DNA barcoding reveals novel insights into pterygophagy and prey selection in distichodontid fishes (Characiformes: Distichodontidae)

Abstract

DNA barcoding was used to investigate dietary habits and prey selection in members of the African-endemic family Distichodontidae noteworthy for displaying highly specialized ectoparasitic fin-eating behaviors (pterygophagy). Fin fragments recovered from the stomachs of representatives of three putatively pterygophagous distichodontid genera (Phago, Eugnathichthys, and Ichthyborus) were sequenced for the mitochondrial gene co1. DNA barcodes (co1 sequences) were then used to identify prey items in order to determine whether pterygophagous distichodontids are opportunistic generalists or strict specialists with regard to prey selection and, whether as previously proposed, aggressive mimicry is used as a strategy for successful pterygophagy. Our findings do not support the hypothesis of aggressive mimicry suggesting instead that, despite the possession of highly specialized trophic anatomies, fin-eating distichodontids are opportunistic generalists, preying on fishes from a wide phylogenetic spectrum and to the extent of engaging in cannibalism. This study demonstrates how DNA barcoding can be used to shed light on evolutionary and ecological aspects of highly specialized ectoparasitic fin-eating behaviors by enabling the identification of prey species from small pieces of fins found in fish stomachs.

Full text

DNA barcoding reveals novel insights into pterygophagy
and prey selection in distichodontid fishes
(Characiformes: Distichodontidae)
Jairo Arroyave & Melanie L. J. Stiassny
Division of Vertebrate Zoology, Department of Ichthyology, American Museum of Natural History, Central Park West at 79th St., New York,
New York 10024
Keywords
Ectoparasitic fin-eating behaviors, mtDNA,
stomach contents, trophic ecology.
Correspondence
Jairo Arroyave, Division of Vertebrate
Zoology, Department of Ichthyology,
American Museum of Natural History,
Central Park West at 79th St., New York,
NY 10024
Tel: +1 212 769 5841, Fax: 212 769 5642;
E-mail: jarroyave@amnh.org
Funding Information
The Department of Ichthyology of the
American Museum of Natural History
(AMNH) through the Axelrod Research
Curatorship provided the bulk of funding for
this study. Additional funding was provided
by the DNA Learning Center (DNALC) of
Cold Spring Harbor Laboratory (CSHL) via its
Urban Barcoding Research Program (UBRP).
Received: 2 October 2014; Revised: 24
October 2014; Accepted: 27 October 2014
doi: 10.1002/ece3.1321
Abstract
DNA barcoding was used to investigate dietary habits and prey selection in
members of the African-endemic family Distichodontidae noteworthy for dis-
playing highly specialized ectoparasitic fin-eating behaviors (pterygophagy). Fin
fragments recovered from the stomachs of representatives of three putatively
pterygophagous distichodontid genera (Phago,Eugnathichthys, and Ichthyborus)
were sequenced for the mitochondrial gene co1. DNA barcodes (co1 sequences)
were then used to identify prey items in order to determine whether pterygo-
phagous distichodontids are opportunistic generalists or strict specialists with
regard to prey selection and, whether as previously proposed, aggressive mim-
icry is used as a strategy for successful pterygophagy. Our findings do not sup-
port the hypothesis of aggressive mimicry suggesting instead that, despite the
possession of highly specialized trophic anatomies, fin-eating distichodontids
are opportunistic generalists, preying on fishes from a wide phylogenetic spec-
trum and to the extent of engaging in cannibalism. This study demonstrates
how DNA barcoding can be used to shed light on evolutionary and ecological
aspects of highly specialized ectoparasitic fin-eating behaviors by enabling the
identification of prey species from small pieces of fins found in fish stomachs.
Introduction
Fishes of the family Distichodontidae, distributed through-
out the freshwaters of much of sub-Saharan Africa and the
Nile River basin, are one of the major groups of the African
freshwater ichthyofauna (Vari 1979; Arroyave et al. 2013).
Although moderate in diversity (~100 spp. arrayed in 15
genera), distichodontids display remarkable variation in
oral anatomy and exhibit a wide array of trophic ecologies,
including detritivory, herbivory, insectivory, piscivory, and
even ectoparasitic fin-eating behaviors (herein referred to
as “pterygophagy”), facilitated by highly specialized jaw
morphologies (Fig. 1). Pterygophagy in distichodontid
fishes, however, has not been investigated beyond the study
that first documented this behavior more than 50 years ago
(Matthes 1961) and two subsequent studies (Matthes 1964;
Roberts 1990). Based on an observed similarity in caudal-
fin coloration and patterning –as revealed by traditional
stomach content analysis –between the ectoparasitic dis-
tichodontids Eugnathichthys eetveldii and E.macroterolepis
and their putative prey Synodontis decorus and Mesoborus
crocodilus, respectively, Roberts (1990) hypothesized that
the barred caudal-fin pattern in pterygophagous distich-
odontids reflects a form of aggressive mimicry, allowing
them to avoid detection by their monospecific prey. Four
distichodontid genera –Eugnathichthys,Belonophago,
Ichthyborus, and Phago –are reportedly ectoparasitic (i.e.,
feeding primarily on fish fins as adults) (Roberts 1990;
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1

