Zoosyst. Evol. 99 (1) 2023, 161-171 | DOI 10.3897/zse.99.98341

Gp Museum ror
BERLIN

Chromatic polymorphism in 7richomycterus albinotatus (Siluriformes,
Trichomycteridae), a mountain catfish from south-eastern Brazil and the
role of colouration characters in trichomycterine taxonomy

Wilson J. E. M. Costa‘, José Leonardo O. Mattos!, Pedro F. Amorim},
Beatrizz O. Mesquita', Axel M. Katz!

1 Laboratory of Systematics and Evolution of Teleost Fishes, Institute of Biology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
https://zoobank. org/D656 BOF 3-BA16-418E-992C-F0D3957C4CEB

Corresponding author: Wilson J. E. M. Costa (wcosta@acd.ufr).br)

Academic editor: Nicolas Hubert # Received 4 December 2022 Accepted 31 January 2023 Published 23 February 2023

Abstract

Colouration is an important tool for systematists inferring species limits and phylogenetic relationships of teleost fishes, but the
use of colouration variation in trichomycterine catfish systematics has generated some controversy. We first report and describe the
occurrence of four, geographically disjunct colour morphs in 7richomycterus albinotatus, endemic to south-eastern Brazil, as well
as ontogenetic colouration change in each morph. A phylogenetic analysis using a cytb fragment (1098 bp) for 23 specimens rep-
resenting all colour morphs and four outgroups did not support any correlation between colour morphs and lineages, with different
colour morphs sharing identical haplotypes. This study indicated that young adult specimens found in lighter habitats had white
and brown to black spots on the flank, whereas similar-sized specimens inhabiting darker habitats had white spots inconspicuous
or absent and dark brown or black spots expanded. Individuals above about 65 mm SL of all populations had flank white marks
less conspicuous or absent and cryptic habits during daylight, contrasting with smaller individuals with white marks and actively
swimming above the substrate. Literature data indicate that ontogenetic colouration and habit changes occur in different trichomyc-
terid lineages. Our data thus show that colouration may be problematic in taxonomical studies, although often being consistently
used to diagnose species and clades. We conclude that colouration should not be discarded a priori as evidence of trichomycterine
relationships and species limits, but should be used with caution in systematic studies, being necessary additional evidence, such as
osteological characters or molecular data.

Key Words

Atlantic Forest, colouration ontogenetic change, mountain biodiversity, Trichomycterinae

Introduction

Colouration has been an important source of characters
for systematists diagnosing species and inferring phylo-
genetic relationships of teleost fishes. Amongst catfishes
of the Trichomycterinae, a diverse Neotropical catfish
trichomycterid subfamily with over 260 species (Fricke et
al. 2022), colouration has been broadly used to diagnose
Species since Eigenmann’s (1918) monographic review of
trichomycterids. Tchernavin (1944), however, did not con-
sider colouration as valid evidence to diagnose species, us-

ing primarily morphometric data for reviewing trichomyc-
terines from the Lake Titicaca and adjacent Andean river
basins, consequently synonymising several species with
different colour patterns. Following Tchernavin’s view,
Arratia and Menu-Marque (1981) and Arratia (1983a)
recorded phenotypic colour variation in southern Ande-
an trichomycterines related to different habitat substrate
types, also synonymising nominal species with different
colour patterns. However, according to Bockmann and
Sazima (2004), intraspecific colour variation in trichomyc-
terids is highly unusual and is useful to diagnose species.

Copyright Costa, W.J.E.M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited.

162

Several authors have used colour patterns to distin-
guish trichomycterine species, with colouration playing
an important role in species diagnoses of most recent
taxonomical papers (e.g. Costa (1992); de Pinna (1992);
Barbosa and Costa (2008); Datovo et al. (2012); Ferrer
and Malabarba (2013); Rizzato and Bichuette (2014);
Teran et al. (2017); Katz and Costa (2022); Costa et al.
(2020a, 2021)). Colour patterns have also been used to
diagnose species groups (Barbosa and Costa 2010a;
Costa et al. 2020b; Vilardo et al. 2020; Costa and Katz
2021). On the other hand, colouration polymorphism
and ontogenetic colour changes have been recorded for
several species (Lima et al. 2008; da Silva et al. 2010;
Nascimento et al. 2017; Ochoa et al. 2017; Costa et al.
2022a), as well as similar homogeneous black coloura-
tion being reported to homoplastically occur in sympat-
ric trichomycterines with cryptic habits during daylight
(Costa et al. 2020b), making species identification based
on colouration problematic.

