Gymnotiform electric fishes of the Tres Fronteras region of the western Amazon

. Gymnotiform electric knifefishes are an important yet undersampled component of the Neotropical aquatic biota. We report on the gymnotiform fauna of the Tres Fronteras region located at the triple border of Brazil, Colombia, and Peru in the biodiverse western Amazon. The presence of at least 33 species of gymnotiforms in the Tres Fronteras region is validated from recent sampling efforts and the review of previously collected materials. A key is provided for the identification of the species that have been collected from the region. We comment on the diversity of habitat utilization and intraspecific colour variation of some species.


Introduction
The Tres Fronteras (in Portuguese, Tríplice Fronteira) refers to the area where the borders of Brazil, Colombia, and Peru meet along the upper Amazon river.This region is home to one of the larger human communities in the western Amazon, with Leticia (Colombia) and Tabatinga (Brazil) nearly merging on the north bank, and Santa Rosa de Yavarí (Peru) partway across the river on an island to the south.Due to the existing infrastructure, increasing population, and high migratory mobility between the three cities, this region has become a hub for local trade and tourism (Oliveira 2006;Lapola et al. 2021).Together, these three cities and the surrounding Indigenous communities have a human population of over 115,000 people (IBGE 2023;INEI 2023), which is steadily increasing and obtains a substantial amount of animal protein from local fish products (Murrieta et al. 2008).This puts pressure on local aquatic resources and provides an urgency for a better understanding of the composition and ecology of the ichthyofauna of this region.
The Tres Fronteras region lies at the heart of the Western Amazonian Endemic Area (Kullander 1986), a biological province with more fish species than any other freshwater region of comparable size on Earth (Tedesco et al. 2017;Cassemiro et al. 2023).There are over 1,000 fish species known from this region with an area of about 1.6 million km 2 and extending from the Andean piedmont of Colombia, Ecuador, and northern Peru to the mouth of the rio Purús in Brazil (Albert et al. 2011;Cassemiro et al. 2023).Although this region is still predominantly undeveloped, it is experiencing unprecedented economic activities, with rapid degradation of its many terrestrial and aquatic biotic recourses (Tagliacollo et al. 2021).
Here we report on the species richness of one ecologically important component of the ichthyofauna in the western Amazon: the weakly electric knifefishes of the order Gymnotiformes.This group is an abundant but often underestimated component of Neotropical aquatic ecosystems.To date, over 270 valid gymnotiform species have been described from throughout the Neotropics (Fricke et al. 2023), with about 94 species and 24 endemic species in the lowland Amazon basin (van der Sleen and Albert 2017).Despite a largely conserved body plan characterized by an elongate anal fin and the absence of dorsal, pelvic, or adipose fins, gymnotiform fishes exhibit a diversity of head and body shapes, and differ in eco-physiology, diet, habitat, and life history.Their ability to produce and detect weak electric fields allows these fishes to communicate, navigate, and find food and mates in nearly every freshwater habitat across the humid lowlands (i.e.below 250 m elevation) of the Neotropical realm (Albert and Crampton 2005).However, many of these species inhabit remote or largely inaccessible and difficult habitats to reach or sample, and these species′ largely cryptic and nocturnal behaviour makes most gymnotiform fishes difficult to collect on a large scale without using specialist equipment and careful planning (Albert and Crampton 2005;Lundberg et al. 2013;Haag et al. 2019).Therefore, gymnotiform fishes have become chronically underreported in most published regional inventories and databases and underrepresented in museum collections (Albert and Reis 2011).
We provide a checklist of the gymnotiforms that inhabit the main channel of the Amazon, associated floodplain lakes, and terra firme (non-floodplain) streams in the Colombian portion of the Tres Fronteras region.This checklist is the result of a multi-year sampling effort conducted by us, using collecting methods that specifically target these fishes and supplemented by specimens from this region deposited by other collectors in natural history museum collections.Such a work conveys the regional diversity of a gymnotiform fauna, provides insights into the ecology, evolution, and sampling of these species, offers resources for freshwater biologists working in the Tres Fronteras region, and advances the complete characterization of the biodiverse western Amazon.