Stiassny et al. 2013), but until the present study, there was
virtually no information regarding the actual prey prefer-
ences of any of them.
Dietary information is critical for an understanding of
community structure, ecological networks, and ecosystem
functioning (Duffy et al. 2007), and can also inform con-
servation efforts for endangered species and/or threatened
ecosystems (Marrero et al. 2004; Crist
obal-Azkarate and
Arroyo-Rodr
ıguez 2007). Approaches to determine the
composition of animal diets include observation of forag-
ing behavior, examination of stomach contents, and fecal
analysis. Other methods such as fatty acid (FA) or stable
isotope (SI) analyses, while capable of providing a sub-
stantive picture of energy and material flow through the
food web, do not have the resolving power to accurately
determine the relative contributions of different prey
items to the diets of predators (Hardy et al. 2010). In
stomach content and fecal analyses, food items are gener-
ally detected and identified either by direct visual inspec-
tion followed by traditional taxonomic identification or
indirectly via DNA-based identification methods (e.g.,
DNA barcoding, DNA fingerprinting). The former
approach, however, is often hampered by extensive prey
digestion rendering only partial/incomplete prey items,
frequently lacking species or even ordinal level diagnostic
characteristics. Most DNA-based identification methods,
on the other hand, allow for the identification and/or dis-
crimination of prey items, often to the species level, even
from partially digested tissue fragments. DNA barcoding,
a molecule-based species identification method that uses
short, standardized gene regions as species tags (e.g., the
mitochondrial co1 gene in animals, rbcL and matK chlo-
roplast genes in land plants), offers an efficient and cost-
effective alternative to determine the identity of prey
items when they are not fully digested but can only be
identified to a broad taxonomic rank (Valentini et al.
2009; Barnett et al. 2010), which is the case with fin frag-
ments found in stomachs of pterygophagous distichodon-
tid fishes (pers. obs.).
To further investigate pterygophagy in distichodontids
and shed some light on evolutionary and ecological aspects
of this highly unusual trophic strategy, DNA barcoding was
used to identify prey species from fin fragments found in
the stomachs of Phago,Eugnathichthys, and Ichthyborus
specimens. Information on prey identity was then used to
determine whether pterygophagous distichodontids are
opportunistic generalists or strict specialists with regard to
prey selection, and to test Roberts’s (1990) hypothesis that
aggressive mimicry is used as a strategy for successful
pterygophagy in distichodontid fishes.
Materials and Methods
Specimen sampling and stomach content
analysis
Fishes used in this study were collected and euthanized
prior to preservation in accordance with recommended
(A)
(B)
(C)
Figure 1. Variation in jaw anatomy in pterygophagous
distichodontids represented in this study by the genera Phago (A),
Eugnathichthys (B), and Ichthyborus (C).
2ª2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Pterygophagy in Distichodontid Fishes J. Arroyave and M. L. J. Stiassny

