages, with the G. cylindricus group representing

the sister clade to all other Gymnotus species (Figure 5). If the nested trans-Andean lineages are

considered to have evolved as a result of Andean uplift approximately 12 mya, then the G.

cylindricus group can be inferred to have evolved much earlier, thus providing a much more

ancient timeframe for dispersal into Central America. More recently, Lovejoy et al. (2010)

proposed that the G. cylindricus group was not the sister lineage to all other Gymnotus species,

but occupies a relatively nested position within Gymnotus (Figure 6). My results agree with this

assessment, although updated divergence time estimates will further clarify the issue.

3 Electric Signal Evolution

3.1 A Complicated History

Five trans-Andean species were examined for their EOD. G. cylindricus and G. maculosus (from

Costa Rica) were both confirmed to be monophasic. G. henni (from Colombia) was also found to

be monophasic. G. ardilai (from Colombia) was found to be tetraphasic and G. panamensis

(from Panama) was found to be triphasic. All trans-Andean taxa, except for G. ardilai, seem to

show an evolutionary reduction in phase number; however, only three of the five examined

trans-Andean species exhibits a monophasic signal as was predicted by the Predator Avoidance

Hypothesis (Stoddard 1999, 2002a, 2002b). Both G. henni and the G. cylindricus + G. maculosus

clade evolved a monophasic signal from a triphasic ancestor.

34

The G. cylindricus group was found to be relatively well-nested in my phylogeny and was not

recovered as the sister group to all other species as suggested by Albert et al. (2004). This

position agrees with the phylogeny of Lovejoy et al. (2010), although it does change the

ancestral EOD state reconstruction in Gymnotus. In my analyses, the plesiomorphic condition in

Gymnotus is the 4+ multiphasic state, while the ancestral condition in Gymnotidae is inferred to

be monophasic (Figure 21 and 22). This suggests that the ancestor of Gymnotus evolved a

multiphasic signal which was subsequently reduced in phase number in other clades. A reduction

in phase number is inferred in the ancestor of the clade consisting of the G3 clade, the G.

cylindricus group, and the G. carapo group, which necessitates a reversal to the ancestral

multiphasic state in at least one clade within the G. carapo group. The optimization also suggests

two independent phase number reductions in cis-Andean clades within the G. carapo group. G.

omarorum is observed to have evolved a triphasic signal from a tetraphasic ancestor, while G.

obscurus evolved a biphasic signal from a triphasic ancestor. This analysis suggests a

complicated history of evolutionary reversals and reductions within this group. Below, I consider

why both cis- and trans-Andean species may evolve reduced phase number from a multiphasic

ancestor and why multiphasic signals are present in trans-Andean species.

These results were not consistent with the prediction of the Predator Avoidance Hypothesis that

all trans-Andean species should have monophasic signals; however, it may be that the

complexity of this system prevents simple patterns of character evolution from being observed.

The many selective pressures influencing electric signal evolution could preclude one selective

pressure from taking precedence above the others. The biology of this group is complex, so

perhaps the fact that the only examples of evolutionary reversal to monophasy are observed in

trans-Andean lineages does support the Predator Avoidance Hypothesis. It is possible that some

lineages are consistent with the Predator Avoidance Hypothesis, while some are subject to

stronger selective pressure from other forces. I have postulated several hypotheses to explain

these patterns.

First, my results may indicate that there are more significant levels of predation in the trans-

Andean region than previously thought. Predation pressure has not been explicitly studied in the

trans-Andean region, so the multiphasic signals of some trans-Andean species could represent

adaptations to avoid electroreceptive predators that are outside the key electroreceptive predator

groups that have been identified in South America. Electric eels are not known from this region,

35

and the diversity of large pimelodid catfish is reduced; however, there could be other

electroreceptive predators present in sufficient abundance to influence EOD evolution. Stoddard

(1999, 2002a, 2002b) has made a compelling case for the strength of predation pressure as an

evolutionary force shaping EOD evolution, so even weak predation pressure may represent a

selective force. As such, until predation pressure in the trans-Andean region is explicitly

quantified, I cannot rule this hypothesis out.

Conversely, if a reduction in phase number is considered to represent an increase in the amount

of low-frequency energy of the electric signal, my results could be reconciled with the Predator

Avoidance Hypothesis. Each trans-Andean clade except G. ardilai exhibits a reduction in EOD

phase number relative to its cis-Andean sister clade, which could represent a reduction of the

anti-predator adaptation of multiphasy in a region where predation pressure is reduced. The loss

of anti-predator behaviour takes time to occur, especially when some predators are removed but

others remain (Blumstein 2002, 2006, Blumstein and Daniel 2005). It is possible that while some

trans-Andean species retain the multiphasic state due to evolutionary persistence, the reduction

in phase number represents the first step towards a reversal to monophasy. This hypothesis

assumes that there is an adaptive benefit to having a low-frequency energy EOD, which is

considered below (Section 3.4).

3.2 G. ardilai may be a recent introduction

G. ardilai is the only trans-Andean species which does not exhibit a reduction in phase number

from its closest cis-Andean sister group. Above, I suggest that perhaps there is additional

predation pressure in certain trans-Andean regions that selects for a multiphasic signal.

Alternately, biogeographic evidence may provide some explanations. There is 0-0.2%

divergence between G. ardilai and G. carapo from the Orinoco. One of the diversification

scenarios suggests that G. ardilai may represent a relatively recent introduction of G. carapo into

the region. If this was the case, G. ardilai would not have been subject to the same selection

pressures as other trans-Andean species throughout its evolutionary history. If it diverged from

G. carapo only very recently, it is not surprising that it still retains the tetraphasic signal.

36

3.3 Mechanisms for a return to monophasy

In order for a reversal to a monophasic state to be possible, there must be physiological

mechanisms that allow for the production of a monophasic EOD from a multiphasic ancestral

state. The most likely mechanism in Gymnotus is a paedomorphic retention of the monophasic

larval EOD. The electric organ of all pulse-type gymnotiforms is derived from hypaxial muscle.

This electric organ remains the same throughout development in Gymnotus (Albert and

Crampton 2005b, Pereira et al. 2007). Monophasic discharges are produced by an action

potential that causes the innervated caudal face of the electrocyte to depolarize, inducing ion

flow through a sodium-potassium pump (Figure 10). This flow of ions causes depolarization of

the next electrocyte and so on, creating a current through the electric organ. In multiphasic

species, subsequent to the depolarization of the caudal face, the rostral face of the electrocyte

will depolarize and create a current in the opposite direction, which creates the next phase that is

opposite in polarity to the initial phase (Hopkins 1988, Stoddard 2002a). Further complexity is

generated by the use of accessory electric organs or special columns of electrocytes that are

unique in their development, sometimes with innervation on both faces (Hopkins 1988). In the

ontogeny of some tetraphasic species, it has been found that the larvae progress from one to four

phases sequentially as they grow. This pattern indicates that EOD phase number phenotype is an

indication of the complexity of the EOD that is achieved during ontogeny (Crampton and

Hopkins 2005, Pereira et al. 2007). Thus, the mechanism for a multiphasic species to evolve to a

monophasic species could simply be the retention of the larval monophasic signal and the loss of

accessory electric organs or the cues that cause additional depolarizations.

3.4 Adaptive significance of low-frequency energy

My results indicate a trend towards reduction in EOD phase number within Gymnotus. If a

reduction in phase number is considered to increase the amount of low-frequency energy in the

EOD, this pattern may suggest that there is an adaptive benefit to having a low-frequency energy

EOD that is most likely related to the two main functions of electrolocation and

electrocommunication.

First, if low-frequency energy has an adaptive benefit for navigation, monophasic species might

be expected to exhibit greater spatial acuity or alternately, command a greater signal space.

There have been no experiments done to date on the differences in spatial acuity of monophasic

37

vs. multiphasic species. Conversely, there has been much research generated on the active signal

space of electric fish. It has been found that EOD amplitude is related to the distance that the

signal is projected from the body (Hopkins 1999, Stoddard and Salazar 2011). All strongly

electric fish exhibit a monophasic discharge, prompting the suggestion that monophasic signals

maximize electrical current output (Stoddard 2002b). A Brachyhypopomus species from the

Amazon has a monophasic signal, which has much greater amplitude than its multiphasic

congeners. In fact, it has such a strong discharge that it has been known to confuse ichthyologists

who mistake the signal for that of an electric eel (Stoddard 1999, 2002a, 2002b). It has been

proposed to be a Batesian mimic of the electric eel; however, no studies have yet been done to

test whether it actually deceives electroreceptive predators.

Higher amplitudes, which are associated with monophasic signals, increase the active signal

space for an individual fish (Hopkins 1999, Stoddard and Salazar 2011). This could be important

both in navigation (increasing the distance at which they can forage, etc.) and communication

(increasing the distance at which they can detect potential mates, etc.). The ability to sense

electric signals within a greater signal space also allows a fish to command a greater territory.

Several Gymnotus species are known to be territorial (Albert and Crampton 2003, 2005a). A

larger territory could potentially help them to command a larger number of high quality

oviposition sites. Access to high quality oviposition sites could influence a female‟s choice in the

wild, suggesting that males which are more successful at acquiring and maintaining a high

quality territory would be more desirable by females (Curtis and Stoddard 2003). Having a

greater signal range could also allow these males the ability to sense when other males try to

trespass or when females enter their territory across greater distances. Male Brachyhypopomus

were found to increase their EOD amplitude much more rapidly when presented with males than

with females (Franchina et al. 2001), suggesting that EOD amplitude is important in dominance

signalling. Stronger signals can also increase stimulation of the electroreceptors essentially

providing a more potent signal to any potential receiver (Stoddard and Salazar 2011).

Greater EOD amplitude and duration may act to improve detection and thus increase the

probability of mating (Stoddard 2002a). Greater amplitude would increase the probability of

detection by increasing signal space, while an increase in duration and pulse rate increases the

chances that male EODs will overlap those of females. Temporal overlap in EOD signals

actually increases the sensitivity of a fish‟s electroreceptors to other EOD signals because the

38

electroreceptors are tuned specifically to detect distortion in the signal (Stoddard 2002a). These

spatially and temporally separated cues allow the fish to distinguish between self-generated and

conspecific-generated EOD signals, effectively separating the functions of electrolocation and

electrocommunication (Aguilera et al. 2001).

Monophasic signals could therefore be more useful for electrolocation and electrocommunication

across longer distances. Trans-Andean species tend to inhabit areas where they are the only

Gymnotus species in the region. Studies of population ecology are lacking; however, it may be

that these species occur in low population densities. In these situations it would be adaptive to

maximize the likelihood of detection by a conspecific by increasing the active signal space. In

territorial species, greater signal range also increases the size of the territory which may provide

access to higher quality foraging sites and oviposition sites to attract females.

Ancestral electric fish are suspected to have had monophasic signals that would have included

more low-frequency energy (Stoddard 2002a, 2002b). Due to the importance of electric courtship

in extant species, there is no reason to suspect that ancient females were not courted in the same

way. Ancient courtship signals would likely have included a large amount of low-frequency

energy. Modern females may have retained their original preference for low-frequency energy in

courtship signals; however, this has not been empirically proven in laboratory studies (Stoddard

2002a). It has been found that increased duration of the second phase in some Brachyhypopomus

species at peak mating times, actually increases the amount of low-frequency energy in the

signal, thus providing some anecdotal support that females may be attracted to low-frequency

energy (Stoddard 2006).

Studies of mating preferences have found that females tend to prefer males of larger body size

and longer body length (Stoddard 2002a, Curtis and Stoddard 2003). In the lab it has been shown

that body size is correlated with the amount of food provided, suggesting that body size in the

wild could be an indicator of foraging ability. EOD amplitude has been positively correlated with

both body size and body length (Franchina et al. 2001, Curtis and Stoddard 2003, Stoddard

2006). Since females are not likely to assess a male‟s body size visually, EOD amplitude could

be a cue that allows females to determine body size and therefore make assessments about a

male‟s quality. These results suggest that females may in fact exhibit a preference for EOD

signals with greater amplitude and duration (Stoddard 2002a, Curtis and Stoddard 2003). At this

39

stage, there is insufficient evidence to discern whether these EOD traits are attractive to females

or if they simply increase the probability of detection. Further lab tests are needed to clarify these

issues of female preference.

The introduction of additional low-frequency energy into the EOD of multiphasic species also

supports the idea that certain EOD signals are indicative of male quality. Males that input more

low-frequency energy in their signals are more susceptible to predation. Low-frequency energy

in courtship signals may have evolved as sexual handicaps, advertising male vigour despite their

conspicuousness to predators (Zahavi 1975). These low-frequency signals could thus be an

honest indicator of a male‟s survival ability. The more low-frequency energy in the courtship

signal, the more conspicuous that male is to predators. If a male can evade predators despite a

large amount of low-frequency energy in his signal, it could indicate that he has superior survival

ability. Male Brachyhypopomus actually emit a specialized signal called a “chirp” during

courtship, which resembles a rapid series of EODs with a significantly reduced or completely

lacking second phase. These signals exhibit significant low-frequency energy which would fall

in the range of the ampullary electroreceptors (Stoddard 2002a). Low-frequency energy has also

been found to be important in courtship in Eigenmannia and Apteronotus (Hopkins 1974,

Hagedorn and Heiligenberg 1985, Stoddard 2006). This low-frequency energy can have serious

consequences, as it has been found that approximately half of the sexually mature males of

Brachyhypopomus pinnicaudatus showed some signs of predation in the form of regenerating

tails, whereas almost all of the females‟ tails were intact. It has also been found that mature

males of Brachyhypopomus diazi actually disappear entirely from streams during breeding

season, while mature females exhibit no such mortality (Stoddard 2002a). It could also be

possible that low-frequency energy is not attractive to females per se, but acts as a cue to induce

spawning (Hagedorn and Heiligenberg 1985, Stoddard 2006). Hagedorn and Heiligenberg (1985)

found that spawning in female Eigenmannia virescens could be stimulated by the electrical

playback of low-frequency energy „chirps‟. In this case, monophasic species would not need to

modify their signal to elicit a courtship response, as their signal would always carry this low-

frequency energy. In a predation-free environment, species may revert to a simpler signal to

accomplish this purpose, rather than retain a more complex signal that requires costly

modifications.

40

Courtship has also been found to represent a significant energy expenditure for gymnotiforms.