Study Area
The hydrology of the Tres Fronteras region is dominated by the whitewater (i.e.sediment and nutrient rich, pH 6.6-7.48,conductivity 105-149 µS/cm) Amazon river, which provides a major habitat and means of dispersal for most aquatic organisms of this region (Schiesari et al. 2003;Hurd et al. 2016).Seasonal fluctuations of the river's water level vary on average by 8.6 m, although extreme fluctuations can be up to 12.1 m (Goulding et al. 2003).This regular flooding creates seasonally inundated floodplain forest and lake habitats, which constitute approximately 2% of the land surface area in the region (Paiva et al. 2013).Above the level of the seasonal inundation exist low-order terra firme streams and small rivers that together constitute most of the length of the regional river network but only a tiny fraction (~0.3%) of the total land surface area (Downing et al. 2012).
Outside Leticia, Colombia, the Yaguarcaca (also spelled Yahuarkaka) floodplain lake system is a series of 21 interconnected lakes intermittently connected to the Amazon and which seasonally inundate the surrounding forest and communities.Multiple blackwater (i.e.acidic, sediment and nutrient poor, pH 5-6, conductivity 52-61 µS/cm) terra firme streams drain into the lakes with a low-amplitude annual flood cycle (Fig. 1).This study reports species collected from the river channel and tributaries, floodplain lakes, and terra firme streams in Colombia which exist along the approximately 50 km stretch of the Amazon that forms the border with Peru between Tabatinga, Brazil and the indigenous community of Macedonia in Colombia (Fig. 2).Collection sites summarized in Table 1.

Methods
Most of the specimens used in this work were collected by us between November 2021 and August 2022.These materials were supplemented by additional specimens collected from the region and previously deposited in the CIACOL (Sinchi) and MCZ (Harvard) collections.Sampling techniques during the 2021-2022 collections depended on demands of the different habitats (Fig. 3).Various sites along the Amazon and its tributaries between Leticia and the Macedonia community were trawled during the early morning and afternoon using a 12-ft shrimp trawl attached to a small outboard-motor vessel.Approximately 100-300 m stretches along sandy beaches, river margins, and river channels were trawled at a time.Trawl length depended heavily on the extent of unobstructed habitat that was available at each site.The Yaguarcaca floodplain lake system outside the city of Leticia was sampled in both the day and night using 6 m beach seine nets with 1/8″ stretch mesh and a series of gill nets from 1¾-4″ mesh size.Gymnotiforms were located and collected from among the abundant floating vegetation composed mostly of Pontederia (Eich hornia) crassipes (Mart.)Solms using a commercially available handheld electronic amplifier similar to that described by Crampton et al. (2007) and a beach seine or dip nets.An aux cable is inserted into the amplifier and the other end is cut with the internal wires exposed and splayed.The cable is attached to a pole and used to probe for electric fish under the water.The electric signals broadcasted in the water are converted to audio using the amplifier allowing for the quick locating and capture of specimens (Fig. 3A).Terra firme stream sites were sampled in the mornings using electronic amplifiers and both seine and dip nets with 1/8″ stretch mesh size.Specimens were euthanized in a solution of diluted clove oil and/or ice, and then fixed in a 10% formalin solution in a flat tray for at least 48 h.Specimens were then washed in water and placed in 70% EtOH for longterm storage.Tissue samples for DNA and stable isotope analyses were extracted from the specimens before formalin fixation and stored in 95% ethanol.All specimens were deposited at the Colección Ictiológica de la Amazonia Colombiana (CIACOL) in Leticia, Colombia.Total length (TL) measured in mm from anteriormost tip of snout to end of tail is reported for all figured specimens.
Distribution and habitat.River channels and margins in the Amazon river basin.
Remarks.Sexually dimorphic, with males having noticeably longer snouts and oral jaws.