guidelines for the use of fishes in research (Nickum et al.
2004), and stress/suffering was ameliorated by minimizing
handling and through the use of anesthetics prior to
euthanasia. Because successful DNA extraction from for-
malin-fixed tissue remains challenging, if not unfeasible
(Chakraborty et al. 2006), only specimens that were pre-
served in 95% EtOH were sampled for this study. A total
of 43 ethanol-preserved individuals (14 Phago, seven Eug-
nathichthys, and 22 Ichthyborus specimens) were dissected
for stomach contents analysis (Table 1). Fin fragments
found in stomachs were isolated, thoroughly cleaned, and
rinsed with distilled water (to avoid contamination with
predator-derived cells/tissues). Each was separately coded
and kept in 95% EtOH. All dissected specimens, except
for those corresponding to the species Ichthyborus ornatus
(whose bodies are deposited in the teaching collection of
the University of Kinshasa, Democratic Republic of
Congo), are cataloged and stored in the ichthyology
Table 1. Specimens sampled for stomach contents analysis and their corresponding co1 barcodes GenBank accession numbers.
Genus Species Catalog # Tissue # GenBank Accession #
Phago P. boulengeri AMNH 259468 AMCC 215881 KP027369
AMNH 259468 AMCC 215880 KP027370
AMNH 259468 AMCC 215879 KP027371
AMNH 259468 AMCC 215878 KP027372
AMNH 259468 AMCC 215877 KP027373
AMNH 259468 AMCC 215876 KP027374
AMNH 259468 AMCC 215875 KP027375
AMNH 259468 AMCC 215874 KP027376
AMNH 259468 AMCC 215873 KP027377
AMNH 259468 AMCC 215872 KP027378
AMNH 259468 AMCC 215727 KP027379
AMNH 260800 AMCC 216764 KP027380
P. intermedius AMNH 255629 AMCC 223226 KP027381
AMNH 255148 AMCC 226195 KP027382
Eugnathichthys E. macroterolepis AMNH 263331 AMCC 227433 KP027383
AMNH 263331 AMCC 227434 KP027384
AMNH 263331 AMCC 227435 KP027385
AMNH 263331 AMCC 227436 KP027386
AMNH 263332 AMCC 227437 KP027387
UKin
1
n/a KP027388
UKin
1
n/a KP027389
Ichthyborus I. quadrilineatus AMNH 257060 AMCC 220511 KP027390
AMNH 257060 AMCC 220512 KP027391
AMNH 257060 t-113-11233 KP027392
I. ornatus UKin
1
T-0188 n/a
T-0189 n/a
T-0190 n/a
T-0191 n/a
T-0192 n/a
T-0193 n/a
T-0194 n/a
T-0195 n/a
T-0196 n/a
T-0197 n/a
T-0198 KP027393
T-0199 n/a
T-0200 KP027394
T-0201 n/a
T-0202 n/a
T-0203 n/a
T-0204 n/a
T-0205 n/a
T-0206 n/a
1
University of Kinshasa (teaching collection), uncataloged.
ª2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3
J. Arroyave and M. L. J. Stiassny Pterygophagy in Distichodontid Fishes

collection of the American Museum of Natural History
(AMNH), available online at the museum’s Vertebrate
Zoology Collection Database (http://entheros.amnh.org/
db/emuwebamnh/index.php).
DNA extraction, amplification, and
sequencing
Total genomic DNA was extracted from both predator
(i.e., pterygophagous distichodontids) and prey items
(i.e., fin fragments found in their stomachs) using DNeasy
Tissue Extraction Kit (Qiagen) following the manufac-
turer’s protocol. DNA extracts were preserved in 95%
EtOH and stored frozen. Amplification and sequencing of
co1 barcodes were carried out using Folmer et al.’s (1994)
universal primers LCO1490 (50-GGTCAACAAATCATAA
AGATATTGG-30) and HCO2198 (50-TAAACTTCAGG
GTGACCAAAAAATCA-30). DNA amplification via poly-
merase chain reaction (PCR) was performed in a 25-lL
volume containing one Ready-To-Go PCR bead (GE
Healthcare), 21 lL of PCR-grade water, 1 lL of each pri-
mer (10 lmol/L), and 2 lL of genomic DNA, under the
following thermal profile: 5-min initial denaturation at
95°C, followed by 35 cycles of denaturation at 95°C for
60 s, annealing at 42°C for 60 s, and extension at 72°C
for 90 s, followed by a 7-min final extension at 72°C.
Double-stranded PCR products were purified using AM-
Pure (Agencourt). Sequencing of each strand of amplified
product was performed in a 5-lL volume containing
1lL of primer (3.2 lmol/L), 0.75 lL of BigDye