Hopkins (1999) estimated the amount of energy expended by electrogenesis, based on current

and voltage of a mormyrid, to represent only about 1% of the daily basal metabolic rate. This

estimate would suggest that the energetic cost of the EOD is quite small; however, this estimate

only considered the standard EOD used for electrolocation. Males of some Brachyhypopomus

species will increase sexually dimorphic characters, such as amplitude, duration, and pulse rate at

peak courtship times and reduce them during non-mating hours in a form of courtship

communication (Hopkins 1999, Silva et al. 1999, Stoddard 2002a, Albert and Crampton 2005a,

Salazar and Stoddard 2008). This modification increases the amount of low-frequency energy in

the signal (Salazar and Stoddard 2008). Increasing amplitude and duration of the EOD increases

the total amount of energy that a fish would need to expend on the EOD (Hopkins 1999,

Stoddard 2002a). In addition, the EOD has been found to be more costly for males than females

of B. pinnicaudatus and to require more energy at night than during the day. The total energetic

expense of the EOD was 3.4% of the energy budget in females, but 11-22.5% of the energy

budget in males (Salazar and Stoddard 2008). This result suggests that the modification or

modulation of the EOD can represent a significant energetic cost to males, which is too costly to

maintain when the benefits are low. Reduction of EOD amplitude and duration during the day

reduced the energetic cost by 38-72% in males, but only 26% in females. Along with the

observation that males will reduce EOD amplitude and duration during social isolation, this

pattern suggests that males will reduce these costly modifications in order to save energy when

they are not likely to accrue any social benefit (Franchina et al. 2001, Salazar and Stoddard 2008,

2009). The base rate for electrogenesis seems to be very small, whereas modification for greater

amplitude or duration can be costly. Monophasic species have no need to modify their signal to

introduce low-frequency energy, so they may be able to allocate their energy budget to growth,

which would provide them with an advantage in territoriality and mating.

Reversal to a monophasic signal may therefore reduce the amount the amount of energy that a

fish is required to spend on costly modulations to introduce low-frequency energy into the signal.

It may also allow a fish to have increased signal amplitude, therefore allowing it to defend a

larger territory and obtain more matings. These selective pressures may become dominant in

areas where predation pressure and species recognition pressures are reduced. It has been

suggested that the increase in phase number allows for more variation in the EOD, increasing the

41

possibilities for species-specific signals; however, most trans-Andean species do not occur in

sympatry with any other Gymnotus species, and therefore are not under selective pressure for

EOD divergence. In these cases where a multiphasic signal is not required, a simple monophasic

signal may be more beneficial to other aspects of the biology of these fish.

3.5 Cis-Andean Reductions in Phase Number

The previous section delineated some hypotheses for why a monophasic signal may be beneficial

in a region where predation pressure is reduced; however, that does not offer an explanation for

why a reduction in phase number is adaptive in regions where predation pressure is high. In two

clades of cis-Andean species there have been independent reductions in phase number. These

individuals would be more susceptible to predation. The social benefits of the low-frequency

energy may outweigh the risk of predation in some species or populations, especially where the

ratio of males to females is high, or there are many different species of electric fish occurring in

sympatry.

These fish may have also evolved other adaptive behaviours that allow them to benefit from this

low-frequency energy while still evading predation. Circadian rhythms in courtship signals allow

males to maximize the benefits of the risky signal by increasing low-frequency energy at peak

mating times when they are most likely to benefit from it, and reducing this low-frequency

energy when the benefits would be low and the risks would be high (Stoddard et al. 2006, 2007,

Salazar and Stoddard 2008). Multiphasic species may also have evolved novel anti-predator

behaviours to assist in evading electroreceptive predation. Stoddard (2006) observed that when

Brachyhypopomus was injected with any solution, they exhibited a 40-60% drop in their EOD

amplitude, which returned to normal after two to five minutes. He suggested that the drop in

amplitude would make it more difficult for an electroreceptive predator to locate the fish as it

escaped and could therefore be considered a behavioural response to what the fish perceived as a

predation attempt. Behavioural responses to avoiding predation may be of critical importance in

these cis-Andean species.

3.6 Abiotic Selective Pressures

The adaptive benefits above all considered biotic selective pressures. There could also be an

influence of abiotic factors on signal evolution. Electric signals are subject to a wide array of

42

abiotic selective pressures, related both to efficient electrolocation and reliable transmission of

communication signals (Crampton and Albert 2006). The electric signal can be affected by

dissolved oxygen, conductivity, temperature, flow, and habitat (Crampton and Albert 2006).

Gymnotiforms, for example, are restricted to freshwater and cannot tolerate any degree of

brackish water because the increased conductivity prevents the proper functioning of the

electrosensory system (Albert 2000, Albert and Crampton 2005b). Adaptation to a particular

habitat has been suggested to constrain electric signal evolution, such as the adaptation of wave-

type species to fast flowing, well-oxygenated environments (Hopkins and Heiligenberg 1978,

Crampton 1998, Crampton and Albert 2006). Albert et al. (2004) found that all trans-Andean

species were restricted to non-floodplain terra firme streams that exhibited low conductivity.

This habitat type could provide an advantage for individuals exhibiting low-frequency energy

EODs. Alternately, the increase in low-frequency energy could constrain species with this signal

type to that particular habitat. Conversely, it has been suggested that physical factors may not

affect the evolution of phase number in Gymnotus at all (Crampton et al. 2011). More studies are

needed to examine the basic biology of these fish before the abiotic factors influencing trans-

Andean EOD evolution can be explained.

3.7 Corollaries

It is important to remember that most of the studies described above in Sections 3.4 and 3.5 have

been conducted using various species of Brachyhypopomus. While the genus also exhibits a

pulse-type signal, it does exhibit certain differences from Gymnotus. For example, Gymnotus

species are not known to modulate their signal during courtship (Crampton and Albert 2006).

Therefore, the evidence presented here would be greatly supplemented by additional studies with

Gymnotus. Recently, G. coatesi and G. curupira have been found to possess sexually dimorphic

signals. Males of these species and those of G. varzea, EODs were observed to contain an

increase of low-frequency energy compared to immature adults and females. Males of G.

arapaima also exhibited more low-frequency energy in their signals than females (Crampton et

al. 2011). More studies are needed to determine if these species exhibit electric courtship signals

as well.

The importance of territoriality in Gymnotus could also affect EOD evolution. Territoriality

could select for more stable EODs. It would be adaptive for a territorial species to be able to

43

recognize individuals, which would require individually distinct EOD components to be

consistent over a period of time. This would likely select against the extreme signal plasticity

that is seen in Brachyhypopomus (Stoddard 2006). Additionally, the tendency for males to have

an increased role in parental care in Gymnotus, could suggest the possibility for male choice as

well as female choice in Gymnotus (Crampton et al. 2011). A two-way mate choice system

would impose an additional set of constraints on the evolution of EOD signals.

Studies examining the differences in energy expenditure and navigational capabilities between

monophasic and multiphasic species are needed. Territoriality could be highly important in

influencing the evolution of EOD parameters, yet there are almost no studies looking at

territorial behaviour in electric fish. There have been some studies completed on dominance

signalling (Hopkins 1974, Hagedorn and Heiligenberg 1985, Stoddard 2002a, Salazar and

Stoddard 2008); however, they have mostly concerned wave-type fish. Studies of the distribution

of electroreceptive predators are also needed. To date, no studies have examined electroreceptive

predation behaviour in the wild, which would greatly inform studies of predator avoidance

adaptations.

It is also critical to remember that many experiments use EOD playbacks to elicit responses from

other electroreceptive fish. Unfortunately, EOD playbacks lack the temporal interactions and

spatial heterogeneity of natural EODs (Curtis and Stoddard 2003). Their simplicity may not

induce the full range of responses from receivers that may be observed in the wild. Experiments

using live fish would be more conclusive. More accurate models of the spatial and temporal

aspects of the EOD could also be used to create more realistic EOD recordings that would

improve the reliability of playback experiments.

4 Future Directions

Future phylogenetic studies on Gymnotus should include more species from the G1 and G2

clades in order to help clarify the position of G. pantherinus. Studies including faster-evolving

genes should also help to clarify relationships between the species complexes within the G.

carapo group. Future work should also endeavour to estimate the timing of divergence of the

trans-Andean lineages. Updated estimates of divergence times will not only assess my

hypothesis that the trans-Andean lineages diverged at different times, but will help to clarify the

time periods and therefore the potential cause for this divergence. These estimates may even help

44

to clarify the timing of dispersal of the G. cylindricus group into Central America. Ideally, future

work will also be able to include both tissues and EOD recordings from the two remaining

unknown trans-Andean species, G. esmeraldas and G. choco.

Further research on the evolution of the electric signal would be greatly enhanced by studies

using live specimens of a more diverse array of species, including Gymnotus and other

gymnotiform species. A greater number of species will help to clarify if most species are

evolving in the same way, or if they have different strategies for coping with the same selection

pressures.

In order to truly evaluate the Predator Avoidance Hypothesis, additional studies on the level of

electroreceptive predation pressure in both cis- and trans-Andean habitats are needed. Studying

electroreceptive predation behaviour in the wild will help to clarify its importance in

gymnotiform communities and will likely uncover interesting anti-predator behaviour in various

gymnotiform species.

Additional work is needed to test the hypothesis that there is an adaptive benefit to low-

frequency energy. Research on the amplitudes, foraging ability, and sensory acuity of

monophasic vs. multiphasic species would be beneficial. This information would be especially

useful when combined with studies of territorial behaviour in gymnotiforms. In addition, a large

amount of work needs to be done to test female preference and the importance of low-frequency

energy in courtship and spawning.

Studies on the energetic costs of different types of electric signals, particularly the difference

between monophasic vs. multiphasic and wave-type vs. pulse-type signals would suggest

whether or not energetic costs play a role in the evolution of electric signals. Finally, clarifying

the effect of abiotic parameters on the electric signal will not only help to clarify how these

factors affect electric signal evolution, but how abiotic factors influence the distribution of

electric fish.

5 Conclusions

This work represents the most complete phylogenetic analysis of Gymnotus to date, almost

doubling the number of species included in the previous molecular phylogeny. My analyses

45

included seven species that have not yet been formally identified, as well as six out of the seven

known trans-Andean species. The resultant phylogenetic hypotheses provide us with a better

understanding of the relationships between the major lineages in Gymnotus. The trans-Andean

species were found to be distributed in four distinct lineages, with the G. cylindricus group

forming a monophyletic clade, composed of all the Central American species. The relative

differences in the amount of divergence between the trans-Andean lineages and their cis-Andean

sister groups suggest that they did not all arise due to the uplift of the Andes as was previously

believed. The monophyletic grouping of all Central American species suggests a single dispersal

event into Central America. Based on these patterns, further divergence time estimates will help

to clarify biogeographic patterns of diversification. Further study is needed to understand the

timing of these events and to trace the patterns of dispersal and vicariance.

My work also provides new information on EOD waveforms for five trans-Andean species, as

well as an analysis of the evolution of phase number. My results suggest that the ancestral state

in Gymnotidae is a monophasic discharge, while the ancestral state in Gymnotus is a 4+

multiphasic state. Not all of the trans-Andean species were found to be monophasic as was

suggested by the Predator Avoidance Hypothesis; however, a general trend of reduction in phase

number was observed. This reduction in phase number could represent an adaptive increase in

low-frequency energy of the EOD signal. Low-frequency energy could be advantageous for

reasons of increased signal space, sexual selection, and energetic costs. Testing these hypotheses

should lay the groundwork for further studies on Gymnotus, such as the variation and evolution

of other aspects of EOD parameters, providing novel implications for species recognition and

mate choice.

46

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54

Table 1. List of specimens included in study.