In the Atlantic Forest of south-eastern Brazil, catfishes
of the genus Trichomycterus Valenciennes, 1832 repre-
sent the fish group with the greatest number of species
adapted to life in mountain rivers, with about 60 val-
id species (Costa et al. 2020a; Costa and Katz 2021).
Amongst these species 1s 7richomycterus albinotatus
Costa, 1992, endemic to the upper section of the Rio Pre-
to drainage, Rio Paraiba do Sul Basin (Costa 1992, 2021),
which drains the Serra da Mantiqueira, a vast mountain
range in south-eastern Brazil with highest peaks reaching
about 2900 m. Juveniles and smaller adults of 7. albinota-
tus are diurnal, being easily observed actively swimming
near the river bottom during daylight (Costa 1992, 2021),
but specimens above 65 mm of standard length (SL) are
never seen swimming during daylight period, thus pre-
sumably considered nocturnal.

Data on distribution and colouration of 7! albinotatus
are still restricted to its original description (Costa 1992).
At that time, 7. al/binotatus was recorded for a short area
of the main course of the upper Rio Preto and adjacent
tributaries (Corrego Véu de Noiva and Ribeiraéo San-
ta Clara) and a colour pattern consisting of alternating
white, black and brown spots on the flank was used to
diagnose this species. In subsequent field studies, more
specimens exhibiting the same characteristics were col-
lected in this area, always in altitudes above about 1200
m above sea level (a.s.1.). More recently, however, spec-
imens similar to 7: albinotatus topotypes, but exhibiting
different flank colouration were found in separate areas
of the same drainage, in altitudes above about 1150 m
a.s.1. Specimens from these geographically disjunct areas
had other morphological characters identical to those ex-
hibited by topotypes of T. albinotatus, including shape,
size and relative position of eye, nares and fins, head
and body proportions and number of fin rays, vertebrae,
odontodes and branchiostegal rays. Interestingly, no
specimen similar to 7. albinotatus was found in slight-
ly lower altitude areas between these sub-drainages, at
about 1100 maz.s.I. or less. The primary objective of this

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Costa, W.J.E.M et al.: Chromatic polymorphism in Trichomycterus albinotatus

study is to first describe the different geographically dis-
junct colour morphs and ontogenetic colouration chang-
es in 7 albinotatus, providing data on their respective
geographical distribution and habitats. Since species di-
versity is often high in mountain regions (Hoorn et al.
2013) and the presence of closely-related species inhab-
iting small neighbouring areas is not a rare phenomenon
amongst mountain organisms (Dimitrov et al. 2012), the
second objective of this study is to check some genet-
ic divergence amongst geographically isolated colour
morphs thorough a phylogenetic analysis using a seg-
ment of the mitochondrial gene cytochrome b (1098 bp)
for 23 specimens from 11 collecting sites, representing
all colour morphs.

Materials and methods

Field methods and specimen collections

Field methods and euthanasia methods were approved by
CEUA-CCS-UFRJ (Ethics Committee for Animal Use
of Federal University of Rio de Janeiro; permit number:
065/18) and collections were made with permits provided
by ICMBio (Instituto Chico Mendes de Conservacao da
Biodiversidade; permit number: 38553-7) and INEA (In-
stituto Estadual do Ambiente; permit number: 037/2018).

Field studies were made during five trips to the upper
section of the Rio Preto drainage (May and July 2015;
July 2017; September and December 2018). During
this period, sporadic collections were also made in the
middle and lower sections of the Rio Preto, where no
specimen of 7. albinotatus was found. Collections of
T: albinotatus were made in 11 collecting sites (Table 1),
in altitudes between 1155 and 1495 m a.s.l., comprising
the Rio Preto main channel and all its main tributaries
(Fig. 1). Specimen collections were made with small dip
nets (40 x 30 cm) during daylight. Euthanasia followed
AVMA Guidelines for the Euthanasia of Animals (Leary
et al. 2020); specimens were euthanised using a buffered
solution of tricaine methane sulphonate (MS-222) at a
concentration of 250 mg/l, for a period of 10 min or
more, until completely ceasing opercular movements.
Just after collection, specimens were observed in small
transparent plastic bottles to record general colour
patterns and fin morphology and then euthanised, except
for usually two or three individuals representing each
collecting locality that were kept alive for about 5—10
hours, photographed in small aquaria and then euthanised
as described above. Most specimens were fixed in 10%
formalin for a period of 10 days and then transferred
to 70% ethanol; specimens to be used in molecular
analyses were fixed and preserved in 98% ethanol,
indicated in the list of material examined (Appendix
1) as ‘DNA’ just after catalogue numbers. Specimens
were later deposited in the ichthyological collection of
the Institute of Biology, Universidade Federal do Rio
de Janeiro, Rio de Janeiro (UFRJ). Geographical names

Zoosyst. Evol. 99 (1) 2023, 161-171

> .| altitude

‘>| ES 2,600 m
/ | 1,800 m_
1,300 m |
| 1,100 m |
| 900m
500m

Figure 1. Geographical distribution of Trichomycterus albinotatus and its colour morphs (CMs): dots, CM1; triangle, CM2; dia-
monds, CM3; squares, CM4. Numbers are collecting sites with DNA samples.