Ready
Reaction Mix, 1 lL of BigDye

buffer, and 2.25 lLof
PCR-grade water. Sequencing reactions consisted of a
2-min initial denaturation at 95°C, followed by 35 cycles
of denaturation at 95°C for 30 s, annealing at 45°C for
60 s, and extension at 72°C for 4 min, followed by a
3-min final extension at 72°C. All sequencing reactions
were purified using CleanSEQ (Agencourt) and electro-
phoresed on an Applied Biosystems 3700 automated
DNA sequencer in the AMNH Molecular Systematics
Laboratories.
Bioinformatics
Contig assemblage and sequence editing were performed
using the software Geneious Pro v7.1.5 (Biomatters, avail-
able from http://www.geneious.com/). Species identifica-
tion (of both predator and prey) was carried out using
barcoding similarity methods based on the match between
the query sequence and the reference sequences deposited
in the Barcode of Life Database (BOLD) and GenBank
using NCBI BLAST (Altschul et al. 1990; Johnson et al.
2008). The best match (“top hit”) was taken as the best
estimate of taxonomic identity, with matches ≥98%
similar assumed to be conspecifics, thus allowing an
admittedly arbitrary, but operational threshold of a 2%
difference between query and reference sequences to
account for intraspecific variation (Jarman et al. 2004). In
those cases where the best estimate of taxonomic identity
was ambiguous (i.e., >2% co1 divergence), available speci-
mens of potential prey species (i.e., species living in
sympatry with the sampled pterygophagous distichodont-
ids) previously unrepresented in GenBank/BOLD databas-
es (Table 2) were sequenced for co1 with the goal of
confirming prey identity to the species level.
Results
Overall, 55 fin fragments were recovered from the stom-
achs of 23 of the 43 sampled specimens, and as expected
it was not possible to visually discern prey species from
fin remains. With the exception of all 19 Ichthyborus
ornatus specimens (which had whole fish, but no fin
fragments in their stomachs) and an individual of
Eugnathichthys macroterolepis (which had stomach con-
tents later identified via co1 barcoding as horn snails), all
remaining stomachs contained between one and five
distinct fin fragments.
DNA barcodes confirmed the species identity of all
individuals of the pterygophagous distichodontid species
investigated in this study (i.e., Phago boulengeri,P. inter-
medius,Eugnathichthys macroterolepis,Ichthyborus quadri-
lineatus, and I. ornatus). Amplification and/or sequencing
of co1 failed in 10 of the 55 fin fragments. The results of
the BLAST search for each of the 45 successfully
sequenced fin fragments are presented in Table 3. The co1
barcodes from a total of 19 fish species in nine families
Table 2. Available specimens of potential prey species (i.e., species
living in sympatry with the sampled pterygophagous distichodontids)
previously unrepresented in GenBank/BOLD databases and sequenced
for co1 with the goal of confirming prey identity to the species/
subspecies level.
Species Catalog # Tissue #
GenBank
Accession #
Chrysichthys
nigrodigitatus
AMNH 263329 AMCC 227431 KP027395
Chrysichthys
ornatus
AMNH 260757 AMCC 215865 KP027396
Oreochromis
lepidurus
AMNH 263330 AMCC 227432 KP027397
Sarotherodon
galilaeus
boulengeri
AMNH 260750 AMCC 215857 KP027398
Tylochromis
lateralis
AMNH 241101 t-031-3016 KP027399
4ª2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Pterygophagy in Distichodontid Fishes J. Arroyave and M. L. J. Stiassny