Speciesa Specimen Voucher Locality

Brachyhypopomus brevirostris 2617 UF 116556 Rio Nanay, Peru

Brachyhypopomus diazi 305 UF 174334 Rio Las Marias, Venezuela

Brachyhypopomus diazi 2408 UF 174334 Rio Albergatón, Venezuela

Brachyhypopomus n. sp. PAL 2432 UF 148572 Rio Palenque, Ecuador

Electrophorus electricus 2026 MZUSP 103218 Lago Secretaria, Brazil

Electrophorus electricus 2619 UF 116585 Rio Nanay, Peru

Gymnotus aff. anguillaris 2091 AUM 36616 Rio Aponwao, Guyana

Gymnotus arapaima 2002 MZUSP 75179 Lago Mamirauá, Brazil

Gymnotus arapaima 2003 MZUSP 103219 Lago Mamirauá, Brazil

Gymnotus ardilai 8175 IAvHP 11511 Rio de Oro, Colombia

Gymnotus ardilai 8186 IAvHP 11510 Rio de Oro, Colombia

Gymnotus bahianus 7244 No Voucher Rio Almada, Brazil

Gymnotus bahianus 7245 No Voucher Rio Almada, Brazil

Gymnotus carapo (CA) 2004 MZUSP 76066 Lago Secretaria, Brazil

Gymnotus carapo (CA) 2030 MZUSP 76066 Lago Secretaria, Brazil

Gymnotus carapo (WA) 2006 UF 131129 Rio Amazonas, Peru

Gymnotus carapo (WA) 2007 UF 131129 Rio Amazonas, Peru

Gymnotus carapo (OR) 2040 UF 174335 Rio Guaratico, Venezuela

Gymnotus carapo (OR) 2041 UF 174335 Rio Guaratico, Venezuela

Gymnotus cataniapo 2062 UF 174330 Rio Atabapo, Venezuela

Gymnotus cataniapo 2063 UF 174332 Rio Cataniapo, Venezuela

Gymnotus chaviro 7357 33715 No Locality

Gymnotus chaviro 7358 33729 No Locality

Gymnotus choco 8209 IAvHP 10646 Rio Atrato, Colombia

Gymnotus coatesi 2042 MCP 34471 Lago Tefé, Brazil

Gymnotus coatesi 2043 MCP 34472 Rio Tefé, Brazil

Gymnotus coropinae (CA) 2010 MZUSP 75188 Lago Tefé, Brazil

Gymnotus coropinae (CA) 2025 MZUSP 60611 Lago Tefé, Brazil

Gymnotus coropinae (GU) 2035 ANSP 179126 Sauriwau River, Guyana

Gymnotus coropinae (GU) 2036 AUM 35848 Sauriwau River, Guyana

Gymnotus coropinae (GU) 2037 ANSP 179127 Mazaruni River, Guyana

Gymnotus coropinae (GU) 2038 ANSP 179127 Mazaruni River, Guyana

Gymnotus curupira 2009 MZUSP 75148 Lago Tefé, Brazil

Gymnotus curupira 2019 UF 122823 Rio Amazonas, Peru

Gymnotus curupira 2021 MZUSP 75146 Lago Tefé, Brazil

Gymnotus curupira 2024 UF 122821 Rio Amazonas, Peru

Gymnotus cylindricus 2092 ROM 84772 Rio Tortuguero, Costa Rica

Gymnotus cylindricus 2093 ROM 84772 Rio Tortuguero, Costa Rica

Gymnotus cylindricus 2094 ROM 84772 Rio Tortuguero, Costa Rica

Gymnotus henni 7276 IAvH-BT 11598 Rio Dagua, Colombia

Gymnotus henni 7277 IAvH-BT 11599 Rio Dagua, Colombia

Gymnotus henni 8189 IMCN 4521 Rio Dagua, Colombia

Gymnotus henni 8193 IMCN 4521 Rio Dagua, Colombia

Gymnotus henni 8207 IAvHP 11359 Colombia

Gymnotus henni 8230 STRI-01589 Rio San Juan, Colombia

Gymnotus henni 8231 STRI-01589 Rio San Juan, Colombia

Gymnotus javari 2020 UF 122824 Rio Amazonas, Peru

Gymnotus jonasi 2016 MZUSP 103220 Rio Solimões, Brazil

Gymnotus jonasi 2471 UF 131410 Rio Ucayali, Peru

Gymnotus maculosus 8126 ROM 89775 Rio Higueron, Costa Rica

55

Gymnotus maculosus 8137 ROM 89778 Rio Montenegro, Costa Rica

Gymnotus maculosus 8169 ROM 89784 Nicoya, Costa Rica

Gymnotus maculosus 8213 STRI-01587 Rio Nosara, Costa Rica

Gymnotus mamiraua 2012 MZUSP 103221 Rio Solimões, Brazil

Gymnotus mamiraua 2013 MCP 29805 Rio Solimões, Brazil

Gymnotus n. sp. 7104 No Voucher Rio Beni, Bolivia

Gymnotus n. sp. 7105 No Voucher Rio Beni, Bolivia

Gymnotus n. sp. CORU 2558 No Voucher Brazil

Gymnotus n. sp. FRIT 7109 No Voucher Tefe, Amazonas, Brazil

Gymnotus n. sp. ITAP 2559 No Voucher Brazil

Gymnotus n. sp. ITAP 7071 No Voucher Parana, Argentina

Gymnotus n. sp. ITAP 7072 No Voucher Parana, Argentina

Gymnotus n. sp. ITAP 7074 No Voucher Parana, Argentina

Gymnotus n. sp. ITAP 7075 No Voucher Parana, Argentina

Gymnotus n. sp. MAMA 7065 No Voucher Parana, Argentina

Gymnotus n. sp. MAMA 7066 No Voucher Parana, Argentina

Gymnotus n. sp. MAMA 7067 No Voucher Parana, Argentina

Gymnotus n. sp. RS1 7088 MNRJ 31520 Lagoa dos Tropeiros, Brazil

Gymnotus n. sp. XING 7305 MNRJ 33642 Xingú-Tapajós, Brazil

Gymnotus obscurus 2017 MZUSP 75155 Lago Mamirauá, Brazil

Gymnotus obscurus 2018 MZUSP 75157 Lago Mamirauá, Brazil

Gymnotus omarorum 7092 No Voucher Uruguay

Gymnotus omarorum 7093 No Voucher Uruguay

Gymnotus panamensis 8018 STRI-7726 Rio Cricamola, Panama

Gymnotus panamensis 8021 ROM 89753 Rio Cricamola, Panama

Gymnotus panamensis 8210 STRI-01579 Rio Cricamola, Panama

Gymnotus pantanal 7076 No Voucher Parana, Argentina

Gymnotus pantherinus 2039 No Voucher Rio Perequê-Açu, Brazil

Gymnotus pantherinus 2945 No Voucher Rio Vermelho, Brazil

Gymnotus pantherinus 7111 MZUSP 87564 Rio Vermelho, Brazil

Gymnotus pedanopterus 2058 UF 174328 Rio Atabapo, Venezuela

Gymnotus pedanopterus 2059 UF 174328 Rio Atabapo, Venezuela

Gymnotus stenoleucus 2060 UF 174329 Rio Atabapo, Venezuela

Gymnotus stenoleucus 2061 UF 174331 Rio Cataniapo, Venezuela

Gymnotus stenoleucus 2064 UF 174329 Rio Atabapo, Venezuela

Gymnotus sylvius

7239 No Voucher

Rio Ribeira de Iguape-Rio Juqueia-Rio São Lourenço,

Brazil

Gymnotus sylvius

7240 No Voucher

Rio Ribeira de Iguape-Rio Juqueia-Rio São Lourenço,

Brazil

Gymnotus tigre 7349 No Voucher Aquarium

Gymnotus tigre 7090 No Voucher Aquarium

Gymnotus ucamara 1927 UF 126184 Rio Ucayali, Peru

Gymnotus ucamara 1950 UF 126184 Rio Ucayali, Peru

Gymnotus varzea 2014 MZUSP 75163 Rio Solimões, Brazil

Gymnotus varzea 2015 MZUSP 75164 Rio Solimões, Brazil

Hypopomus artedi 2232 ANSP 179505 Mazaruni River, Guyana

Rhamphichthys rostratus 2632 UF 116575 Rio Amazonas, Peru

Sternopygus astrabes 2203 No Voucher Lago Tefé, Brazil

Sternopygus macrurus 2639 UF 117121 Rio Nanay, Peru

a Drainage abbreviations: OR, Orinoco; CA, Central Amazon; WA, Western Amazon; GU, Guyanas.

56

Table 2. List of primers used for amplification and sequencing of the cyt b, 16S, RAG2, and Zic1 genes.

Primer Name Primer Sequence (5’–3’) Source

GLUDG.L CGAAGCTTGACTTGAARAACCAYCGTTG Palumbi et al. 1991

CytbR (CB6THR-H) CTCCGATCTTCGGATTACAAG Palumbi et al. 1991

H15573 AATAGGAAGTATCATTCGGGTTTGATG Meyer 1993

16Sar CGCCTGTTTATCAAAAACAT Palumbi 1996

16Sbr CCGGTCTGAACTCAGATCACGT Palumbi 1996

RAG2F1 TTTGGRCARAAGGGCTGGCC Lovejoy and Collette 2001

RAG2R6 TGRTCCARGCAGAAGTACTTG Lovejoy and Collette 2001

RAG2GYF ACAGGCATCTTTGGKATTCG Lovejoy et al. 2010

RAG2GYR TCATCCTCCTCATCTTCCTC Lovejoy et al. 2010

Zic1_F9 GGACGCAGGACCGCARTAYC Li et al. 2007

Zic1_R967 CTGTGTGTGTCCTTTTGTGRATYTT Li et al. 2007

Zic1_intF TCCTCGAACGTGGTGAACAG This study

Zic1_intR TTCGGGTTAGTTAGTTGCTCCGG This study

57

Table 3. Summary of EOD recordings.

Species Specimen EOD Temperature

(°C)

Conductivity

(µScm-1

)

Drainage Basin

Gymnotus panamensis 8015 2010-02-18-15 26.8 54.9 Cricamola, Panama

Gymnotus panamensis 8016 2010-02-18-16 27.2 56.2 Cricamola, Panama

Gymnotus panamensis 8017 2010-02-18-17 27.2 56.7 Cricamola, Panama

Gymnotus panamensis 8018 2010-02-18-18 27 59.2 Cricamola, Panama

Gymnotus panamensis 8019 2010-02-18-19 27.1 56.5 Cricamola, Panama

Gymnotus panamensis 8021 2010-02-18-21 27.2 56.6 Cricamola, Panama

Gymnotus panamensis 8022 2010-02-18-22 27.2 56.5 Cricamola, Panama

Gymnotus panamensis 8023 2010-02-18-23 27.2 56.7 Cricamola, Panama

Gymnotus maculosus 8119 2010-04-20-119 26.8 49.5 Bebedero, Costa Rica

Gymnotus maculosus 8120 2010-04-20-120 27 49.1 Bebedero, Costa Rica

Gymnotus maculosus 8121 2010-04-20-121 26.8 51.2 Bebedero, Costa Rica

Gymnotus maculosus 8122 2010-04-20-122 26.8 51.1 Bebedero, Costa Rica

Gymnotus maculosus 8123 2010-04-20-123 26.8 51.1 Bebedero, Costa Rica

Gymnotus maculosus 8124 2010-04-20-124 26.8 51.3 Bebedero, Costa Rica

Gymnotus maculosus 8125 2010-04-20-125 26.9 48.8 Bebedero, Costa Rica

Gymnotus maculosus 8126 2010-04-20-126 26.9 49.3 Bebedero, Costa Rica

Gymnotus maculosus 8127 2010-04-20-127 26.8 49.8 Bebedero, Costa Rica

Gymnotus maculosus 8128 2010-04-20-128 26.9 49.1 Bebedero, Costa Rica

Gymnotus maculosus 8129 2010-04-20-129 26.8 51.3 Bebedero, Costa Rica

Gymnotus maculosus 8130 2010-04-20-130 26.8 51.3 Bebedero, Costa Rica

Gymnotus maculosus 8131 2010-04-20-131 26.8 49.3 Bebedero, Costa Rica

Gymnotus maculosus 8132 2010-04-20-132 26.8 51 Bebedero, Costa Rica

Gymnotus maculosus 8133 2010-04-20-133 27 47.7 Bebedero, Costa Rica

Gymnotus maculosus 8134 2010-04-20-134 26.8 50.5 Bebedero, Costa Rica

Gymnotus maculosus 8135 2010-04-20-135 26.8 49.6 Bebedero, Costa Rica

Gymnotus maculosus 8136 2010-04-20-136 26.8 49.3 Bebedero, Costa Rica

Gymnotus maculosus 8137 2010-04-22-137 27.2 59.8 Bebedero, Costa Rica

Gymnotus maculosus 8138 2010-04-22-138 27.2 59.7 Bebedero, Costa Rica

Gymnotus maculosus 8139 2010-04-22-139 27.2 59.6 Bebedero, Costa Rica

Gymnotus maculosus 8140 2010-04-22-140 27.2 59.9 Bebedero, Costa Rica

Gymnotus maculosus 8141 2010-04-22-141 27.2 60 Bebedero, Costa Rica

Gymnotus maculosus 8142 2010-04-22-142 27.2 59.9 Bebedero, Costa Rica

Gymnotus maculosus 8143 2010-04-22-143 27.1 59.7 Bebedero, Costa Rica

Gymnotus maculosus 8144 2010-04-22-144 27.2 59.6 Bebedero, Costa Rica

Gymnotus maculosus 8145 2010-04-22-145 27.2 59.6 Bebedero, Costa Rica

Gymnotus maculosus 8146 2010-04-22-146 27.1 59.8 Bebedero, Costa Rica

Gymnotus maculosus 8147 2010-04-22-147 27.2 60 Bebedero, Costa Rica

Gymnotus maculosus 8148 2010-04-22-148 27.2 59.5 Bebedero, Costa Rica

Gymnotus cylindricus 8149 2010-04-22-149 27.2 59.5 Lake Arenal, Costa Rica

Gymnotus cylindricus 8150 2010-04-22-150 26.9 59.7 Lake Arenal, Costa Rica

Gymnotus cylindricus 8151 2010-04-22-151 27.2 59.5 Lake Arenal, Costa Rica

Gymnotus cylindricus 8152 2010-04-22-152 27 59.6 Lake Arenal, Costa Rica

Gymnotus cylindricus 8153 2010-04-22-153 26.9 59.4 Lake Arenal, Costa Rica

Gymnotus cylindricus 8154 2010-04-22-154 27.2 59.5 Lake Arenal, Costa Rica

Gymnotus cylindricus 8155 2010-04-23-155 26.9 65.8 San Carlos, Costa Rica

Gymnotus cylindricus 8156 2010-04-23-156 26.9 65.7 San Carlos, Costa Rica

Gymnotus cylindricus 8157 2010-04-23-157 26.9 65.7 San Carlos, Costa Rica

Gymnotus cylindricus 8158 2010-04-23-158 26.8 65.9 San Carlos, Costa Rica

Gymnotus cylindricus 8159 2010-04-23-159 26.8 65.9 San Carlos, Costa Rica

Gymnotus cylindricus 8160 2010-04-24-160 26.9 65.8 Bebedero, Costa Rica

58

Gymnotus cylindricus 8161 2010-04-24-161 26.8 66.3 Bebedero, Costa Rica

Gymnotus cylindricus 8162 2010-04-24-162 26.9 66.1 Bebedero, Costa Rica

Gymnotus cylindricus 8163 2010-04-24-163 26.9 66.3 Bebedero, Costa Rica

Gymnotus cylindricus 8164 2010-04-24-164 26.9 66.7 Bebedero, Costa Rica

Gymnotus cylindricus 8165 2010-04-24-165 26.9 66.5 Bebedero, Costa Rica

Gymnotus cylindricus 8166 2010-04-24-166 26.8 66.9 Sarapiquí, Costa Rica

Gymnotus maculosus 8167 2010-04-25-167 26.8 68.6 Tempisque, Costa Rica

Gymnotus maculosus 8168 2010-04-25-168 26.8 68.6 Tempisque, Costa Rica

Gymnotus maculosus 8169 2010-04-25-169 26.8 68.6 Tempisque, Costa Rica

Gymnotus ardilai 8172 2010-06-14-172 26.7 126.6 Magdalena, Colombia

Gymnotus ardilai 8173 2010-06-14-173 26.2 122 Magdalena, Colombia

Gymnotus ardilai 8174 2010-06-14-174 26.2 153.4 Magdalena, Colombia

Gymnotus ardilai 8175 2010-06-15-175 26.8 122.9 Magdalena, Colombia

Gymnotus ardilai 8179 2010-06-16-179 27.9 113.4 Magdalena, Colombia

Gymnotus ardilai 8180 2010-06-16-180 28.2 113.3 Magdalena, Colombia

Gymnotus ardilai 8181 2010-06-16-181 26.7 112 Magdalena, Colombia

Gymnotus ardilai 8182 2010-06-17-182 27.2 153.2 Magdalena, Colombia

Gymnotus ardilai 8183 2010-06-17-183 27 152.8 Magdalena, Colombia

Gymnotus ardilai 8184 2010-06-17-184 27.2 129.8 Magdalena, Colombia

Gymnotus ardilai 8185 2010-06-17-185 27.1 130.1 Magdalena, Colombia

Gymnotus ardilai 8186 2010-06-17-186 27 153 Magdalena, Colombia

Gymnotus henni 8187 2010-06-23-187 27.2 52.9 Dagua, Colombia

Gymnotus henni 8188 2010-06-23-188 27.1 53.2 Dagua, Colombia

Gymnotus henni 8189 2010-06-23-189 27.1 53.1 Dagua, Colombia

Gymnotus henni 8191 2010-06-23-191 27.1 53.5 Dagua, Colombia

Gymnotus henni 8193 2010-06-23-193 27 53.6 Dagua, Colombia

59

Figure 1: Family level relationships of the order Gymnotiformes after Stoddard (2002a).

60

Figure 2: Geographic distribution of gymnotiform species with delineation of biogeographic regions. Modified from

Albert et al. (2004). Distribution in the trans-Andean region is shaded in red, and in the cis-Andean region in blue.

Abbreviations: EA, Amazon Basin east of Purus Arch and all tributaries below fall-line of Guyana Shield (2 985 000

km2). GU, Guyanas – Orinoco Basin, including island of Trinidad and Upper Negro drainages above fall line (1 843

000 km2). MA, Atlantic and Pacific slopes of Middle America from the Motagua to Tuyra Basins (393 000 km2).