are according to the regional popular use in Portuguese
(e.g. Corrego, Ribeirio, Rio), making easier recognition
of localities in the field and avoiding common equivocal
generalisations when tentatively translating them to
English. A complete list of specimens analysed appears
in the Appendix 1.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from muscular tis-
sue of the right side of the caudal peduncle using the
DNeasy Blood & Tissue Kit (Qiagen), according to the
manufacturer’s protocol. The analyses included a frag-
ment of the mitochondrial encoded gene cytochrome b
(CYTB), which have been successfully used to delimit
trichomycterine species (Costa and Katz 2021). Amplifi-
cation was made through the polymerase chain reaction
(PCR) method, using the primers Cytb Siluri F and Cytb
Siluri R (Villa-Verde et al. 2012). Double-stranded PCR
amplifications were performed in 60 ul reactions with
reagents at the following concentrations: 5x GreenGo-
Taq Reaction Buffer (Promega), 1 mM MgCl, 1 mM
of each primer, 75 ng of total genomic DNA, 0.2 mM
of each dNTP and 1 U of standard Taq polymerase or
Promega GoTaq Hot Start polymerase. The thermocy-
cling profile was as follows: initial denaturation for 2
min at 94-95 °C; 35 cycles of denaturation for 1 min at
94°C, annealing for 1 min at 48.0—60.0 °C and extension
for 1—2 min at 72 °C; and terminal extension for 4 min
at 72 °C. Negative controls were used to check on con-
taminations. The PCR products were then purified using
the Wizard SV Gel and PCR Clean-Up System (Prome-
ga). Sanger Sequencing reactions were made by Mac-

Table 1. Collecting sites, coordinates, altitude and UFRJ cat-
alogue numbers of specimens of 7richomycterus albinotatus
used in the molecular analysis. All localities are situated
in the upper Rio Preto drainage, Rio Paraiba do Sul Basin,
south-eastern Brazil.

Collecting sites Coordinates and altitude Voucher
number
Rio Preto main channel
CS1 Cachoeira do 22°19'54'S, 44°36'57'W, 1495m = 11659
Escorrega
CS2 Rio Preto, Marombal 22°19'33"S, 44°35'27'W, 1305m 9873,
11976
CS3 Rio Preto, Maromba 2 =. 22°19'33"S, 44°36'11"W, 1250 m_ 11661-2,
10476
Ribeirao Santa Clara subdrainage
CS4 Cachoeira Santa Clara 22°18'53"S, 44°35'45'"W, 1215m_ = 11665
Corrego da Cruzes subdrainage
CS5 Corrego das Cruzes 22°20'16'S, 44°35'21"W, 1195 m = 12035
Rio do Marimbondo subdrainage
CS6 Cachoeira do 22°21'41°S, 44°35'15'W, 1435m_ 11932,
Marimbondo 11983
Corrego Alcantilado subdrainage
CS7 Corrego Alcantilado 1 = 22°17'36'S, 44°33'39"W, 1320 m_ = 11658,
11663
CS8 Corrego Alcantilado 2. = 22°17'33'S, 44°33'32'W, 1295m 11664
CS9 Cachoeira da Muralha = 22°17'38"S, 44°33'26"W, 1170m_ = 11964
CS10 Corrego Alcantilado 3. 22°17'48"S, 44°33'19'"W, 1155m_ = 11963
Ribeirao das Flores subdrainage
CS11 Cachoeira da Prata 22°14'48'S, 44°31'19'W, 1220m_ 11931

rogen Inc. (South Korea). Sequencing chromatograms
and sequences were assessed using MEGA 7 (Kumar
et al. 2016). The generated DNA sequences were trans-
lated into amino acids residues to verify the absence of
premature stop codons or indels using the programme
MEGA 7. A list of GenBank access numbers 1s available
in Online Resource 2.

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164 Costa, W.J.E.M et al.: Chromatic polymorphism in Trichomycterus albinotatus