Table 3. Results of the BLAST search for each of the 45 successfully sequenced fin fragments retrieved from the stomachs of the pterygophagous
distichodontid species sampled in this study.
Genus Species Catalog # Fin Fragment ID Best Match (“Top Hit”) Family, Order % Similarity
Phago P. boulengeri AMNH 259468 215881-a Brycinus imberi Alestidae, Characiformes 100
AMNH 259468 215879-a Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.4
215879-b Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.4
215879-c Hemichromis bimaculatus Cichlidae, Perciformes 94.3
215879-e Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.4
AMNH 259468 215878-a Synodontis contracta Mochokidae, Siluriformes 98.6
215878-b Synodontis nigriventris Mochokidae, Siluriformes 98.0
215878-c Synodontis nigriventris Mochokidae, Siluriformes 98.0
AMNH 259468 215877-a Phago boulengeri Distichodontidae, Characiformes 100
215877-b Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.2
215877-c Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.2
AMNH 259468 215876-b Tylochromis polylepis
2
Cichlidae, Perciformes 97.5
215876-c Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.4
215876-d Tylochromis polylepis
2
Cichlidae, Perciformes 97.1
AMNH 259468 215875-a Phago boulengeri Distichodontidae,
Characiformes
100
215875-c Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.2
AMNH 259468 215874-a Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.4
215874-b Sarotherodon galilaeus
1
Cichlidae, Perciformes 99.4
AMNH 259468 215873-a Synodontis nigriventris Mochokidae, Siluriformes 98.0
215873-b Phago boulengeri Distichodontidae, Characiformes 100
215873-c Brycinus comptus Alestidae, Characiformes 100
215873-d Brycinus comptus Alestidae, Characiformes 100
AMNH 259468 215872-a Phago boulengeri Distichodontidae, Characiformes 100
215872-b Synodontis nigriventris Mochokidae, Siluriformes 98.7
AMNH 259468 215727-a Chrysichthys nigrodigitatus Claroteidae, Siluriformes 92.5
215727-b Chrysichthys nigrodigitatus Claroteidae, Siluriformes 92.5
AMNH 260800 216764-a Heterotis niloticus Arapaimidae,
Osteoglossiformes
100
216764-b Heterotis niloticus Arapaimidae,
Osteoglossiformes
100
216764-c Heterotis niloticus Arapaimidae,
Osteoglossiformes
100
P. intermedius AMNH 255629 223226-a Alestopetersius sp. “mbuji” Alestidae, Characiformes 99.2
223226-b Alestopetersius sp. ‘mbuji” Alestidae, Characiformes 99.2
Eugnathichthys E. macroterolepis AMNH 263331 227433-a Chrysichthys ornatus
3
Claroteidae, Siluriformes 97.7
AMNH 263331 227435-a Chrysichthys ornatus
3
Claroteidae, Siluriformes 97.1
AMNH 263331 227436-a Awaous ocellaris Gobiidae, Perciformes 88.6
AMNH 263332 227437-a Trachinotus goreensis Carangidae, Perciformes 100
227437-b Chrysichthys auratus
4
Claroteidae, Siluriformes 96.7
UKin uncat. UK-1-a Trachinotus goreensis Carangidae, Perciformes 100
UK-1-b Oreochromis mossambicus
5
Cichlidae, Perciformes 96.9
UKin uncat. UK-2-a Chrysichthys auratus
4
Claroteidae, Siluriformes 96.7
UK-2-b Chrysichthys ornatus
3
Claroteidae, Siluriformes 96.9
Ichthyborus I. quadrilineatus AMNH 257060 220511-a Chrysichthys auratus Claroteidae, Siluriformes 93.3
220511-b Chrysichthys auratus Claroteidae, Siluriformes 93.3
220512-c Synodontis annectens Mochokidae, Siluriformes 99.6
220512-d Ichthyborus quadrilineatus Distichodontidae,
Characiformes
99.7
113-11233-a Hepsetus odoe Hepsetidae, Characiformes 88.5
1
Confirmed as subspecies Sarotherodon galilaeus boulengeri (>99.7% co1 similarity).
2
Confirmed as Tylochromis lateralis (99.3% co1 similarity).
3
Confirmed as Chrysichthys ornatus (>99.2% co1 similarity).
4
Confirmed as Chrysichthys nigrodigitatus (99.7% co1 similarity).
5
Confirmed as Oreochromis lepidurus (99.9% co1 similarity).
ª2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 5
J. Arroyave and M. L. J. Stiassny Pterygophagy in Distichodontid Fishes