NE, coastal drainages of northeast Brazil including Parnaíba, Piaui, São Francisco and Jequitinhonha Basins (1 357

000 km2). NW, Northwestern South America including the Magdalena and Maracaibo Basins, and the north slope of

Venezuela (471 000 km2). PA, Paraguay-Paraná Basin including Dulce-Salí and Salado Basins of Argentina (3 185

000 km2). PS, Pacific Slope of Colombia and Ecuador, from Baudó to Guayaquil Basins, including the Atrato

(Caribbean) Basin (200 000 km2). SE, coastal drainages of southeast Brazil and Uruguay from the Docé to Lagoa

Mirim Basins (628 000 km2). WA, Amazon Basin west of Purus Arch, below about 500 m elevation (3 556 000

km2).

61

Figure 3: Type-locality map for 35 described Gymnotus species modified from Albert et al. (2004).

62

Figure 4: Distribution map for trans-Andean Gymnotus species. Arrows indicate position of highly localized

distributions.

63

Figure 5: Morphological Hypothesis for Gymnotus after Albert et al. (2004). Trans-Andean Gymnotus species are

shown in red.

64

Figure 6: Molecular Hypothesis for Gymnotus after Lovejoy et al. (2010). Trans-Andean Gymnotus species are

shown in red.

65

Figure 7: Electrostatic field of Gymnotiformes after Stoddard (2002a). The blue circle represents a resistive object,

which diverts the electric field and the red circle represents a conductive object which concentrates the electric field.

The green portion on the fish represents the bilateral pair of electric organs that extend along the ventral portion of

the body.

66

Figure 8: Spectral sensitivity of the two types of electroreceptor cells in Gymnotiformes after Stoddard (2002a).

67

Figure 9: Pulse and Wave type signal discharges after Stoddard and Markham (2008). On the left, the waveforms are

represented in voltage over time. On the right are examples of power spectra for the corresponding signal type. Pulse

type discharges are characterized by one to six phases (i.e. deviations from the 0V baseline) of alternating polarity

punctuated by brief periods of silence. In contrast, wave type discharges are characterized by a pattern of one to four

phases recurring in a continuous cycle.

68

Figure 10: Voltage-time waveforms of monophasic and multiphasic signals modified from Stoddard (2002b).

69

Figure 11: Electric organ discharge production after Stoddard (2002a). Electrocytes are arranged within tubes placed

in series to sum the voltage and the tubes are stacked in parallel to sum the current. A) An action potential causes

depolarization of the caudal face of the electrocyte, which causes sodium ions to flow towards the anterior portion of

the electric organ. This results in the head-positive first phase of the EOD. B) This current triggers a second action

potential in the rostral face of the electrocyte, causing sodium ions to flow towards the posterior portion of the

electric signal. This results in the head-negative second phase of the EOD. C) When both faces depolarize in

sequence, a biphasic signal is the result. Triggering of accessory electric organs and specialized electrocytes is

responsible for the production of additional phases.

70

Figure 12: Voltage/time waveform of both the first phase and the full EOD of Brachyhypopomus pinnicaudatus and

their corresponding power spectrum plotted over the spectral sensitivity of ampullary electroreceptors. Modified

from Stoddard (2002b). All known electroreceptive predators possess ampullary electroreceptors that are maximally

sensitive around 30Hz. The full EOD waveform has much less energy in the range of the ampullary receptors.

Multiphasic signals should be relatively cryptic to electroreceptive predators.

71

Figure 13: Collecting localities in Panama and Costa Rica. Blue pins represent localities for G. cylindricus, yellow

pins represent localities for G. maculosus, and the green pin represents the locality for G. panamensis.

72

Figure 14: Collecting localities in Colombia. The red pin represents the locality for G. henni and the white pin

represents the localities for G. ardilai.

73

Figure 15: Strict consensus phylogeny of 422 most parsimonious trees showing Gymnotus relationships, based on

the combined analysis of cyt b, 16S, RAG2, and Zic1 genes (3807 characters, 5037 steps, CI=0.47, RI=0.85).

Numbers above nodes represent bootstrap values. Trans-Andean Gymnotus species are shown in red.

74

Figure 16: Maximum Likelihood phylogeny showing Gymnotus relationships, based on the combined analysis of cyt

b, 16S, RAG2, and Zic1 genes. Numbers above nodes represent bootstrap values. Trans-Andean Gymnotus species

are shown in red.

75

Figure 17: Bayesian phylogeny showing Gymnotus relationships, based on the combined analysis of cyt b, 16S,

RAG2, and Zic1 genes. Numbers above nodes represent posterior probabilities. Trans-Andean Gymnotus species are

shown in red.

76

Figure 18: Results of Maximum Parsimony analyses of individual mtDNA and RAG2 datasets. The strict consensus

tree of Zic1 was not sufficiently resolved and is not shown. Redundant individuals of the same species have been

pruned from the trees. Trans-Andean Gymnotus species are shown in red.

77

Figure 19: Results of Maximum Likelihood analyses of individual mtDNA, RAG2, and Zic1 datasets. Redundant

individuals of the same species have been pruned from the trees. Trans-Andean Gymnotus species are shown in red.

78

Figure 20: Electric organ discharges of five species of trans-Andean Gymnotus visualized as voltage over time

waveforms.

79

Figure 21: Maximum Likelihood Optimization of electric organ discharge (EOD) phase number of Gymnotus

species using the total evidence Maximum Likelihood consensus phylogeny. Ancestral state reconstructions are

represented as proportional likelihoods. Trans-Andean Gymnotus species are shown in red.

80

Figure 22: Parsimony Optimization of electric organ discharge (EOD) phase number of Gymnotus species using the

total evidence Maximum Likelihood consensus phylogeny. Ancestral states were assessed from eight equally

parsimonious reconstructions. Ancestral states are shown above the equivocal nodes in proportion to their

appearance in the eight trees. Trans-Andean Gymnotus species are shown in red. Note that this tree shows the most

parsimonious character state reconstruction for the unknown EODs of G. n. sp. XING, G. bahianus, G. choco, G. n.

sp. RSI.

81

Appendices

Appendix A: 16S Alignment.

FORMAT DATATYPE = DNA INTERLEAVE GAP = - MISSING = ?;

MATRIX

Brachyhypopomus_brevirostris_2617 -------------------------------------GAGGTCCCGCCTGCCCAGTGAC-

Brachyhypopomus_diazi_2408 -----------------------------------AGGAGGTCCCGCCTGCCCAGTGAC-

Brachyhypopomus_diazi_305 ---------------------------------------------------CCAGTGAC-

Brachyhypopomus_n._sp._PAL_2432 -------------------------------------GAGGTCCCGCCTGCCCAGTGAC-

Electrophorus_electricus_2026 --------------CTTCTGT---AACCTATATATAGGAGGTCCTGCCTGCCCAGTGAA-

Electrophorus_electricus_2619 ---AAAAACATCGCCTTCTGT---AACCTATATATAGGAGGTCCTGCCTGCCCAGTGAA-

Gymnotus_aff._anguillaris_2091 ----------------------------AGTACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_arapaima_2002 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_arapaima_2003 ------------------------------------------------------------

Gymnotus_ardilai_8175 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_ardilai_8186 --CAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_bahianus_7244 --CAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_bahianus_7245 --CAAAAACATCGCCTCTCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_carapo_(CA)_2004 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_carapo_(CA)_2030 ------------------------AATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_carapo_(OR)_2040 -----------------------------ATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_carapo_(OR)_2041 -----------------------------ATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_carapo_(WA)_2006 ------------------------AATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_carapo_(WA)_2007 ------------------------AATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_cataniapo_2062 ------------------------AACACACACATAGGAGGTCCTGCCTGCCCGGTGAC-

Gymnotus_cataniapo_2063 ----AAAACATCGCCTCCCGC--AAACACACACATAGGAGGTCCTGCCTGCCCGGTGAC-

Gymnotus_chaviro_7357 ----AAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_chaviro_7358 ---------------------------------------------------CCAGTGAC-

Gymnotus_choco_8209 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_coatesi_2042 ------AACATCGCCTCTTGC--AAATTAATACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_coatesi_2043 ---------------TCTTGC--AAATTAATACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_coropinae_(CA)_2010 -------------------------ATTAATACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_coropinae_(CA)_2025 ---------------TCTTGC--AAATTAATACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_coropinae_(GU)_2035 -----------------TTGC--AAGTTA-CACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_coropinae_(GU)_2036 -------------------------AGTTACACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_coropinae_(GU)_2037 -------------------------AGTTACACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_coropinae_(GU)_2038 -----------------------------ACACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_curupira_2009 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_curupira_2019 ------------------------------------GGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_curupira_2021 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_curupira_2024 ----AAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_cylindricus_2092 ------------------------------------GGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_cylindricus_2093 ------------------------------------GGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_cylindricus_2094 ------------------------------------GGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_henni_7276 --CAAAAACATCGCCTCCCGA--AAATCAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_henni_7277 --CAAAAACATCGCCTCCCGA--AAATCAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_henni_8189 ---AAAAACATCGCCTCCCGA--AAATCAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_henni_8193 ---AAAAACATCGCCTCCCGA--AAATCAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_henni_8207 ---AAAAACATCGCCTCCCGC--AAATCAATACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_henni_8230 --CAAAAACATCGCCTCCCGA--AAATCAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_henni_8231 --CAAAAACATCGCCTCCCGA--AAATCAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_javari_2020 ---------------------------------------GGTCCTGCCTGCCCGGTGACG

Gymnotus_jonasi_2016 -----------------------------ACACAT-AGAGGTCCTGCCTGCCCAGTGACA

Gymnotus_jonasi_2471 ---------------------------------------GGTCCTGCCTGCCCAGTGACA

Gymnotus_maculosus_8126 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_maculosus_8137 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_maculosus_8169 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_maculosus_8213 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_mamiraua_2012 ------------------------AA-TAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_mamiraua_2013 ATCA-AAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._7104 --CAAAAACATCGCCTCCCGC--AAATTAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._7105 --CAAAAACATCGCCTCCCGC--AAATTAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._CORU_2558 -TCAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._FRIT_7109 ---AAAAACATCGCCTCCCGC--AAACTAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._ITAP_2559 -TCAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._ITAP_7071 ------------------------------------GGAGGTCCTGCCTGCCCAGTGAC-

82

Gymnotus_n._sp._ITAP_7072 --CAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._ITAP_7074 --CAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._ITAP_7075 --------------------C--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._MAMA_7065 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._MAMA_7066 --CAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._MAMA_7067 ------------------------------------------CCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._RS1_7088 -TCAAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_n._sp._XING_7305 ---AAAAACATCGCCTCCCGC---AATTAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_obscurus_2017 ATCAAAAACATCGCCTCCCGC--AAATCAATGTATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_obscurus_2018 ------------------------------------GGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_omarorum_7092 ---AAAAACATCGCCTCCCGC--AAATCAATACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_omarorum_7093 ------------------------------------------CCTGCCTGCCCAGTGAC-

Gymnotus_panamensis_8018 --CAAAAACATCGCCTCCCGC--AAATTAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_panamensis_8021 ---AAAAACATCGCCTCCCGC--AAATTAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_panamensis_8210 ---AAAAACATCGCCTCCCGC--AAATTAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_pantanal_7076 -TCAAAAACATCGCCTCCCGC--AAATTAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_pantherinus_2039 ----------------------------AACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_pantherinus_2945 ---AAAAACATCGCCTCCTGC--AAA-TAACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_pantherinus_7111 --CAAAAACATCGCCTCCTGC--AAAT-AACACATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_pedanopterus_2058 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_pedanopterus_2059 -------------------------------------GAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_stenoleucus_2060 ------AACATCGCCTCTTGC--AAATTAACACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_stenoleucus_2061 --------------CTCTTG----AATTAACACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_stenoleucus_2064 ------------------------AATTAACACATAAGAGGTCCTGCCTGCCCGGTGACG

Gymnotus_sylvius_7239 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_sylvius_7240 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_tigre_7090 --CAAAAACATCGCCTCCCGC--AAACCAGTATATGGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_tigre_7349 -TCAAAAACATCGCCTCCCGC--AAACCAGTATATGGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_ucamara_1927 ---AAAAACATCGCCTCCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_ucamara_1950 ----------------CCCGC--AAATCAATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_varzea_2014 ----------------------------AATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Gymnotus_varzea_2015 -----------------------------ATATATAGGAGGTCCTGCCTGCCCAGTGAC-

Hypopomus_artedi_2232 -------------------------------------GAGGTCCCGCCTGCCCAGTGAC-

Rhamphichthys_rostratus_2632 -------------------------------------GAGGTCCTGCCTGCCCGGTGACT

Sternopygus_astrabes_2203 ---AAAAACATCGCCTCCCGCAAAACTCAATGTATAGGAGGTCCTGCCTGCCCAGTGACT

Sternopygus_macrurus_2639 -----------------------AAATCAATGTATAGGAGGTCCTGCCTGCCCAGTGACT

Brachyhypopomus_brevirostris_2617 CACTGTTT-AACGGCCGCGGTATTTTAACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Brachyhypopomus_diazi_2408 CACTGTTT-AACGGCCGCGGTATTTTAACCGTGCAAAGGTAGCGCAATCACTTGTCCTTT

Brachyhypopomus_diazi_305 CACTGTTT-AACGGCCGCGGTATTTTAACCGTGCAAAGGTAGCGCAATCACTTGTCCTTT

Brachyhypopomus_n._sp._PAL_2432 CCCTGTTC-AACGGCCGCGGTATTTTAACCGTGCAAAGGTAGCGCAATCACTTGTCCTTT

Electrophorus_electricus_2026 ---TATTA-AACGGCCGCGGTATTTTGACCGTGCAAAGGTAGCGCAATCACTTGCCCCTT

Electrophorus_electricus_2619 ---TATTA-AACGGCCGCGGTATTTTGACCGTGCAAAGGTAGCGCAATCACTTGCCCCTT

Gymnotus_aff._anguillaris_2091 AACAATTTTAACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_arapaima_2002 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_arapaima_2003 ----------------------TCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_ardilai_8175 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_ardilai_8186 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_bahianus_7244 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_bahianus_7245 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_carapo_(CA)_2004 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_carapo_(CA)_2030 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_carapo_(OR)_2040 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_carapo_(OR)_2041 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_carapo_(WA)_2006 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_carapo_(WA)_2007 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_cataniapo_2062 AACAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_cataniapo_2063 AACAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_chaviro_7357 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_chaviro_7358 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_choco_8209 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_coatesi_2042 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_coatesi_2043 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_coropinae_(CA)_2010 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_coropinae_(CA)_2025 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_coropinae_(GU)_2035 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_coropinae_(GU)_2036 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_coropinae_(GU)_2037 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_coropinae_(GU)_2038 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_curupira_2009 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_curupira_2019 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_curupira_2021 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_curupira_2024 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