Phylogenetic analysis

A phylogenetic analysis was performed using a CYTB
fragment, 1098 bp, for 23 specimens of 7 albinotatus from
11 collecting sites (CS 1—11), representing all colour morphs
(CM1-4). Outgroups included single sequences of four
congeners, including 7richomycterus vitalbrazili Vilardo,
Katz & Costa, 2020, the sister group of 7! albinotatus,
the only other member of the subgenus Humboldtglanis
Costa, 2021; Trichomycterus brasiliensis Litken, 1874 and
Trichomycterus mirissumba Costa, 1992, two species of
the subgenus Cryptocambeva Costa, 2021, the sister group
of Humboldtglanis, and Trichomycterus auroguttatus
Costa, 1992, a member of the subgenus Psammocambeva
Costa, 2021, the sister group of the clade comprising
Cryptocambeva plus Humboldtglanis. The data were
partitioned according to codon positions; the best-fitting
models of molecular evolution for each partition was
found using the Bayesian Information Criterion (BIC) of
ModelFinder (Kalyaanamoorthy et al. 2017), implemented
in IQ-TREE 1.6.11 (Nguyen et al. 2015), which found
the K2P+FQ+I+G4 model for the 1 codon position, the
TN+F+I model for 2" codon position and the GTR+FI+G4
model for the 3 codon position. The dataset was analysed
using Maximum Likelihood (ML) methods implemented
in IQ-TREE, with two methods for assessing the reliability
of internal branches: the Shimodaira-Hasegawa-like
procedure support (SH-aLRT; Guindon et al. (2010)) and
the ultrafast bootstrap support (UFBoot; Minh et al. (2013);
Hoang et al. (2017)), using 1000 replicates and default
parameters. Genetic distances were calculated to illustrate
genetic diversity amongst taxa using Kimura 2-parameter
(K2P) model (Kimura 1980) in MEGA 7.

Results

Colour morphs

Four colour morphs were found, as below described (see
Fig. 1 for geographical distribution of colour morphs).

Colour morph 1 (CM1). Description: In specimens
between about 30 and 60 mm SL (Fig. 2B—E), flank pale
brownish-yellow, darker above longitudinal body mid-
line; two horizontal rows of rounded to horizontally elon-
gated brown to black spots, alternated with similar-shaped
white spots, sometimes with faint golden iridescence.
Horizontal row of pale brown spots irregularly shaped on
ventral part of flank, sometimes inconspicuous. In larger
adult specimens above about 65 mm SL (Fig. 2A), dark
spots on flank enlarged and coalesced to form vermicu-
late pattern; light marks paler.

Distribution and habitat: Areas above 1150 m a.s.l.
of the upper course of the Rio Preto, the Ribeirao Santa
Clara and Corrego das Cruzes drainage (CS1-6; Fig. 1).
Rivers and streams about 2-15 m wide, deepest areas
about 5 m deep, running in broad valleys, making the
riverine environment highly exposed to sunlight. Water
clear, hyaline. Common presence of large rocks favours

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formation of small pools where most specimens were
concentrated (Fig. 3A, B). Bottom with light grey rocks
and multicolour gravel, predominating white, light grey
and light brownishyellow colours.

Colour morph 2 (CM2). Description: Similar to CM1,
except for white marks irregularly shaped and dark marks
highly coalesced even in smaller specimens (Fig. 2F). In
a single specimen found above 65 mm SL, white spots
absent (Fig. 2G).

Distribution and habitat: Upper Rio do Marimbondo,
altitude about 1435 m a.s.l. (CS6; Fig. 1). River about
5—10 m wide, deepest areas about 3 m deep, running in
broad valley, riverine environment highly exposed to
sunlight. Water clear, greenish hyaline. Common pres-
ence of large rocks favours formation of small pools be-
low waterfalls where most specimens were concentrated
(Fig. 3C). Bottom predominantly with light grey rocks of
different sizes.

Colour morph 3 (CMS3). Description: Similar to
CM1, but white spots on body mid-line highly coalesced,
usually with intense golden iridescence (Fig. 2H).

Distribution and habitat: Rio das Flores subdrainage at
about 1220 m a.s.l. (Fig. 1; CS11). Rivers about 5-10 m
wide, deepest areas about 2 m deep, running in broad val-
ley, riverine environment highly exposed to sunlight. Wa-
ter clear, yellowish hyaline. Rocks small, rare small wa-
terfalls where most specimens are concentrated (Fig. 3E).
Bottom predominantly with small light grey rocks, light
grey to light yellow gravel and sand.

Colour morph 4 (CM&4). Description: Flank and head
pale yellow, with dark brown to black spots (Fig. 2I—J);
flank dark spots highly coalesced in larger specimens
(Fig. 2J), forming interconnected vermiculate pattern,
flank white spots almost inconspicuous in smaller speci-
mens (Fig. 21), always absent in specimens above about
65 mm SL (Fig. 2J).

Distribution and habitat: Upper section of the Cor-
rego do Alcantilado (CS7-10; Fig. 1), at altitudes be-
tween 1155 and 1320 ma.s.l. Stream about 2—5 m wide,
deepest areas barely reaching about 2 m deep, running
in narrow and steep valley, with numerous small water-
falls (Fig. 3D); riverine environment poorly exposed to
sunlight. Water clear, yellowish hyaline. Bottom predom-
inantly composed of dark grey rocks.