Compsaraia samueli Albert & Crampton, 2009
Figure 5C New records.COLOMBIA -Amazonas • río Amazonas, frente a la comunidad de Nazaret; 04°07′44″S, 070° 03′09″W; 66 m elev.; 12.IX.2021;K.T. Remarks.Compsaraia samueli is known for extreme sexual dimorphism of the length of the oral jaw; however, no adult males with sexually dimorphic elongate snouts have yet been collected from the Tres Fronteras region.Adult males are aggressive and engage in agonistic behaviour and appear not to feed much during this terminal phase.Furthermore, not all reproductive males or large males have elongate oral jaws, suggesting this may be a facultative trait.Identification.Body with few or no surface chromatophores, pinkish in life, pale yellow in preserved specimens; adult body size small, rarely more than 160 mm TL; face short, with slightly superior mouth and robust oral jaws; eyes minute, covered with layer of skin; electric organ truncated posterior to anal fin allowing hemal spines to be visible; precaudal vertebrae 13 or 14.

Distribution and habitat.
Common in large river chan nels of the Amazon river and its major tributaries.
Remarks.Believed to be the most abundant species of gymnotiform in the main channels of the Amazon river basin.
Identification.Body with few or no surface chromatophores, pinkish in life, pale yellow in preserved specimens; face short, with terminal mouth and robust oral jaws; electric organ and caudal peduncle elongate posterior to anal fin; precaudal vertebrae 14-16.
Distribution and habitat.River channels in the Amazon and Orinoco river basins.
Remarks.The type locality of S. orthos is in the Orinoco river basin, and although populations are known from both the Orinoco and Amazon river basins, a review of the genus was unable to find consistent morphometric differences to distinguish the two populations, which are currently treated as a single species.
Remarks.This species was found very commonly in trawls along the margins of the Amazon.Adult males are aggressive and engage in agonistic towards conspecific males.Not all reproductive males or the largest males have sexually dimorphic external teeth, which suggests this is a facultative trait.Females are not known to develop the external teeth seen in some males.Many adult males with and without external teeth exhibit scratch marks on the head and anterior body region, presumably resulting from agonistic contests.Only one male displaying sexual dimorphism was collected during our sampling, and no others are known from collections in this region (Fig. 6).Identification.Body grey in life, with purple along pterygiophores; preserved specimens brown.Scales absent along mid-dorsum as far as origin of electroreceptive dorsal organ; snout long, downturned, tubular, gracile, with mouth small; fins pale, with dark margins; eye covered with layer of skin; caudal fin present.

References
Type locality.Rio Solimões, downstream from the mouth of the río Purus, Amazonas, Brazil.
Distribution and habitat.Widespread along the main stem of the Amazon river from Iquitos, Peru to Pará, Brazil.
References.De Santana and Vari 2010.
Distribution and habitat.Lowlands of the Amazon river basin.Type locality.Paraná do Paracuúba, near mouth of the rio Negro and entrance to Lago Janauari, Amazonas, Brazil.

Remarks. We regard
Distribution and habitat.Floating meadows and river margins of Amazon and its tributaries.
Remarks.This species was found to be highly abundant in the floating meadows of the Yaguarcaca lake system during seasons of both high and low water.No secondary sexual dimorphism known.
References. Sullivan et al. 2013;Crampton et al. 2016.Identification.Distinguished from other toothless knifefishes (Hypopomidae + Rhamphichthyidae) by the following: adult body size small, up to 116 mm TL, oblique pigment bands 13-15, dark, with irregular margins present along body surface to end of anal fin; dark oblique band over middle of head through eye; anal-fin membrane hyaline; mouth small, terminal; snout short, less than one-third head length, anal-fin origin posterior to pectoral-fin base, supra-pectoral electric organs paired, extending onto back of head behind eyes.
Distribution and habitat.Low-conductivity creeks and streams in nearly all major drainages of the Amazon, Orinoco, and Guiana basins.