83

Gymnotus_cylindricus_2092 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_cylindricus_2093 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_cylindricus_2094 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_henni_7276 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_henni_7277 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_henni_8189 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_henni_8193 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_henni_8207 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_henni_8230 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_henni_8231 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_javari_2020 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_jonasi_2016 AATAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_jonasi_2471 AATAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_maculosus_8126 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_maculosus_8137 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_maculosus_8169 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_maculosus_8213 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_mamiraua_2012 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_mamiraua_2013 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._7104 AACAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._7105 AACAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._CORU_2558 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._FRIT_7109 AACTGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._ITAP_2559 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._ITAP_7071 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._ITAP_7072 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._ITAP_7074 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._ITAP_7075 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._MAMA_7065 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._MAMA_7066 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._MAMA_7067 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._RS1_7088 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_n._sp._XING_7305 AACAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_obscurus_2017 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_obscurus_2018 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_omarorum_7092 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_omarorum_7093 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_panamensis_8018 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_panamensis_8021 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_panamensis_8210 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_pantanal_7076 AACAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_pantherinus_2039 AACAATTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_pantherinus_2945 AACAATTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_pantherinus_7111 AACAATTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_pedanopterus_2058 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_pedanopterus_2059 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_stenoleucus_2060 AATAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_stenoleucus_2061 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_stenoleucus_2064 AACAGTTC-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTCTT

Gymnotus_sylvius_7239 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_sylvius_7240 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_tigre_7090 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_tigre_7349 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_ucamara_1927 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_ucamara_1950 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_varzea_2014 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Gymnotus_varzea_2015 AATAGTTT-AACGGCCGCGGTATCCTGACCGTGCGAAGGTAGCGCAATCACTTGTCTTTT

Hypopomus_artedi_2232 CACTGTTT-AACGGCCGCGGTATTTTAACCGTGCAAAGGTAGCGCAATCACTTGTCCTTT

Rhamphichthys_rostratus_2632 ATTAGTTT-AACGGCCGCGGTATTTTGACCGTGCAAAGGTAGCGCAATCACTTGTCCTTT

Sternopygus_astrabes_2203 GTCAGTTT-AACGGCCGCGGTATTTTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Sternopygus_macrurus_2639 ATTAGTTT-AACGGCCGCGGTATTTTGACCGTGCAAAGGTAGCGCAATCACTTGTCTTTT

Brachyhypopomus_brevirostris_2617 AAATGAAGACCTGTATGAATGGCATAACGAGGGCTCTACTGTCTCCCCTTTCAAGTCAGT

Brachyhypopomus_diazi_2408 AAATGAAGACCTGTATGAATGGCATAACGAGGGCTTTACTGTCTCCCCTTTCAAGTCAGT

Brachyhypopomus_diazi_305 AAATGAAGACCTGTATGAATGGCATAACGAGGGCTTTACTGTCTCCCCTTTCAAGTCAGT

Brachyhypopomus_n._sp._PAL_2432 AAATGAAGACCTGTATGAATGGCATAACGAGGGCTTTACTGTCTCCCCTTTCAAGTCAGT

Electrophorus_electricus_2026 AATTAGGGGCCTGTATGAATGGCTAGACGAAGGCCCAACTGTCTCCCTTTTCAAATCAGT

Electrophorus_electricus_2619 AATTAGGGGCCTGTATGAATGGCTAGACGAAGGCCCAACTGTCTCCCTTTTCAAATCAGT

Gymnotus_aff._anguillaris_2091 AAATAAAGACCCGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_arapaima_2002 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_arapaima_2003 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_ardilai_8175 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_ardilai_8186 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

84

Gymnotus_bahianus_7244 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_bahianus_7245 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_carapo_(CA)_2004 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_carapo_(CA)_2030 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_carapo_(OR)_2040 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_carapo_(OR)_2041 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_carapo_(WA)_2006 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_carapo_(WA)_2007 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_cataniapo_2062 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_cataniapo_2063 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_chaviro_7357 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_chaviro_7358 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_choco_8209 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_coatesi_2042 AAATAGGGACCTGTATGAATGGCAAAACGAAGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_coatesi_2043 AAATAGGGACCTGTATGAATGGCAAAACGAAGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_coropinae_(CA)_2010 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_coropinae_(CA)_2025 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_coropinae_(GU)_2035 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_coropinae_(GU)_2036 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_coropinae_(GU)_2037 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_coropinae_(GU)_2038 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_curupira_2009 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_curupira_2019 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_curupira_2021 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_curupira_2024 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_cylindricus_2092 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_cylindricus_2093 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_cylindricus_2094 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_henni_7276 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACTAGTCAAT

Gymnotus_henni_7277 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACTAGTCAAT

Gymnotus_henni_8189 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACTAGTCAAT

Gymnotus_henni_8193 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACTAGTCAAT

Gymnotus_henni_8207 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACTAGTCAAT

Gymnotus_henni_8230 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACTAGTCAAT

Gymnotus_henni_8231 AAATAAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACTAGTCAAT

Gymnotus_javari_2020 AAATAGGGACCTGTATGAATGGCAAAACGAAGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_jonasi_2016 AAATAGAGACCTGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_jonasi_2471 AAATAGAGACCTGTATGAATGGCAAAACGAGGGCTTAGCTGTCTCCCTTTACAAGTCAGT

Gymnotus_maculosus_8126 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_maculosus_8137 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_maculosus_8169 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_maculosus_8213 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_mamiraua_2012 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_mamiraua_2013 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._7104 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._7105 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._CORU_2558 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._FRIT_7109 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._ITAP_2559 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._ITAP_7071 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._ITAP_7072 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._ITAP_7074 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._ITAP_7075 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._MAMA_7065 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._MAMA_7066 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._MAMA_7067 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._RS1_7088 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_n._sp._XING_7305 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_obscurus_2017 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_obscurus_2018 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_omarorum_7092 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_omarorum_7093 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_panamensis_8018 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_panamensis_8021 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_panamensis_8210 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAAT

Gymnotus_pantanal_7076 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_pantherinus_2039 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTTCAAGTCAGT

Gymnotus_pantherinus_2945 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTTCAAGTCAGT

Gymnotus_pantherinus_7111 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTTCAAGTCAGT

Gymnotus_pedanopterus_2058 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

ymnotus_pedanopterus_2059 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_stenoleucus_2060 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_stenoleucus_2061 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

85

Gymnotus_stenoleucus_2064 AAATAGGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_sylvius_7239 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_sylvius_7240 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_tigre_7090 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACCAGTCAGT

Gymnotus_tigre_7349 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACCAGTCAGT

Gymnotus_ucamara_1927 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_ucamara_1950 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_varzea_2014 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Gymnotus_varzea_2015 AAATAAGGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCTTTACAAGTCAGT

Hypopomus_artedi_2232 AAATGAGGACCTGTATGAATGGCACCACGAGGGCTTTACTGTCTCCCTTTTCAAGTCAGT

Rhamphichthys_rostratus_2632 AAATGAGGACCTGTATGAAAGGCAAAACGAGGGCTTTACTGTCTCCCCATTCAAGTCAGT

Sternopygus_astrabes_2203 AAATGAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCCTCTCCAGTCAGT

Sternopygus_macrurus_2639 AAATGAAGACCTGTATGAATGGCAAAACGAGGGCTTAACTGTCTCCCCTTTCCAGTCAAT

Brachyhypopomus_brevirostris_2617 GAAATTGATCTGCCCGTGCAGAAGCGGACATAAAAATATAAGACGAGAAGACCCTTTGGA

Brachyhypopomus_diazi_2408 GAAATTGATCTGCCCGTGCAGAAGCGGACATAAAAATATAAGACGAGAAGACCCTTTGGA

Brachyhypopomus_diazi_305 GAAATTGATCTGCCCGTGCAGAAGCGGACATAAAAATATAAGACGAGAAGACCCTTTGGA

Brachyhypopomus_n._sp._PAL_2432 GAAATTGATCTGCCCGTGCAGAAGCGAGCATAAGAATATAAGACGAGAAGACCCTTTGGA

Electrophorus_electricus_2026 TAAATTGATCTACCCGTGCAGAAGCAGGTATTCACCTACAAGACGAGAAGACCCTTTGGA

Electrophorus_electricus_2619 TAAATTGATCTACCCGTGCAGAAGCAGGTATTCACCTACAAGACGAGAAGACCCTTTGGA

Gymnotus_aff._anguillaris_2091 GAAATTGACCTGTCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_arapaima_2002 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_arapaima_2003 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_ardilai_8175 GAAATTGACCTGCCCGTGCAGATGCGGACATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_ardilai_8186 GAAATTGACCTGCCCGTGCAGATGCGGACATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_bahianus_7244 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_bahianus_7245 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_carapo_(CA)_2004 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_carapo_(CA)_2030 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_carapo_(OR)_2040 GAAATTGACCTGCCCGTGCAGATGCGGACATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_carapo_(OR)_2041 GAAATTGACCTGCCCGTGCAGATGCGGACATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_carapo_(WA)_2006 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_carapo_(WA)_2007 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_cataniapo_2062 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_cataniapo_2063 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_chaviro_7357 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_chaviro_7358 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_choco_8209 GAAATTGACCTGCCCGTGCAGATGCGGACATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_coatesi_2042 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_coatesi_2043 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_coropinae_(CA)_2010 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATATTACAAGACGAGAAGACCCTTTGGA

Gymnotus_coropinae_(CA)_2025 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATATTACAAGACGAGAAGACCCTTTGGA

Gymnotus_coropinae_(GU)_2035 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATAGTACAAGACGAGAAGACCCTTTGGA

Gymnotus_coropinae_(GU)_2036 GAAATTGATCTGCCCGTGCAGATGCGGGCACAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_coropinae_(GU)_2037 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATAGTACAAGACGAGAAGACCCTTTGGA

Gymnotus_coropinae_(GU)_2038 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATAGTACAAGACGAGAAGACCCTTTGGA

Gymnotus_curupira_2009 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_curupira_2019 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_curupira_2021 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_curupira_2024 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_cylindricus_2092 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_cylindricus_2093 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_cylindricus_2094 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_henni_7276 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_henni_7277 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_henni_8189 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_henni_8193 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_henni_8207 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_henni_8230 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_henni_8231 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_javari_2020 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATGATACAAGACGAGAAGACCCTTTGGA

Gymnotus_jonasi_2016 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_jonasi_2471 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_maculosus_8126 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_maculosus_8137 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_maculosus_8169 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_maculosus_8213 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_mamiraua_2012 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_mamiraua_2013 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._7104 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._7105 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._CORU_2558 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._FRIT_7109 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

86

Gymnotus_n._sp._ITAP_2559 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._ITAP_7071 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._ITAP_7072 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._ITAP_7074 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._ITAP_7075 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._MAMA_7065 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._MAMA_7066 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._MAMA_7067 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGAC-AGAAGACCCTTTGGA

Gymnotus_n._sp._RS1_7088 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_n._sp._XING_7305 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATATTACAAGACGAGAAGACCCTTTGGA

Gymnotus_obscurus_2017 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_obscurus_2018 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_omarorum_7092 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_omarorum_7093 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_panamensis_8018 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_panamensis_8021 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_panamensis_8210 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_pantanal_7076 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_pantherinus_2039 GAAATTGACCTGCCCGTGCAGATGCGGACATGATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_pantherinus_2945 GAAATTGACCTGCCCGTGCAGATGCGGACATGATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_pantherinus_7111 GAAATTGACCTGCCCGTGCAGATGCGGACATGATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_pedanopterus_2058 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTATGGA

Gymnotus_pedanopterus_2059 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTATGGA

Gymnotus_stenoleucus_2060 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATTATACAAGACGAGAAGACCCTTTGGA

Gymnotus_stenoleucus_2061 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATTATACAAGACGAGAAGACCCTTTGGA

Gymnotus_stenoleucus_2064 GAAATTGACCTGCCCGTGCAGATGCGGGCACAATTATACAAGACGAGAAGACCCTTTGGA

Gymnotus_sylvius_7239 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_sylvius_7240 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_tigre_7090 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_tigre_7349 GAAATTGACCTGCCCGTGCAGATGCGGGCATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_ucamara_1927 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_ucamara_1950 GAAATTGACCTGCCCGTGCAGATGCGGGCATAACAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_varzea_2014 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Gymnotus_varzea_2015 GAAATTGACCTGCCCGTGCAGATGCGGACATAATAATACAAGACGAGAAGACCCTTTGGA

Hypopomus_artedi_2232 GAAATTGATCTGCCCGTGCAGAAGCGGACATAAAAATATAAGACGAGAAGACCCTTTGGA

Rhamphichthys_rostratus_2632 GAAATTGATCTGCCCGTGCAGAAGCGGACATAATTATACAAGACGAGAAGACCCTTTGGA

Sternopygus_astrabes_2203 GAAATTGATCTACCCGTGCAGAAGCGGGTATAAAGATACAAGACGAGAAGACCCTTTGGA

Sternopygus_macrurus_2639 GAAATTGATCTACCCGTGCAGAAGCGGGTATAAAAATACAAGACGAGAAGACCCTTTGGA

Brachyhypopomus_brevirostris_2617 GCTTAAGATAT-AAGCCAACTATGTTAATAGGCTCACCAACTCAGCCCTAAACTCAATAG

Brachyhypopomus_diazi_2408 GCTTAAGATAT-AAGCCAACTACGTTAATAAGCCCCCTAACCCAGCTTTAAACTCAATAG

Brachyhypopomus_diazi_305 GCTTAAGATAT-AAGCCAACTACGTTAATAAGCCCCC-AACCCAGCTTTAAACTCAATAG

Brachyhypopomus_n._sp._PAL_2432 GCTTAAGATAT-AAGCCAACTACGTTAATAAACTTATTAAACAAGTCTTAAACTCAATAG

Electrophorus_electricus_2026 GCTTAAGATTA-AAGTCATCTACATTAATAAGTTAC----ACTTAAACCAAGTA----AA

Electrophorus_electricus_2619 GCTTAAGATTA-AAGTCATCTACATTAATAAGTTAC----ACTTAAACCAAGTA----AA

Gymnotus_aff._anguillaris_2091 GCTTAAGACAC-AAGTCAACTATGTTAATAATTTGTCAC-ACCTAAATTAAACT--ATAA

Gymnotus_arapaima_2002 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_arapaima_2003 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_ardilai_8175 GCTTAAGACAT-AAGCCAACTATGTTAATAACCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_ardilai_8186 GCTTAAGACAT-AAGCCAACTATGTTAATAACCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_bahianus_7244 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_bahianus_7245 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_carapo_(CA)_2004 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_carapo_(CA)_2030 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_carapo_(OR)_2040 GCTTAAGACAT-AAGCCAACTATGTTAATAACCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_carapo_(OR)_2041 GCTTAAGACAT-AAGCCAACTATGTTAATAACCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_carapo_(WA)_2006 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_carapo_(WA)_2007 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_cataniapo_2062 GCTTAAGACAC-AAACCAACTATGTTAATAATTTACCCC-ACCTAAATTAAACT--GTAA