Phylogeny

The phylogenetic analysis highly supported 7. albinotatus
as a unique lineage, with maximum values for both
SH-aLRT and UFBoot (Fig. 4). The tree topology did
not support any correlation between colour morphs and
lineages, with colour morphs not forming unique lin-
eages. Genetic distances amongst haplotypes were low
(0.1-0.4%). The eleven haplotypes found amongst the
23 sequenced individuals were scattered in different lo-
calities. H1 was the most common haplotype, present in
seven analysed specimens collected in seven separate lo-
calities and exhibited all the four colour morphs.

Zoosyst. Evol. 99 (1) 2023, 161-171 165

a ———

. gy 2 ott eh tapi Ser

Figure 2. Colour morphs (CMs) of Trichomycterus albinotatus, with respective collecting sites (CSs) and collection catalogue num-
bers: CM1 (a-e); CM2 (f-g); CM3 (h); CM4 (i-j). a. CS3, UFRJ 11062, 66.8 mm SL; b. CS3, UFRJ 11062, 40.5 mm SL; e. CS3,
UFRJ 11976, 44.3; d. CS5, UFRJ 12035, 44.9 mm SL; e. CS5, UFRJ 12035, 46.2 mm SL; f. CS6, UFRJ 12064, 45.2 mm SL; g. CS6,
UFRJ 12064, 67.0 mm SL; h .CS11, UFRJ 12052, 49.2 mm SL; i. CS9, UFRJ 11670, 44.8 mm SL; j. CS10, UFRJ 12063, 71.5 mm SL.

Discussion
Distribution of colour morphs

High levels of intraspecific colouration polymorphism
have been recorded in taxonomical papers on some
trichomycterines, including both discrete and indiscrete
colouration variation in syntopic specimens (e.g. Arra-
tia et al. (1978); Sarmento-Soares et al. (2005); Lima et
al. (2008)). On the other hand, according to Arratia and
Menu-Marque (1981) and Arratia (1983a), some Andean
trichomycterine species exhibit distinct colour morphs
in disjunct habitats with different river substrates. In the
present study, we found specimens of 7. albinotatus ex-
hibiting different colour morphs in disjunct areas of the
upper Rio Preto drainage, with slightly different eco-
logical conditions (Fig. 2; Table 2). In the most extreme
case (compare Fig. 2C with Fig. 2I for specimens about
45 mm SL and Fig. 2A with Fig. 2J for specimens about
65-70 mm SL), specimens found in habitats with light
substrate, situated in wide valleys highly exposed to sun-
light, had a colour pattern consisting of alternating white
and brown to black spots (CM1, Fig. 2A—C). Contrast-
ingly, specimens found in a stream with dark grey rocks
on the bottom, situated in a narrow steep valley weakly
exposed to sunlight were darker, with dark spots elongat-
ed and coalesced and white spots inconspicuous or absent
(CM4, Fig. 2I—J). It is remarkable that, during daylight
field studies, CM1 specimens when swimming close to
multicolour gravel substrate were often almost invisible,

showing that CM1 may favour camouflage in this kind of
habitat. These data suggest that the existence of different
colour morphs may be related to specific kinds of habitat,
a hypothesis to be tested only after detailed inventory on
physical and chemical factors of the riverine habitats be-
ing available.

Ontogenetic colouration and habitat change

In all colour morphs of 7: al/binotatus, white marks on
the flank were less conspicuous or had completely dis-
appeared, whereas dark marks have expanded their
extent in individuals above 65 mm SL (Fig. 2A, G, J).
This ontogenetic colour change may be related to field
evidence indicating that specimens about 60 mm or less
are typically diurnal, whereas larger ones are apparently
nocturnal. The presence of rows of alternating dark and
light spots on the body side is a common colour pattern
occurring in diurnal trichomycterids living in sandy or
gravel substrate, which contribute to making them little
visible for predators (Zuanon and Sazima 2004). For ex-
ample, in 7: auroguttatus, a typical diurnal psammophilic
Species sympatric to 7? albinotatus (Costa 2021), there
are alternating dark brown and golden spots on the flank
(Costa 1992). On the other hand, different species having
cryptic habits during daylight have dark colouration and
no bright mark (Costa et al. 2020b). A similar ontogenetic
change was recorded for 7° vitalbrazili, the sister group of
T. albinotatus (Vilardo et al. 2020).

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166

Table 2. List of specimens used in the molecular analy-
sis, with respective voucher number in the ichthyological
collection of the Biology Institute, Federal University of Rio
de Janeiro (UFRJ) and GenBank accession numbers. In speci-
mens of 7: albinotatus, CS means collecting site (see Table 1);

CM, colour morph; H, haplotype.