Remarks.
No known sexual dimorphism.It is said that this species is a leaf mimic, often lying on one side on stream floors amongst leaf litter.
References.De Santana and Crampton 2011;Sazima et al. 2006.Identification.Distinguished from other toothless knifefishes (Hypopomidae + Rhamphichthyidae) by its medium adult body size (270 mm TL), tan base colour with 8-10 irregular pigment bars and over body surface with a notable discontinuity at the lateral line and crescent-shaped pigment patches on anal-fin membrane; a short rounded snout about one-third head length; analfin origin anterior to pectoral-fin base; paired suprapectoral electric organs that extend onto the back of the head behind the eyes; and paired mental electric organs that extend along the ventral margin of the head.

Rhamphichthys pantherinus
Type locality.Mouth of the rio Negro, Brazil.
Distribution and habitat.Floodplains and river channels and margins in the Amazon, Orinoco, and Essequibo river basins.
Remarks.Both the light and dark colour morphs of S. elegans have been collected from the Tres Fronteras region.Mainly consumes arthropod and fish zooplankton and forms large interspecies schools in the large river channels with other planktivorous gymnotiforms (e.g.Adontosternarchus spp.and Rhabdolichops spp.).
References.Crampton et al. 2004 Identification.Body pinkish white in life, cream to pale yellow when preserved; snout conical; anal-fin rays mostly branched, anal-fin membrane with black distal margin; no caudal fin; caudal appendage sometimes thick and club-like.
Distribution and habitat.River channels and margins in the lowland Amazon, Orinoco, and Essequibo river basins.
References.Dutra et al. 2014.Identification.Body large (up to 480 mm TL), opaque, and pearlescent in life with slight countershading, yellow-tan when preserved; body deeper and thicker than sympatric congeners; snout more squared in lateral profile and larger eye than in E. loretana; opercle with golden sheen; distinct black blotch over posterior ventral portion of body cavity; anal-fin rays mostly branched, anal-fin membrane with black margin; no caudal fin.
Distribution and habitat.Apparently widespread in the rivers of the Amazon, Orinoco, and Essequibo river basins.In the western and central Amazon basins, E. limbata inhabits whitewater river margins.Cár denas-Campo leg.; ethanol preservation; 1 specimen; CIACOL 6750.

References
Identification.Body small (up to 250 mm TL) and gracile, semi-translucent in life, lightly pigmented with four dark-grey stripes extending from behind head to tail; head darkly pigmented; fins hyaline; anal-fin rays mostly branched; no caudal fin.
Distribution and habitat.Margins of rivers and streams in the western Amazon.

Eigenmannia nigra
Distribution and habitat.Rivers and lakes in the Amazon and Orinoco river basins.In the western and central Amazon basins, E. nigra inhabits river margins and floodplain oxbow lakes.
Remarks.This record represents a considerable range extension for this species, and the first validated collection in the western Amazon since its original description.
Remarks.Likely an undescribed species that is similar in appearance to E. macrops from the upper Potaro river drainage in Guyana.This species is seemingly specialized to large river habitats with apparently low tolerance for capture and maintenance in captivity.
Identification.Body semitranslucent, pinkish-cream colour without black lines or markings in life, preserved specimens yellow; superior mouth; eye covered with layer of skin; fins hyaline; deep transparent electric organ visible along the tail.
Distribution and habitat.River channels and margins of large rivers in the Amazon and Orinoco river basins.
Remarks.This species is seemingly specialized to large river habitats with low tolerance for capture and maintenance in captivity.
References.Lundberg and Mago-Leccia 1986.Identification.Body semitranslucent, pinkish cream colour without black lines or markings and yellow in preserved specimens; terminal mouth; eye covered with layer of skin; fins hyaline; narrow transparent electric organ visible along tail.
Distribution and habitat.River channels and margins of large rivers in the Amazon and Orinoco river basins.
Remarks.This species is seemingly specialized to large river habitats with low tolerance for capture and maintenance in captivity. References.