Gymnotus_cataniapo_2063 GCTTAAGACAC-AAACCAACTATGTTAATAATTTACCCC-ACCTAAATTAAACT--GTAA

Gymnotus_chaviro_7357 GCTTAAGACAT-AAGCCAACTATGTTAATAATCTACC--TATCTAGATTAAACT--GTAA

Gymnotus_chaviro_7358 GCTTAAGACAT-AAGCCAACTATGTTAATAATCTACC--TATCTAGATTAAACT--GTAA

Gymnotus_choco_8209 GCTTAAGACAT-AAGCCAACTATGTTAATAACCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_coatesi_2042 GCTTAAAACAC-AAGCCACCCACGTCAATAGCCTTAAT-AACCTAGACTAAACA--ACAG

Gymnotus_coatesi_2043 GCTTAAAACAC-AAGCCACCCACGTCAATAGCCTTAAT-AACCTAGACTAAACA--ACAG

Gymnotus_coropinae_(CA)_2010 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAGT-AACCCAGACTAAACA--ACAA

Gymnotus_coropinae_(CA)_2025 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAGT-AACCCAGACTAAACA--ACAA

Gymnotus_coropinae_(GU)_2035 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAGT-AACCCAGACTAAACA--ACAA

Gymnotus_coropinae_(GU)_2036 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAGT-AACCCAGACTAAACA--ACAA

Gymnotus_coropinae_(GU)_2037 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAGT-AACCCAGACTAAACA--ACAA

Gymnotus_coropinae_(GU)_2038 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAGT-AACCCAGACTAAACA--ACAA

Gymnotus_curupira_2009 GCTTAAGACAT-AAGTCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

Gymnotus_curupira_2019 GCTTAAGACAT-AAGTCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

87

Gymnotus_curupira_2021 GCTTAAGACAT-AAGTCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

Gymnotus_curupira_2024 GCTTAAGACAT-AAGTCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

Gymnotus_cylindricus_2092 GCTTAAGATAT-AAGCCAACTATGTTAATAGTCTATTCATATATAAACTAAACT--GTAA

Gymnotus_cylindricus_2093 GCTTAAGATAT-AAGCCAACTATGTTAATAGTCTATTCATATATAAACTAAACT--GTAA

Gymnotus_cylindricus_2094 GCTTAAGATAT-AAGCCAACTATGTTAATAGTCTATTCATATATAAACTAAACT--GTAA

Gymnotus_henni_7276 GCTTAAGACAC-AAGCCAACTATGTTAATAGCCTTCA--CACCCAGACTAAACT--GTAA

Gymnotus_henni_7277 GCTTAAGACAC-AAGCCAACTATGTTAATAGCCTTCA--CACCCAGACTAAACT--GTAA

Gymnotus_henni_8189 GCTTAAGACAC-AAGCCAACTATGTTAATAGCCTTCA--CACCCAGACTAAACT--GTAA

Gymnotus_henni_8193 GCTTAAGACAC-AAGCCAACTATGTTAATAGCCTTCA--CACCCAGACTAAACT--GTAA

Gymnotus_henni_8207 GCTTAAGACAC-AAGCCAACTATGTTAATAGCCTTTA--CACCCAGACTAAACT--GTAA

Gymnotus_henni_8230 GCTTAAGACAC-AAGCCAACTATGTTAATAGCCTTCA--CACCCAGACTAAACT--GTAA

Gymnotus_henni_8231 GCTTAAGACAC-AAGCCAACTATGTTAATAGCCTTCA--CACCCAGACTAAACT--GTAA

Gymnotus_javari_2020 GCTTAAAACAC-AAGCCACCCACGTCAATAGCCTTAAT-AACCTAGACTAAACA--ACAG

Gymnotus_jonasi_2016 GCTTAAGACAT--AGCCACCCGCGTTAATAGCTTGTAT-AACTCAGACTAAACA--ACAA

Gymnotus_jonasi_2471 GCTTAAGACAT--AGCCACCCGCGTTAATAGCTTGTAT-AACTCAGGCTAAACA--ACAA

Gymnotus_maculosus_8126 GCTTAAGATAT-AAGCCAACTATGTTAATAGTCTATTCATATATAAACTAAACT--GTAA

Gymnotus_maculosus_8137 GCTTAAGATAT-AAGCCAACTATGTTAATAGTCTATTCATATATAAACTAAACT--GTAA

Gymnotus_maculosus_8169 GCTTAAGATAT-AAGCCAACTATGTTAATAGTCTATTCATATATAAACTAAACT--GTAA

Gymnotus_maculosus_8213 GCTTAAGATAT-AAGCCAACTATGTTAATAGTCTATTCATATATAAACTAAACT--GTAA

Gymnotus_mamiraua_2012 GCTTAAGACAC-AAGCCAACTATGTTAATAACCTATT--TAAGTAGACTAAACT--GTAA

Gymnotus_mamiraua_2013 GCTTAAGACAC-AAGCCAACTATGTTAATAACCTATT--TAAGTAGACTAAACT--GTAA

Gymnotus_n._sp._7104 GCTTAAGATAT-AAGCCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

Gymnotus_n._sp._7105 GCTTAAGATAT-AAGCCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

Gymnotus_n._sp._CORU_2558 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_n._sp._FRIT_7109 GCTTAAGACAC-AAGCCAACTATGTTAATAATTTACCTCGACCTAAATTAAACT--GTAA

Gymnotus_n._sp._ITAP_2559 GCTTAAGACAC-AAGCCAACTATGTTAATAACCTATT--TAAGTAGGCTAAACT--GTAA

Gymnotus_n._sp._ITAP_7071 GCTTAAGACAC-AAGCCAACTATGTTAATAACCTATT--TAAGTAGGCTAAACT--GTAA

Gymnotus_n._sp._ITAP_7072 GCTTAAGACAC-AAGCCAACTATGTTAATAACCTATT--TAAGTAGGCTAAACT--GTAA

Gymnotus_n._sp._ITAP_7074 GCTTAAGACAC-AAGCCAACTATGTTAATAACCTATT--TAAGTAGGCTAAACT--GTAA

Gymnotus_n._sp._ITAP_7075 GCTTAAGACAC-AAGCCAACTATGTTAATAACCTATT--TAAGTAGGCTAAACT--GTAA

Gymnotus_n._sp._MAMA_7065 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_n._sp._MAMA_7066 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_n._sp._MAMA_7067 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_n._sp._RS1_7088 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_n._sp._XING_7305 GCTTAAGACAT-AAGCCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

Gymnotus_obscurus_2017 GCTTAAGACAT-AAGCCAACTATGTTAATAATTTACC--TACCTAAATTAAACT--GTAA

Gymnotus_obscurus_2018 GCTTAAGACAT-AAGCCAACTATGTTAATAATTTACC--TACCTAAATTAAACT--GTAA

Gymnotus_omarorum_7092 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTGTT--TAAATAGGCTAAACT--GTAA

Gymnotus_omarorum_7093 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTGTT--TAAATAGG-TAAACT--GTAA

Gymnotus_panamensis_8018 GCTTAAGACATTAAGCCAACTATGTTAATAATTTATACATACATAAACTAAACT--GTAA

Gymnotus_panamensis_8021 GCTTAAGACATTAAGCCAACTATGTTAATAATTTATACATACATAAACTAAACT--GTAA

Gymnotus_panamensis_8210 GCTTAAGACATTAAGCCAACTATGTTAATAATTTATACATACATAAACTAAACT--GTAA

Gymnotus_pantanal_7076 GCTTAAGATAT-AAGCCAACTATGTTAATAATTTATT--TATCTAAATTAAACT--GTAA

Gymnotus_pantherinus_2039 GCTTAAGACAC-AAGCCAATTATGTTAATAGCCTAAA--CACCTAGACTAAACT--ATAC

Gymnotus_pantherinus_2945 GCTTAAGACAC-AAGCCAATTATGTTAATAGCCTAAA--TACCTAGACTAAACT--ATAC

Gymnotus_pantherinus_7111 GCTTAAGACAC-AAGCCAATTATGTTAATAGCCTAAA--TACCTAGACTAAACT--ATAC

Gymnotus_pedanopterus_2058 GCTTAAGACAC-AAGCCAACCATGTCAATAATTTACA--CACCTAAATTAAACT--ATAA

Gymnotus_pedanopterus_2059 GCTTAAGACAC-AAGCCAACCATGTCAATAATTTACA--CACCTAAATTAAACT--ATAA

Gymnotus_stenoleucus_2060 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAAT-AACCCAGACTAAACA--ACAA

Gymnotus_stenoleucus_2061 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAAT-AACCCAGACTAAACA--ACAA

Gymnotus_stenoleucus_2064 GCTTAAAACAC-AAGCCACCCACGTCAATGGTCTTAAT-AACCCAGACTAAACA--ACAA

Gymnotus_sylvius_7239 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_sylvius_7240 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGGCTAAACT--GTAA

Gymnotus_tigre_7090 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTACA--CACCTAGACTAAACT--GCAA

Gymnotus_tigre_7349 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTACA--CACCTAGACTAAACT--GCAA

Gymnotus_ucamara_1927 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_ucamara_1950 GCTTAAGACAT-AAGCCAACTATGTTAATAGCCTATT--TAAATAGCCTAAACT--GTAA

Gymnotus_varzea_2014 GCTTAAGACAT-AAGCCAACTATGTTAATAATCTACC--TATCTAGATTAAACT--GTAA

Gymnotus_varzea_2015 GCTTAAGACAT-AAGCCAACTATGTTAATAATCTACC--TATCTAGATTAAACT--GTAA

Hypopomus_artedi_2232 GCTTAAGACAT-CAACCAACTATATTAATAGGCTCACCAACCGAGACTTAAACTCAATAG

Rhamphichthys_rostratus_2632 GCTTAAGACAC-AAGCCAACTATGTTAATAAACCATA-TAACCTGGCTTAAACTAAATAG

Sternopygus_astrabes_2203 GCTTAAGACCT-AAGCCACCTATGTTAATAGTACAAA-TAAACCAAACTAAACTAAATAG

Sternopygus_macrurus_2639 GCTTAAGACCT-AAACCACCTATGTTAATAATCTA---CAAACTAGTTTAAACTAAATAG

Brachyhypopomus_brevirostris_2617 TC-ATGGCCCAC---ATCTTCGGTTGGGGCGACAATGGAGAAAAACAAAGCCTCCACGCG

Brachyhypopomus_diazi_2408 TT-ATGGCCCAC---ATCTTCGGTTGGGGCGACAATGGAGGAAAACAAAGCCTCCACGCG

Brachyhypopomus_diazi_305 TT-ATGGCCCAC---ATCTTCGGTTGGGGCGACAATGGAGGAAAACAAAGCCTCCACGCG

Brachyhypopomus_n._sp._PAL_2432 CT-GTGGCCCGT---ATCTTCGGTTGGGGCGACAATGGAGGAAAACAAAGCCTCCACGTG

Electrophorus_electricus_2026 TA-CTGACCTAC---ATCTTCGGTTGGGGCGACCACGGGGGAAAACTAAGCCCCCATGAA

Electrophorus_electricus_2619 TA-CTGACCTAC---ATCTTCGGTTGGGGCGACCACGGGGGAAAACTAAGCCCCCATGAA

Gymnotus_aff._anguillaris_2091 CA-CTGACCCCCCCCGTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGCG

Gymnotus_arapaima_2002 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_arapaima_2003 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

88

Gymnotus_ardilai_8175 CA-CTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGAG

Gymnotus_ardilai_8186 CA-CTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGAG

Gymnotus_bahianus_7244 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_bahianus_7245 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_carapo_(CA)_2004 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_carapo_(CA)_2030 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_carapo_(OR)_2040 CA-CTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGAG

Gymnotus_carapo_(OR)_2041 CA-CTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGAG

Gymnotus_carapo_(WA)_2006 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_carapo_(WA)_2007 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_cataniapo_2062 CA-CTGGCCATA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_cataniapo_2063 CA-CTGGCCATA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_chaviro_7357 CA-CTGGCCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGTA

Gymnotus_chaviro_7358 CA-CTGGCCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGTA

Gymnotus_choco_8209 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGAG

Gymnotus_coatesi_2042 CA-CTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_coatesi_2043 CA-CTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_coropinae_(CA)_2010 CA-TTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_coropinae_(CA)_2025 CA-TTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_coropinae_(GU)_2035 CA-TTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_coropinae_(GU)_2036 CA-TTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_coropinae_(GU)_2037 CA-TTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_coropinae_(GU)_2038 CA-TTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_curupira_2009 CA-CTGACCTAA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGAG

Gymnotus_curupira_2019 CA-CTGACCTAA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGAG

Gymnotus_curupira_2021 CA-CTGACCTAA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGAG

Gymnotus_curupira_2024 CA-CTGACCTAA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGAG

Gymnotus_cylindricus_2092 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_cylindricus_2093 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGAG

Gymnotus_cylindricus_2094 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGAG

Gymnotus_henni_7276 CATCTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_henni_7277 CATCTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_henni_8189 CATCTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_henni_8193 CATCTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_henni_8207 CATCTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_henni_8230 CATCTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_henni_8231 CATCTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_javari_2020 CA-CTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_jonasi_2016 TA-CTGGC-TCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATGAAACCTCCATGCA

Gymnotus_jonasi_2471 TA-CTGGC-TCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATGAAACCTCCATGCA

Gymnotus_maculosus_8126 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_maculosus_8137 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_maculosus_8169 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_maculosus_8213 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_mamiraua_2012 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAGAACAAAGCCTCCACGCG

Gymnotus_mamiraua_2013 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAGAACAAAGCCTCCACGCG

Gymnotus_n._sp._7104 CA-CTGGTCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGCG

Gymnotus_n._sp._7105 CA-CTGGTCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGCG

Gymnotus_n._sp._CORU_2558 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_n._sp._FRIT_7109 CA-CTGGCCGCC---GTCTTCGGTTGGGGCGACCATGGAGTAAAACAAAACCTCCCTGCG

Gymnotus_n._sp._ITAP_2559 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_n._sp._ITAP_7071 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAGAACAAAACCTCCACGCG

Gymnotus_n._sp._ITAP_7072 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAGAACAAAACCTCCACGCG

Gymnotus_n._sp._ITAP_7074 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAGAACAAAACCTCCACGCG

Gymnotus_n._sp._ITAP_7075 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_n._sp._MAMA_7065 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_n._sp._MAMA_7066 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_n._sp._MAMA_7067 CA-CTGG-CTCA---GTCTTCGG-TGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_n._sp._RS1_7088 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_n._sp._XING_7305 CA-CTGGCCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGCG

Gymnotus_obscurus_2017 CA-CTGGCCTAA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGCG

Gymnotus_obscurus_2018 CA-CTGGCCTAA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGCG

Gymnotus_omarorum_7092 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_omarorum_7093 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCACGCG

Gymnotus_panamensis_8018 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCATGCG

Gymnotus_panamensis_8021 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCATGCG

Gymnotus_panamensis_8210 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACGAAACCTCCATGCG

Gymnotus_pantanal_7076 CA-CTGGTCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGCG

Gymnotus_pantherinus_2039 CA-CTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGCAAAACAAAACCTCCATGAG

Gymnotus_pantherinus_2945 CA-CTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGCAAAATAAAACCTCCATGAG

Gymnotus_pantherinus_7111 CA-CTGGCCCCA---GTCTTCGGTTGGGGCGACCACGGAGCAAAATAAAACCTCCATGAG

Gymnotus_pedanopterus_2058 CA-CTGGCCCTC---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

Gymnotus_pedanopterus_2059 CA-CTGGCCCTC---GTCTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCTCCATGCG

89

Gymnotus_stenoleucus_2060 CA-CTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_stenoleucus_2061 CC-CTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_stenoleucus_2064 CA-CTGGCC-CA---GTTTTCGGTTGGGGCGACCACGGAGTAAAATAAAACCCCCATGCA

Gymnotus_sylvius_7239 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_sylvius_7240 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_tigre_7090 CA-CTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGCAAAACAAAACCTCCATGCG

Gymnotus_tigre_7349 CA-CTGGCCCTA---GTCTTCGGTTGGGGCGACCACGGAGCAAAACAAAACCTCCATGCG

Gymnotus_ucamara_1927 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_ucamara_1950 CA-CTGGCCTCA---GTCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCACGCG

Gymnotus_varzea_2014 CA-CTGGCCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGTA

Gymnotus_varzea_2015 CA-CTGGCCTAA---ATCTTCGGTTGGGGCGACCACGGAGTAAAACAAAACCTCCATGTA

Hypopomus_artedi_2232 AC-CTGGCCCCCC--GTCTTCAGTTGGGGCGACGATGGAGAAAAATAAAGCCTCCACGCA

Rhamphichthys_rostratus_2632 AA-CTGGCCCCC---GTCTTCGGTTGGGGCGACCGCGGGGGAAAACAAAGCCCCCACGTG

Sternopygus_astrabes_2203 TC-CTGACCCAA---GTCTTCGGTTGGGGTGACCGCGGGGGAAAACAAAGCCCCCACGTG

Sternopygus_macrurus_2639 CA-TTGGCCCAA---GTCTTCGGTTGGGGTGACCGCGGGGGAAAACAAAGCCCCCATGTG

Brachyhypopomus_brevirostris_2617 GCACGGGACTTC--CCCAAAAATCAAGAGCAACCCCTCTAAATCTCAGAACCTCTGACCA

Brachyhypopomus_diazi_2408 GTATGGGATACC--CCCAAAAATCAAGAGCAACCCCTCTAAATCTCAGAACCTCTGACCT

Brachyhypopomus_diazi_305 GTATGGGGTACC--CCCAAAAATCAAGAGCAACCCCTCTAAATCTCAGAACCTCTGACCT

Brachyhypopomus_n._sp._PAL_2432 GTATGGGACTAC--CCCAAAAATCAAGAGCAACCCCTCTAAATCTCAGAACCTCTGACCT

Electrophorus_electricus_2026 GAAAGATAGACC--CTTCTAAACCTAGAAAGACATTTCTAAGTCGCAGAACATCTGACTA

Electrophorus_electricus_2619 GAAAGATAGACC--CTTCTAAACCTAGAAAGACATTTCTAAGTCGCAGAACATCTGACTA

Gymnotus_aff._anguillaris_2091 GATAGGGCACA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_arapaima_2002 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_arapaima_2003 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_ardilai_8175 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_ardilai_8186 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_bahianus_7244 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_bahianus_7245 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_carapo_(CA)_2004 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_carapo_(CA)_2030 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_carapo_(OR)_2040 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_carapo_(OR)_2041 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_carapo_(WA)_2006 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_carapo_(WA)_2007 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_cataniapo_2062 GATAGGGCATA---CCCTAAAACCAAGAAAGACACTTCCAAGCCCCAGAACATCTGACCT

Gymnotus_cataniapo_2063 GATAGGGCATA---CCCTAAAACCAAGAAAGACACTTCCAAGCCACAGAACATCTGACCT

Gymnotus_chaviro_7357 GATAGGGAATA---TCCTAAAACCAAGAGAAACATCTCCAAGTCACAGAATATCTGACTA

Gymnotus_chaviro_7358 GATAGGGAATA---TCCTAAAACCAAGAGAAACATCTCCAAGTCACAGAATATCTGACTA

Gymnotus_choco_8209 GATAGGGAATA---TCCTAAAACCAAGAGAAACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_coatesi_2042 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_coatesi_2043 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_coropinae_(CA)_2010 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_coropinae_(CA)_2025 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_coropinae_(GU)_2035 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_coropinae_(GU)_2036 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_coropinae_(GU)_2037 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACT-

Gymnotus_coropinae_(GU)_2038 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACT-

Gymnotus_curupira_2009 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_curupira_2019 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_curupira_2021 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_curupira_2024 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_cylindricus_2092 GATAGGGAAAAA--TCCTAAAATCAAGAGAGACATCTCCAAATCACAGAATATCTGACCA

Gymnotus_cylindricus_2093 GATAGGGAAAAA--TCCTAAAATCGAGAGAGACATCTCCAAATCACAGAATATCTGACCA

Gymnotus_cylindricus_2094 GATAGGGAAAAA--CCCTAAAATCGAGAGAGACATCTCCAAATCACAGAATATCTGACCA

Gymnotus_henni_7276 GATAAGGATATA--TCCTAAAACTAAGAAAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_henni_7277 GATAAGGATATA--TCCTAAAACTAAGAAAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_henni_8189 GATAAGGATATA--TCCTAAAACTAAGAAAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_henni_8193 GATAAGGATATA--TCCTAAAACTAAGAAAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_henni_8207 GATAGGAATATA--TCCTAAAACTAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_henni_8230 GATAAGGATATA--TCCTAAAACTAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_henni_8231 GATAAGGATATA--TCCTAAAACTAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_javari_2020 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_jonasi_2016 GACAGGGAATA---CCCTAAAACCAAGAAAGACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_jonasi_2471 GACAGGGAATA---CCCTAAAACCAAGAAAGACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_maculosus_8126 GATAGGGAAAAA--TCCTAAAATCAAGAGAGACATCTCCAAATCACAGAATATCTGACCA

Gymnotus_maculosus_8137 GATAGGGAAAAA--TCCTAAAATCAAGAGAGACATCTCCAAATCACAGAATATCTGACCA

Gymnotus_maculosus_8169 GATAGGGAAAAA--TCCTAAAATCAAGAGAGACATCTCCAAATCACAGAATATCTGACCA

Gymnotus_maculosus_8213 GATAGGGAAAAA--TCCTAAAATCAAGAGAGACATCTCCAAATCACAGAATATCTGACCA

Gymnotus_mamiraua_2012 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_mamiraua_2013 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_n._sp._7104 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_n._sp._7105 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

90

Gymnotus_n._sp._CORU_2558 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_n._sp._FRIT_7109 GATAGGACATA---CCCTAAAACCAAGAAAGACACTTCTAAGCCACAGAACATCTGACCT

Gymnotus_n._sp._ITAP_2559 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_n._sp._ITAP_7071 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_n._sp._ITAP_7072 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_n._sp._ITAP_7074 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_n._sp._ITAP_7075 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_n._sp._MAMA_7065 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_n._sp._MAMA_7066 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_n._sp._MAMA_7067 GATAGGGAATA---TCCTAAAACCAAGA-AGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_n._sp._RS1_7088 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_n._sp._XING_7305 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_obscurus_2017 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_obscurus_2018 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_omarorum_7092 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_omarorum_7093 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_panamensis_8018 GATAGGGAAAAA--TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_panamensis_8021 GATAGGGAAAAA--TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_panamensis_8210 GATAGGGAAAAA--TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_pantanal_7076 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAATATCTGACCA

Gymnotus_pantherinus_2039 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_pantherinus_2945 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_pantherinus_7111 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_pedanopterus_2058 GATTAGGACACA--TCCTAAAACCAAGAGAGACACCTCCAAGCCACAGAACATCTGACCA

Gymnotus_pedanopterus_2059 GATTAGGACACA--TCCTAAAACCAAGAGAGACACCTCCAAGCCACAGAACATCTGACCA

Gymnotus_stenoleucus_2060 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_stenoleucus_2061 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_stenoleucus_2064 GACAGGGAATA---CCCTAAAACCAAGAAAAACATTTCTAAGTCACAGAACATCTGACTA

Gymnotus_sylvius_7239 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_sylvius_7240 GATAGGGAATA---TCCTAAAACCAAGAGAGACATCTCCAAGTCACAGAACATCTGACCA

Gymnotus_tigre_7090 GATAGGGAATA---CCCTAAAACTAAGAGATACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_tigre_7349 GATAGGGAATA---CCCTAAAACTAAGAGATACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_ucamara_1927 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_ucamara_1950 GATAGGGAATA---CCCTAAAACCAAGAGAGACATCTCCAAGCCACAGAACATCTGACCA

Gymnotus_varzea_2014 GATAGGGAATA---TCCTAAAACCAAGAGAAACATCTCCAAGTCACAGAATATCTGACTA

Gymnotus_varzea_2015 GATAGGGAATA---TCCTAAAACCAAGAGAAACATCTCCAAGTCACAGAATATCTGACTA

Hypopomus_artedi_2232 GCATGGAATTTT--CCTAAAA-TCAAGAGTAACCCCTCAAAACCTCAGAATCTCTGACCT

Rhamphichthys_rostratus_2632 GAGTGAGGATATTACCTTACAACCAAGAGAGACCCCTCTAAGTCACAGAACTTCTGACCA

Sternopygus_astrabes_2203 GAATGGGGCCAACCCCC-AAAACCATGAGAGACATCTCTAAGTCGCAGAACATCTGACCA

Sternopygus_macrurus_2639 GAACGGGGACAGCCCCCTAAAACCAAGAGAGACATCTCTAAGCCCCAGAACATCTGACCA

Brachyhypopomus_brevirostris_2617 AATA-GATCCGGCTCCT--CGCCGATCAACGGACCAAGTTACCCTAGGGATAACAGCGCA

Brachyhypopomus_diazi_2408 AACA-GATCCGGCTCCT--CGCCGATCAACGGACCAAGTTACCCTAGGGATAACAGCGCA

Brachyhypopomus_diazi_305 AACA-GATCCGGCTCCT--CGCCGATCAACGGACCAAGTTACCCTAGGGATAACAGCGCA

Brachyhypopomus_n._sp._PAL_2432 AATA-GATCCGGCCCCC--CGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Electrophorus_electricus_2026 AAA--GATCCGTTTTTA---TACGACCAGCGAACCAAGTTACCCAAGGGATAACAGCGCA

Electrophorus_electricus_2619 AAA--GATCCGTTTTTA---TACGACCAGCGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_aff._anguillaris_2091 CATA-GATCCGGCCTTC-CAGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_arapaima_2002 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_arapaima_2003 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_ardilai_8175 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_ardilai_8186 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_bahianus_7244 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_bahianus_7245 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_carapo_(CA)_2004 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_carapo_(CA)_2030 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_carapo_(OR)_2040 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_carapo_(OR)_2041 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_carapo_(WA)_2006 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_carapo_(WA)_2007 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_cataniapo_2062 TATACGATCCGGCCTTT-AGGCCGATCAGCGGACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_cataniapo_2063 TATACGATCCGGCCTTT-AGGCCGATCAGCGGACCAAGTTACCCTAGGGATAACAGCGC-

Gymnotus_chaviro_7357 TATA-GATCCGGCCCTT-CAGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_chaviro_7358 TATA-GATCCGGCCCTT-CAGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_choco_8209 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_coatesi_2042 TATA-GATCCGGCCTTC-CGGCCGATCAACGAACCAAGTTACCCCAGGGATAACAGCGCA

Gymnotus_coatesi_2043 TATA-GATCCGGCCTTC-CGGCCGATCAACGAACCAAGTTACCCCAGGGATAACAGCGCA

Gymnotus_coropinae_(CA)_2010 TACA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_coropinae_(CA)_2025 TACA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_coropinae_(GU)_2035 TACA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_coropinae_(GU)_2036 TACA-GATCCGGCCCTC-CGGCCGATCAGCGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_coropinae_(GU)_2037 -ACA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_coropinae_(GU)_2038 -ACA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

91

Gymnotus_curupira_2009 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_curupira_2019 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_curupira_2021 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_curupira_2024 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_cylindricus_2092 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_cylindricus_2093 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_cylindricus_2094 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_henni_7276 CAT--GATCCGGCCCCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_henni_7277 CAT--GATCCGGCCCCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_henni_8189 CAT--GATCCGGCCCCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_henni_8193 CAT--GATCCGGCCCCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_henni_8207 CAT--GATCCGGCCCCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_henni_8230 CAT--GATCCGGCCCCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_henni_8231 CAT--GATCCGGCCCCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_javari_2020 TATA-GATCCGGCCTTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_jonasi_2016 CACA-GATCCGGCCTCT-TGGCCGATCAACGAACCAAGTTACCCCAGGGATAACAGCGCA

Gymnotus_jonasi_2471 CACA-GATCCGGCCTCT-TGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_maculosus_8126 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_maculosus_8137 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_maculosus_8169 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_maculosus_8213 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_mamiraua_2012 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_mamiraua_2013 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._7104 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._7105 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._CORU_2558 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._FRIT_7109 CACATGACCCGGCCTTTTAGGCCGATCAACGGACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_n._sp._ITAP_2559 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._ITAP_7071 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._ITAP_7072 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._ITAP_7074 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._ITAP_7075 TATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._MAMA_7065 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._MAMA_7066 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._MAMA_7067 AATA-GATCCGGCCTCC-CGG-C-AT-AACGAACCAAGTT-CCCAAGGGATAACAGCGCA