Voucher Species Accession
number numbers
9873.1 Trichomycterus albinotatus CS2CM1 H2 MT459172
10476.1 Trichomycterus albinotatus CS3CM1H1 MT459173
11662.1 Trichomycterus albinotatus CS3 CM1 H3 MT459174
11663.1 Trichomycterus albinotatus CS7CM4H1 MT459175
11664.1 Trichomycterus albinotatus CS8 CM4 H2 MT459176
11658.2 Trichomycterus albinotatus CS7CM4H2 MT459177
11659.2 Trichomycterus albinotatus CS1CM1 H4 MT459178
11665.1 Trichomycterus albinotatus CS5 CM1 H2 MT459179
11665.2 Trichomycterus albinotatus CS5 CM1 H5 MT459180
11931.1 Trichomycterus albinotatus CS11CM3H6 MT459181
1193:1,2 Trichomycterus albinotatus CS11CM3H1 MT459182
11932.2 Trichomycterus albinotatus CS6 CM2H2 MT459183
11932.3 Trichomycterus albinotatus CS6 CM2H2 MT459184
11963.1 Trichomycterus albinotatus CS10CM4H8 MT459185
11963.2 Trichomycterus albinotatus CS10CM4H7 #MT459186
11963.3 Trichomycterus albinotatus CS10CM4 H1 MT459187
11963.4 Trichomycterus albinotatus CS10CM4H8 M1459188
11964.1 Trichomycterus albinotatus CS9 CM4 H9 MT459189
11964.2 Trichomycterus albinotatus CS9 CM4H1 MT459190
11976.1 Trichomycterus albinotatus CS2CM1H10 MT459191
11976.2 Trichomycterus albinotatus CS2CM1 H11 MT459192
11983.1 Trichomycterus albinotatus CS6 CM2H1 MT459193
12035.1 Trichomycterus albinotatus CS5 CM1H1 MT459194
11968.2 — Trichomycterus auroguttatus MT470410
10642 Trichomycterus brasiliensis MT470418
11672.2 Trichomycterus mirissumba MT470411

Little is still known about natural history of

trichomycterines; therefore, it is not possible to infer
securely if the synchronic change of colouration and
period of activity in 7 albinotatus and T: vitalbrazili
corresponds to a unique evolutionary event or if it is
a widespread condition amongst trichomycterines.
Miranda-Ribeiro (1905) first reported conspicuous
differences in the colouration of juvenile and adult
specimens of trichomycterines. Since then, several papers
have provided evidence of ontogenetic colouration change
in Trichomycterus and the closely-related genus Cambeva
(Costa 1992; Barbosa and Costa 2008, 2010b; da Silva
et al. 2010; Ferrer and Malabarba 2013; Reis et al. 2020;
Costa et al. 2021, 2022b). Amongst these studies, only
da Silva et al. (2010) have demonstrated that small and
large individuals of Cambeva iheringi (Eigenmann, 1917)
besides having a distinct colour pattern, are also found
in different habitats. According to these authors, spotted
juveniles of C. iheringi live partially or totally buried
in light sandy substrate, whereas dark-coloured larger
adults are found in areas with dark stones. Arratia (1983b)
reported juveniles and small adults of three Chilean
trichomycterines living in shallower and more exposed
habitats than larger adults, but no mention was made of
possible ontogenetic change in colouration. Furthermore,
recent studies in species of the trichomycterid subfamilies

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Costa, W.J.E.M et al.: Chromatic polymorphism in Trichomycterus albinotatus

Copionodontinae and ‘Trichogeninae indicate — that
ontogenetic changes in the activity period is followed by
colouration change (Sazima 2004; Zanata and Primitivo
2013). Sazima (2004) noted that juveniles and small adults
of Trichogenes longipinnis Britski & Ortega, 1983 are more
active at the daytime, whereas large adults with darker
colours are mainly seen at night. Similarly, Zanata and
Primitivo (2013) reported diurnal nektonic habits in small
juveniles of Copionodon pecten de Pinna, 1992, as well as
nocturnal benthonic habits in sub-adults and adults, which
may exhibit a darker coloured phenotype. Therefore, since
copionodontines and trichogenines together form a clade
distantly related to trichomycterines (Katz et al. 2018), it is
possible that synchronic change of colouration and habits
during ontogeny have occurred independently in the clade
comprising copionodontines and trichogenines and in the
clade comprising 7’ albinotatus and T: vitalbrazili.

Role of colouration in trichomycterine
taxonomy

This study firstly reported different colour morphs of
T. albinotatus inhabiting geographically disjunct areas,
which could induce taxonomists to recognise them as dif-
ferent species. Although the occurrence of geographically
disjunct colour morphs is possibly uncommon amongst
trichomycterids, data on colouration polymorphism pro-
vided by Arratia & Menu-Marque (1981) and Arratia
(1983a) suggest that colouration may be vary according
to local environmental conditions in different trichomyc-
terid lineages, attesting against the use of colouration
characters in trichomycterid systematics.