and four orders were identified as being identical or fairly
similar to those from the fin fragments found in the
examined stomachs, with most barcode matches being
>99% similar. Barcodes from fin fragments found in a
single Ichthyborus and four Phago specimens BLASTed to
conspecifics (i.e., I.quadrilineatus and P.boulengeri,
respectively), suggesting a not infrequent occurrence of
cannibalism among some pterygophagous lineages.
The BLASTing of co1 barcodes from 15 of the 45 fin
fragments resulted in best matches (“top hit”) that were
<98% similar, and therefore, whose best estimate of taxo-
nomic identity could only be made above the species
level. Although some prey species were not represented in
either the BOLD or the GenBank databases, in all cases
match percentages to query sequences were still sufficient
to at least confidently assign prey items to genus (or fam-
ily in the case of the horn snails recovered from one Eug-
nathichthys specimen). In eight of the 15 instances of
questionable identification, species identity was later con-
firmed using co1 barcodes generated in this study from
potential prey species collected in sympatry with the sam-
pled pterygophages. Likewise, all prey items initially iden-
tified as Sarotherodon galilaeus were confirmed as
subspecies S. galilaeus boulengeri using co1 barcodes previ-
ously unrepresented in databases (Table 3).
Discussion
Pterygophagous distichodontids –represented in this
study by members of the genera Phago,Eugnathichthys,
and Ichthyborus –prey on fishes from a wide phylogenetic
spectrum that includes at least nine teleostean families
(Arapaimidae, Alestidae, Distichodontidae, Hepsetidae,
Claroteidae, Mochokidae, Carangidae, Gobiidae, and Cic-
hlidae) from four orders (Osteoglossiformes, Characifor-
mes, Siluriformes, and Perciformes). These findings
suggest that the ecological strategy involved in distich-
odontid pterygophagy is one of prey generalization rather
than specialization (contra Roberts (1990)). Interestingly,
in these fishes, a notably high degree of morphological
and behavioral specialization underpins a highly special-
ized feeding modality, which in turn facilitates the utiliza-
tion of a wide spectrum of potential prey. Although the
trade-offs between specialization and generalization are
complex and multifactorial (Hawkins 1994; Thompson
1994), ecological models have shown that the more
polyphagous the predator, the less vulnerable it is to scar-
city and/or extinction of a particular prey species (Mon-
toya et al. 2006). The present finding that Phago
boulengeri from the Congo River basin feeds on the fins
of Heterotis niloticus, a species native to the Sahelo-Suda-
nese region (Daget 1984), and only recently (year 1960)
introduced into the Congo basin (FAO 2005), further
reinforces the idea that pterygophagy in distichodontids
facilitates opportunistic feeding on a wide range of avail-
able prey regardless of historical context.
The findings of this study further indicate that adult
Eugnathichthys macroterolepis, although primarily pterygo-
phagous can, on occasion, exploit alternative food
resources. The stomach of one individual collected near
the mouth of the Congo River contained numerous mol-
lusks identified as horn snails (family Potamididae) via
DNA barcoding. Interestingly, these snails were intact but
devoid of shells implying that E. macroterolepis used its
strong jaws (Fig. 1B) to grasp the exposed foot of each
snail to twist it out of its shell before consumption, pre-
sumably in a manner analogous to that of the Lake Victo-
rian “snail shelling” cichlids (Greenwood 1973). Similarly,
our results indicate that at least one species of Ichthybo-
rus,I. ornatus, is not an obligate pterygophage, as all 19
specimens examined here had intact, or partially digested,
fishes distending their stomachs. Belonophago is the only
pterygophagous distichodontid genus not included in the
current study due to lack of available ethanol-preserved
material. However, observation of aquarium-held speci-
mens of Belonophago tinanti indicates that it is an obligate
pterygophage feeding exclusively on caudal fins from a
wide range of species, although prey preferences in wild
populations remain to be determined.
Our results indicate that at least two species of pterygo-
phagous distichodontids (i.e., Phago boulengeri and
Ichthyborus quadrilineatus) engage in cannibalism. This
unanticipated finding underscores the manifestly oppor-
tunistic prey selection strategy of fin-eating distichodon-
tids, allowing them to feed on any accessible resources,
even members of their own species. We note in this
regard that examination of the caudal fins of over 70 pre-
served specimens of P. boulengeri held in the AMNH
collection reveals a high proportion (>20%) of fins show-
ing clear evidence of attack. The damaged fins character-
istically are missing a discrete block of fin rays that
appear to have been cleanly sheared off (Fig. 2). While it
is not possible to ascertain whether all of these Phago
specimens were subject to intraspecific attack, or attack
by other sympatric pterygophagous distichodontids, such
a high incidence of fin damage in the species is notewor-
thy. Although cannibalism in fishes is widespread and has
been documented in numerous families from across the
teleost tree of life (Smith and Reay 1991), most known
instances represent filial cannibalism, in which adults
consume all or part of their own offspring (Manica
2002). The present study appears to be the first to report
the occurrence of ectoparasitic cannibalism by pterygo-
phagous fishes.
In an early study investigating fin-eating behavior in
distichodontid fishes, Roberts (1990) proposed that
6ª2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
Pterygophagy in Distichodontid Fishes J. Arroyave and M. L. J. Stiassny