Discussion
This work represents the first gymnotiform-targeted regional survey in the Amazon basin.It highlights the high phenotypic and taxonomic diversity of these fishes in the lowland western Amazon.However, the geographic ranges and taxonomic limits of many species remain poorly understood, largely due to limited sampling and the relatively few experts working with the group.Most major gymnotiform data from the western and central Amazon have been collected in the vicinity of Iquitos (Peru) and Tefé and Manaus (Brazil).The Tres Fronteras represented a large gap in the sampling of gymnotiforms along the length of the Amazon river.
Although the presence of 33 species has been validated from this region, an additional 27 species are expected based on known occurrences further upstream and downstream; however, they have not yet been recorded in between near Leticia (Table 3).We suggest several reasons for this discrepancy, including the possibility of naturally disjunct distributions or adult modal localities or breeding sites, an incomplete taxonomy of the fauna resulting in inaccurate ranges of species, or incomplete sampling of the region.We also report on intraspecific phenotypic diversity within several gymnotiform species collected in the Tres Fronteras region, including Parapteronotus hase mani, Sternarchorhamphus muelleri, and Steatogenys elegans (Fig. 9).Individuals of P. hasemani were previously known to present shades of brown to black, but here we record an individual with bright-orange colouration and report that specimens of all pigment varieties are present in the same region, perhaps suggesting that there is not population structure of body colour in this species.Parapteronotus hasemani is known to display sexual dimorphism of the head, with males developing elongated oral jaws; however, there is no indication the diversity of colour is sexually dimorphic as well, as no dimorphic males were collected and a gradation of colour patterns were observed.Sternarchor hamphus is a monotypic genus which varies in colour from dark purple to pinkish white.Specimens with dark and moderate colouration were collected together in the main channel of the Amazon.One specimen (CIACOL 6621) markedly changed colour from medium grey to pinkish-white approximately 45 minutes after capture, which suggests that external pigmentation may reflect stress or condition of the animal.Additionally, we report the collection of both light and dark morphs of Steatogenys elegans from the same stretch of the whitewater Amazon channel.This species is highly variable in colour pattern across its range, and this is thought to be due to water type or the presence of cryptic species (Cooke et al. 2014).The taxonomic status of these colour morphs is currently unknown, but our brief examination of the specimens was unable to find additional morphological characters to separate them as distinct species.

Figure 1 .
Figure 1.Representative freshwater aquatic habitats in the Tres Fronteras region.A. North bank of río Amazonas between Leticia, Colombia, and the community of Macedonia.B. Caño Arara near its confluence with the río Amazonas.C. Yaguarcaca lake system.D. Quebrada La Arenosa.E. Port of Leticia, Colombia, during season of low water.

Figure 2 .
Figure 2. Map of the Tres Fronteras region with major localities sampled during the 2021-2022 field seasons indicated by yellow circles.Circles may indicate more than one collection effort.

Figure 3 .
Figure3.Select methods used to collect gymnotiform knifefishes in the field.A. J.D. Bogotá-Gregory demonstrates the use of a handheld electric amplifier to locate electrogenic fishes in a mat of aquatic vegetation with the help of J. Montalvo and sons at the Yaguarcaca lake system.B. R.J. Torgersen removes a doradid catfish from the trawl net after sampling the deep channel of the Amazon river.C. K.T. Torgersen uses a dipnet in a small creek near a road crossing outside Leticia.D. K.T. Torgersen and J.D. Bogotá-Gregory deploy a trawl net in the main channel of the Amazon.

Table 1 .
Summary of major sites sampled during the 2021-2022 field collections.

Site Locality description Latitude Longitude 13
South bank of isla Corea −04.0639 −070.120814Channel of tributary south of isla Corea −04.0664 −070.115615Amazon River at Zaragoza community −03.

Table 2 .
List of gymnotiform species collected and/or verified from the Tres Fronteras region.Habitat abbreviations for multiple habitats include river channel (RC), terra firme (TF), and floodplain (FP).

2005; Torgersen and Albert 2022 Figure 4. Venn
diagrams of habitat type of gymnotiform species in the Tres Fronteras region.Numbers indicate numbers of species known to occur in each distinct habitat type.Platyurosternarchus macrostoma is the species known to inhabit all three habitats.

Table 3 .
List of gymnotiform species expected, but not yet reported from the Tres Fronteras region.Habitat abbreviations for multiple habitats include river channel (RC), terra firme (TF), and floodplain (FP).