Similarly, the use of colouration alone to distinguish
sympatric species may induce misidentifications. For
example, sympatric juveniles of 7. itatiayae Miranda
Ribeiro, 1906 and 7. nigroauratus Barbosa & Costa,
2008, two species belonging to different subgenera (Costa
2021), have similar colouration, making it difficult to
distinguish them in the field (Barbosa and Costa 2008), as
well as the non-closely related, sympatric T. immaculatus
(Eigenmann & Eigenmann, 1898) and 7. quintus Costa,
2020 that share a similar overall black colouration,
are only distinguishable under careful examination
(Costa et al. 2020b).

These data do not diminish the value of colour pat-
tern characters to diagnose species and trichomycter-
ine clades. For example, even with the occurrence of
chromatic polymorphism herein reported for 7? albino-
tatus, the presence of white marks on the flank of ju-
veniles uniquely shared by 7! albinotatus and T. vital-
brazili amongst trichomycterines, clearly corroborated
monophyly of this clade formally named as subgenus
Humboldtglanis Costa, 2021, which 1s highly supported
in a phylogenetic study integrating morphology and DNA
sequences (Costa 2021). Similarly, apomorphic colour
patterns are shared by trichomycterine clades corroborat-
ed by both osteological and molecular data (Costa 2021;

Zoosyst. Evol. 99 (1) 2023, 161-171

Figure 3. Habitats of Trichomycterus albinotatus: a. Upper Rio Preto, the type locality of 7’ albinotatus, b. Upper Corrego das
Cruzes; c. Cachoeira do Marimbondo, upper Rio do Marimbondo subdrainage; d. Cachoeira da Muralha, upper Corrego do
Alcantilado subdrainage; e. Cachoeira das Antas, upper Ribeirao das Flores subdrainage.

zse.pensoft.net

168

88/65

85/80 }

100/100

0.02

Costa, W.J.E.M et al.: Chromatic polymorphism in Trichomycterus albinotatus

‘9873.1 Trichomycterus albinotatus CS2/CM1/H2
11932.2 Trichomycterus albinotatus CS6/CM2/H2
11932.3 Trichomycterus albinotatus CS6/CM2/H2
11665.1 Trichomycterus albinotatus CS4/CM1/H2
11664.1 Trichomycterus albinotatus CS8/CM4/H2
11658.2 Trichomycterus albinotatus CS7/CM3/H2
11931.1 Trichomycterus albinotatus CS11/CM3/H2
11659.2 Trichomycterus albinotatus CS1/CM1/H4
11963.1 Trichomycterus albinotatus CS10/CM4/H1
11963.1 Trichomycterus albinotatus CS10/CM4/H8
11976.2 Trichomycterus albinotatus CS2/CM1/H3
11963.2 Trichomycterus albinotatus CS10/CM4/H7
10476.1 Trichomycterus albinotatus CS3/CM1/H1
11964.2 Trichomycterus albinotatus CS9/CM4/H1
11931.2 Trichomycterus albinotatus CS11/CM3/H1
11983.1 Trichomycterus albinotatus CS6/CM2/H1
11963.3 Trichomycterus albinotatus CS10/CM4/H1
11665.2 Trichomycterus albinotatus CS4/CM1/H5
11964.1 Trichomycterus albinotatus CS9/CM4/H9
11663.1 Trichomycterus albinotatus CS7/CM4/H1
12035.1 Trichomycterus albinotatus CS5/CM1/H1
11976.1 Trichomycterus albinotatus CS2/CM1/H10
11662.1 Trichomycterus albinotatus CS3/CM1/H3

12125.1 Trichomycterus vitalbrazili

1167.2 Trichomycterus mirissumba
10642.1 Trichomycterus brasiliensis

Figure 4. Phylogenetic relationships amongst 23 specimens of Trichomycterus albinotatus and three outgroups, inferred by
Maximum Likelihood analysis performed in IQ-TREE using a cyt b fragment 1098 bp. Numbers close to nodes are support values:
SH-aLRT support (%) and ultrafast bootstrap support (%). CS1—11, collecting sites 1-12; CM1-4, colour morphs 1-4; H1I-11,
haplotypes 1-11. The most external outgroup is not represented in the figure.

Costa and Katz 2021), thus demonstrating that colour
patterns may be effectively used for diagnosing tricho-
mycterine species and groups. These data in fact show
that colouration should not be discarded a priori as ev-
idence of species limits or phylogenetic relationships in
trichomycterines, as well as colouration alone should not
be used to infer species limits, being necessary to rein-
force species delimitation hypotheses using additional
morphological evidence (e.g. osteological characters) or
molecular data.