Figure 2. Characteristically damaged fins in
Phago specimens victims of pterygophagy.
Scale bars represent 1 cm.
Figure 3. Citharinoid phylogeny (modified
after Arroyave et al. 2013), with the
distichodontid “J clade” highlighted and
pterygophagous lineages indicated by red
circles.
ª2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 7
J. Arroyave and M. L. J. Stiassny Pterygophagy in Distichodontid Fishes

aggressive mimicry is used as a strategy for successful
pterygophagy in Eugnathichthys. While aggressive mimicry
appears to be the preferred strategy in the few lepidopha-
gous and pterygophagous freshwater fishes so far investi-
gated (Hori and Watanabe 2000; Sazima 2002), in the
case of the distichodontids investigated here our results
do not support that hypothesis. The striking-barred color-
ation and patterning of the caudal fins of Eugnathichthys
eetveldii and E. macroterolepis first noted by Roberts
(1990) is recognized here as a character diagnostic of a
clade of distichodontid fishes (designated the “J clade” by
Arroyave et al. (2013), p. 11, fig. 4), and no other distich-
odontids share this feature (Fig. 3). While the “J clade”
does include all pterygophagous genera, it also includes
three genera with members that are either piscivores
(Mesoborus) or insectivores (Hemistichodus and Micros-
tomatichthyoborus). The topology of Arroyave et al.’s
(2013) distichodontid tree (Fig. 3) suggests that this cau-
dal patterning is likely an exaptation (sensu Gould and
Vrba (1982)) rather than an adaptation for aggressive
mimicry. The results of this study therefore suggest that
Roberts’s (1990) findings (i.e., similar caudal coloration
between predator and prey) are simply coincidental. The
fact that none of the prey species identified in the present
study (with the exception of the cannibalized individuals)
display a caudal-barring pattern or coloration similar to
that found in their pterygophagous predators further
refutes the notion that fin-eating distichodontids are uti-
lizing aggressive mimicry as a strategy for successful
pterygophagy.
Although highly unusual, pterygophagy in teleost fishes
is not exclusive to distichodontids and has been docu-
mented in a few other groups, such as piranhas of the
genus Serrasalmus (Northcote et al. 1986, 1987; Nico and
Taphorn 1988), blennies of the genus Aspidonotus (Eibl-
Eibesfeldt 1959; Randall and Randall 1960; Kuwamura
1983), and cichlids of the genera Docimodus (Ribbink
1984) and Genyochromis (Ribbink et al. 1983). Neverthe-
less, information on predator-prey interactions for most
of these is virtually nonexistent, and the present study
represents the first assessment of prey preferences in a
group of highly specialized pterygophagous fishes.
Although dietary studies such as the one presented here
are primarily qualitative, basic knowledge of species-level
interactions between predators and prey constitutes the
very first step in determining more precise food-web
characterizations in complex tropical freshwater ecosys-
tems.
Acknowledgments
We are grateful to the following institutions, programs,
and individuals for their support, financial, and otherwise:
the Department of Ichthyology of the American Museum
of Natural History (AMNH) through the Axelrod
Research Curatorship, which provided the bulk of fund-
ing for this study; the DNA Learning Center (DNALC) of
Cold Spring Harbor Laboratory (CSHL) via its Urban
Barcoding Research Program (UBRP) provided additional
funding and facilitated the participation of high school
students Jason Cruz and Paul Jorge (City College Acad-
emy of the Arts) who assisted with data generation in the
early stages of the study. Finally, we extend our particular
thanks to Tobit Liyandja (University of Kinshasa) for his
efforts to collect additional specimens of Ichthyborus and
Eugnathichthys for this study.
Conflict of Interest
None declared.
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J. Arroyave and M. L. J. Stiassny Pterygophagy in Distichodontid Fishes