Gymnotus_n._sp._RS1_7088 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_n._sp._XING_7305 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_obscurus_2017 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_obscurus_2018 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_omarorum_7092 AATA-GATCCGGCCTCC-CGGCCGATCAACGGACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_omarorum_7093 AATA-GATCCGGCCTCC-CGGCCGATCAACGGACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_panamensis_8018 TATA-GATCCGGCCTTC-GGACCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_panamensis_8021 TATA-GATCCGGCCTTC-GGACCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_panamensis_8210 TATA-GATCCGGCCTTC-GGACCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_pantanal_7076 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_pantherinus_2039 TAT--GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_pantherinus_2945 TAT--GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_pantherinus_7111 TAT--GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_pedanopterus_2058 CATA-GATCCGGCCTTT-CAGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_pedanopterus_2059 CATA-GATCCGGCCTTT-CAGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_stenoleucus_2060 TACA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_stenoleucus_2061 TATA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_stenoleucus_2064 TACA-GATCCGGCCCTC-CGGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Gymnotus_sylvius_7239 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_sylvius_7240 AATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_tigre_7090 CATA-GATCCGGCC-CC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_tigre_7349 CATA-GATCCGGCC-CC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_ucamara_1927 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_ucamara_1950 CATA-GATCCGGCCTCC-CGGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_varzea_2014 TATA-GATCCGGCCCTT-CAGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Gymnotus_varzea_2015 TATA-GATCCGGCCCTT-CAGCCGATCAACGAACCAAGTTACCCAAGGGATAACAGCGCA

Hypopomus_artedi_2232 TACA-GATCCGGCCCC----GCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Rhamphichthys_rostratus_2632 GAAA-GATCCGGCCCTT--AGCCGATCAACGAACCAAGTTACCCTAGGGATAACAGCGCA

Sternopygus_astrabes_2203 AAA--GATCCGGCCAC-AAAGCCGATTAACGGACCAAGTTACCCTAGGGATAACAGCGCA

Sternopygus_macrurus_2639 AAAA-GATCCGGCTACTAAAGCCGATTAACGGACCAAGTTACCCTAGGGATAACAGCGCA

Brachyhypopomus_brevirostris_2617 ATCCCCTTTCAGAGTTCCTATCGACGAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Brachyhypopomus_diazi_2408 ATCCCCTTCTAGAGTTCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Brachyhypopomus_diazi_305 ATCCCCTTCTAGAGTTCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Brachyhypopomus_n._sp._PAL_2432 ATCCCCTTCTAGAGTTCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Electrophorus_electricus_2026 ATCCTCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Electrophorus_electricus_2619 ATCCTCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_aff._anguillaris_2091 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

92

Gymnotus_arapaima_2002 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_arapaima_2003 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_ardilai_8175 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_ardilai_8186 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_bahianus_7244 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_bahianus_7245 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_carapo_(CA)_2004 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_carapo_(CA)_2030 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_carapo_(OR)_2040 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_carapo_(OR)_2041 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_carapo_(WA)_2006 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_carapo_(WA)_2007 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_cataniapo_2062 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_cataniapo_2063 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_chaviro_7357 ATCCCCTTCCAGAGTCCCTATCGAC-AGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_chaviro_7358 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_choco_8209 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coatesi_2042 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coatesi_2043 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coropinae_(CA)_2010 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coropinae_(CA)_2025 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coropinae_(GU)_2035 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coropinae_(GU)_2036 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coropinae_(GU)_2037 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_coropinae_(GU)_2038 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_curupira_2009 ATCCCCTTCCAGAGTCCCTATCAACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_curupira_2019 ATCCCCTTCCAGAGTCCCTATCAACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_curupira_2021 ATCCCCTTCCAGAGTCCCTATCAACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_curupira_2024 ATCCCCTTCCAGAGTCCCTATCAACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_cylindricus_2092 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_cylindricus_2093 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_cylindricus_2094 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_henni_7276 ATCCCCTTCCAGAGTCCTTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_henni_7277 ATCCCCTTCCAGAGTCCTTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_henni_8189 ATCCCCTTCCAGAGTCCTTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_henni_8193 ATCCCCTTCCAGAGTCCTTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_henni_8207 ATCCCCTTCCAGAGCCCTTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_henni_8230 ATCCCCTTCCAGAGTCCTTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_henni_8231 ATCCCCTTCCAGAGTCCTTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_javari_2020 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_jonasi_2016 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_jonasi_2471 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_maculosus_8126 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_maculosus_8137 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_maculosus_8169 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_maculosus_8213 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_mamiraua_2012 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_mamiraua_2013 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._7104 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._7105 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._CORU_2558 ATCCCCTTTCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._FRIT_7109 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._ITAP_2559 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._ITAP_7071 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._ITAP_7072 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._ITAP_7074 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._ITAP_7075 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._MAMA_7065 ATCCCCTTTCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._MAMA_7066 ATCCCCTTTCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._MAMA_7067 ATCCCCTTTCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._RS1_7088 ATCCCCTTTCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_n._sp._XING_7305 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_obscurus_2017 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_obscurus_2018 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_omarorum_7092 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_omarorum_7093 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_panamensis_8018 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_panamensis_8021 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_panamensis_8210 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_pantanal_7076 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_pantherinus_2039 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_pantherinus_2945 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_pantherinus_7111 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

93

Gymnotus_pedanopterus_2058 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_pedanopterus_2059 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_stenoleucus_2060 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_stenoleucus_2061 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_stenoleucus_2064 ATCCCCTCCCAGAGTCCATATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_sylvius_7239 ATCCCCTTTCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_sylvius_7240 ATCCCCTTTCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_tigre_7090 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_tigre_7349 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_ucamara_1927 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_ucamara_1950 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_varzea_2014 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Gymnotus_varzea_2015 ATCCCCTTCCAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Hypopomus_artedi_2232 ATCCCCTTCTAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Rhamphichthys_rostratus_2632 ATCCCCTCCGAGAGTCCCTATCGACAAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Sternopygus_astrabes_2203 ATCCCCTCCCAGAGTCCCTATCGACGAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Sternopygus_macrurus_2639 ATCCCCTCTCAGAGTCCCTATCGACGAGGGGGTTTACGACCTCGATGTTGGATCAGGACA

Brachyhypopomus_brevirostris_2617 TCCTAATGGTGCAGCCGCTATTAAGGGTT-------------------------------

Brachyhypopomus_diazi_2408 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGT------------------------

Brachyhypopomus_diazi_305 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGT------------------------

Brachyhypopomus_n._sp._PAL_2432 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGT----------------------------

Electrophorus_electricus_2026 TCCTAATGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Electrophorus_electricus_2619 TCCTAATGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_aff._anguillaris_2091 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTC---------

Gymnotus_arapaima_2002 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTG--

Gymnotus_arapaima_2003 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_ardilai_8175 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_ardilai_8186 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTA------

Gymnotus_bahianus_7244 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTAC-----

Gymnotus_bahianus_7245 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTAC-----

Gymnotus_carapo_(CA)_2004 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

Gymnotus_carapo_(CA)_2030 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_carapo_(OR)_2040 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_carapo_(OR)_2041 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_carapo_(WA)_2006 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

Gymnotus_carapo_(WA)_2007 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

Gymnotus_cataniapo_2062 TCCTAATGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_cataniapo_2063 TCCTAATGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_chaviro_7357 TCC---------------------------------------------------------

Gymnotus_chaviro_7358 TCCTAATGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_choco_8209 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGA-

Gymnotus_coatesi_2042 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_coatesi_2043 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

Gymnotus_coropinae_(CA)_2010 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_coropinae_(CA)_2025 TCCTATTGGTGCAGCCGCTATTAAGGGTTC-T----------------------------

Gymnotus_coropinae_(GU)_2035 TCCTATTGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTC---------

Gymnotus_coropinae_(GU)_2036 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_coropinae_(GU)_2037 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_coropinae_(GU)_2038 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

Gymnotus_curupira_2009 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_curupira_2019 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_curupira_2021 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCC--------

Gymnotus_curupira_2024 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_cylindricus_2092 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_cylindricus_2093 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTG-------------------------

Gymnotus_cylindricus_2094 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTG-------------------------

Gymnotus_henni_7276 TCCTAATGGTGTAACCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_henni_7277 TCCTAATGGTGTAACCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_henni_8189 TCCTAATGGTGTAACCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_henni_8193 TCCTAATGGTGTAACCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_henni_8207 TCCTAATGGTGTAACCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_henni_8230 TCCTAATGGTGTAACCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_henni_8231 TCCTAATGGTGTAACCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTA------

Gymnotus_javari_2020 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_jonasi_2016 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_jonasi_2471 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_maculosus_8126 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGA-

Gymnotus_maculosus_8137 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGA-

Gymnotus_maculosus_8169 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTA------

Gymnotus_maculosus_8213 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTG--

Gymnotus_mamiraua_2012 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_mamiraua_2013 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

94

Gymnotus_n._sp._7104 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._7105 TCCTAATGGTGCAGCCGCTA-TAAGGGTTCG-----------------------------

Gymnotus_n._sp._CORU_2558 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._FRIT_7109 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._ITAP_2559 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._ITAP_7071 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._ITAP_7072 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._ITAP_7074 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCC--------

Gymnotus_n._sp._ITAP_7075 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTA------

Gymnotus_n._sp._MAMA_7065 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._MAMA_7066 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._MAMA_7067 TCCTAATGGTGCAGCCGCTATTAAGGGTTC------------------------------

Gymnotus_n._sp._RS1_7088 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_n._sp._XING_7305 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_obscurus_2017 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_obscurus_2018 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_omarorum_7092 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_omarorum_7093 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_panamensis_8018 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACA----

Gymnotus_panamensis_8021 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_panamensis_8210 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_pantanal_7076 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_pantherinus_2039 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_pantherinus_2945 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_pantherinus_7111 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_pedanopterus_2058 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGT----------------------------

Gymnotus_pedanopterus_2059 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGT----------------------------

Gymnotus_stenoleucus_2060 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_stenoleucus_2061 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_stenoleucus_2064 TCCTATTGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_sylvius_7239 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_sylvius_7240 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_tigre_7090 TCCTAATGGTGCAGCCGCTATTAAAGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_tigre_7349 TCCTAATGGTGCAGCCGCTATTAAAGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_ucamara_1927 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Gymnotus_ucamara_1950 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

Gymnotus_varzea_2014 TCCTAATGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGAT

Gymnotus_varzea_2015 TCCTAATGGTGTAGCCGCTATTAAGGGTTCGTTTGTTCAACGAT----------------

Hypopomus_artedi_2232 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTT---------------------------

Rhamphichthys_rostratus_2632 CCCTAATGGTGCAGCCGCTATTAAGGG---------------------------------

Sternopygus_astrabes_2203 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGCCCTACGTGAT

Sternopygus_macrurus_2639 TCCTAATGGTGCAGCCGCTATTAAGGGTTCGTTTGTTCAACGATTAAA------------

Brachyhypopomus_brevirostris_2617 --

Brachyhypopomus_diazi_2408 --

Brachyhypopomus_diazi_305 --

Brachyhypopomus_n._sp._PAL_2432 --

Electrophorus_electricus_2026 --

Electrophorus_electricus_2619 CT

Gymnotus_aff._anguillaris_2091 --

Gymnotus_arapaima_2002 --

Gymnotus_arapaima_2003 CT

Gymnotus_ardilai_8175 CT

Gymnotus_ardilai_8186 --

Gymnotus_bahianus_7244 --

Gymnotus_bahianus_7245 --

Gymnotus_carapo_(CA)_2004 --

Gymnotus_carapo_(CA)_2030 --

Gymnotus_carapo_(OR)_2040 CT

Gymnotus_carapo_(OR)_2041 CT

Gymnotus_carapo_(WA)_2006 --

Gymnotus_carapo_(WA)_2007 --

Gymnotus_cataniapo_2062 --

Gymnotus_cataniapo_2063 --

Gymnotus_chaviro_7357 --

Gymnotus_chaviro_7358 CT

Gymnotus_choco_8209 --

Gymnotus_coatesi_2042 --

Gymnotus_coatesi_2043 --

Gymnotus_coropinae_(CA)_2010 CT

Gymnotus_coropinae_(CA)_2025 --

Gymnotus_coropinae_(GU)_2035 --

Gymnotus_coropinae_(GU)_2036 --

95

Gymnotus_coropinae_(GU)_2037 CT

Gymnotus_coropinae_(GU)_2038 --

Gymnotus_curupira_2009 CT

Gymnotus_curupira_2019 CT

Gymnotus_curupira_2021 --

Gymnotus_curupira_2024 --

Gymnotus_cylindricus_2092 CT

Gymnotus_cylindricus_2093 --

Gymnotus_cylindricus_2094 --

Gymnotus_henni_7276 CT

Gymnotus_henni_7277 CT

Gymnotus_henni_8189 CT

Gymnotus_henni_8193 CT

Gymnotus_henni_8207 CT

Gymnotus_henni_8230 CT

Gymnotus_henni_8231 --

Gymnotus_javari_2020 CT

Gymnotus_jonasi_2016 CT

Gymnotus_jonasi_2471 CT

Gymnotus_maculosus_8126 --

Gymnotus_maculosus_8137 --

Gymnotus_maculosus_8169 --

Gymnotus_maculosus_8213 --

Gymnotus_mamiraua_2012 CT

Gymnotus_mamiraua_2013 --

Gymnotus_n._sp._7104 CT

Gymnotus_n._sp._7105 --

Gymnotus_n._sp._CORU_2558 CT

Gymnotus_n._sp._FRIT_7109 CT

Gymnotus_n._sp._ITAP_2559 CT

Gymnotus_n._sp._ITAP_7071 CT

Gymnotus_n._sp._ITAP_7072 CT

Gymnotus_n._sp._ITAP_7074 --

Gymnotus_n._sp._ITAP_7075 --

Gymnotus_n._sp._MAMA_7065 CT

Gymnotus_n._sp._MAMA_7066 CT

Gymnotus_n._sp._MAMA_7067 --

Gymnotus_n._sp._RS1_7088 CT

Gymnotus_n._sp._XING_7305 CT

Gymnotus_obscurus_2017 CT

Gymnotus_obscurus_2018 CT

Gymnotus_omarorum_7092 CT

Gymnotus_omarorum_7093 CT

Gymnotus_panamensis_8018 --

Gymnotus_panamensis_8021 CT

Gymnotus_panamensis_8210 CT

Gymnotus_pantanal_7076 CT

Gymnotus_pantherinus_2039 --

Gymnotus_pantherinus_2945 CT

Gymnotus_pantherinus_7111 CT

Gymnotus_pedanopterus_2058 --

Gymnotus_pedanopterus_2059 --

Gymnotus_stenoleucus_2060 --

Gymnotus_stenoleucus_2061 --

Gymnotus_stenoleucus_2064 --

Gymnotus_sylvius_7239 CT

Gymnotus_sylvius_7240 CT

Gymnotus_tigre_7090 CT

Gymnotus_tigre_7349 CT

Gymnotus_ucamara_1927 --

Gymnotus_ucamara_1950 --

Gymnotus_varzea_2014 CT

Gymnotus_varzea_2015 --

Hypopomus_artedi_2232 --

Rhamphichthys_rostratus_2632 --

Sternopygus_astrabes_2203 CT

Sternopygus_macrurus_2639 --