Acknowledgements

We are grateful to M.A. Barbosa, C.P. Bove, B.B. Cos-
ta, E. Henschel and S. Lopes for assistance during field
expeditions and to L.M. da Cunha for providing permis-
sion to collect fish in Sitio Cachoeiras do Alcantilado.
A former version of this paper has benefitted from crit-

zse.pensoft.net

icisms provided by two anonymous reviewers. Thanks
are also due to Heok Hee Ng for suggestions provided
in the present version. Instituto Chico Mendes de Con-
servacéo da Biodiversidade and Instituto Estadual do
Ambiente provided collecting permits. This work was
supported by Conselho Nacional de Desenvolvimento
Cientifico e Tecnologico (CNPq; grant 304755/2020-6
to WJEMC), Funda¢aéo Carlos Chagas Filho de Amparo
a Pesquisa do Estado do Rio de Janeiro (FAPERJ; grant
E-26/201.213/2021 to WJEMC, E-26/202.005/2020 to
AMK and E-26/202.327/2018 to JLM) and Coorde-
nacao de Aperfeicoamento de Pessoal de Nivel Superi-
or (CAPES; grant 88882.425731/2019-01 to BOM and
grant 88887.466724/2019-00 to PFA). This study was
also supported by CAPES (Finance Code 001) through
Programa de Poés-Gradua¢ao em Biodiversidade e Bio-
logia Evolutiva /UFRJ, Programa de Pos-Graduacao em
Genética/UFRJ and Programa de P0os-Graduacaéo em
Zoologia, Museu Nacional/UFRJ.

Zoosyst. Evol. 99 (1) 2023, 161-171

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Zoosyst. Evol. 99 (1) 2023, 161-171

Appendix 1

List of specimens, with respective catalogue numbers of
the ichthyological collection of the Institute of Biology,
Federal University of Rio de Janeiro (UFRJ). DNA, means
specimens fixed and preserved in 98% ethanol; other spec-
imens were fixed in 10% formalin and then preserved in
70% ethanol. All specimens collected in the Rio Preto
drainage, Rio Paraiba do Sul basin, south-eastern Brazil.
Rio de Janeiro State: Itatiaia Municipality: — UFRJ
10476, 2 (DNA); Rio Preto about 650 m above the village
of Maromba, 22°19'33"S, 44°36'11"W, about 1240 m asl
(type locality); W. J. E. M. Costa et al., 3 May 2015. —
UFRJ 11062, 4; UFRJ 11976, 5 (DNA); same locality as
anterior; W. J. E. M. Costa et al., 27 Sep. 2018. — UFRJ
11661, 1 (DNA), UFRJ 11662, 1 (DNA), UFRJ 11668, 6;
same locality as anterior; W. J. E. M. Costa et al., 25 Jul.
2017. — UFRJ 9873, 2 (DNA); Rio Preto, about 930 m
below Cachoeira do Escorrega, 22°19'33"S, 44°35'27"W,
about 1305 m asl; same locality as UFRJ 8579; W. J. E.
M. Costa & C. P. Bove, 2 Feb. 2014. - UFRJ 10485, 3;
UFRJ 11667, 5; UFRJ 11659, 3 (DNA); Rio Preto about
100 m above Cachoeira do Escorrega, 22°19'54"S, 44°36
57"W, about 1495 m asl; W. J. E. M. Costa et al., 25 Jul.
2017. Resende Municipality: — UFRJ 12035, 12 (DNA);
UFRJ 12060, 7; Corrego das Cruzes close to the end of
Vale das Cruzes road, 22°20'16"S, 44°35'21"W, about
1200 m asl; W. J. E. M. Costa et al., 2 Nov. 2018. -UFRJ
11932, 3 (DNA); UFRJ 12064, 7; UFRJ 11983, 1 (DNA);
Poco do Marimbondo, Rio Marimbondo, 20°21'41"S,

171

44°35'15"W, about 1435 m asl; W. J. E. M. Costa et al.,
27 Sep. 2018. Minas Gerais State: Bocaina de Minas
Municipality: UFRJ 11665, 5 (DNA); Cachoeira Santa
Clara, 22°18'53"S, 44°35'45"W, about 1215 m asl; W. J.
E. M. Costa et al., 24 Jul. 2017. - UFRJ 11666, 15, Ri-
beirao Santa Clara, 22°18'58"S, 44°35'29"W, about 1160
m; W. J. E. M. Costa et al., 25 Jul. 2017.— UFRJ 11931, 3
(DNA); UFRJ 12061, 7; Cachoeira da Prata, Rio da Prata,
22°14'48"S, 44°31'19"W, about 1220 m asl; W. J. E. M.
Costa et al., 26 Sep. 2018. — UFRJ 12059, 3; Cachoeira
das Antas, 22°16'52"S, 44°32'02"W, about 1090 m asl; P.
F Amorim & B. Mesquita, 1 Sep. 2018. — UFRJ 12063, 4;
Corrego do Alcantilado, 22°17'47"S, 44°33'19"W, about
1155 masl; W. J. E. M. Costa et al., 25 Sep. 2018. —-UFRJ
11664, 1 (DNA); UFRJ 11670, 3; Corrego do Alcantilado
about 300 m from Cachoeira do Alcantilado, 22°17'33"S,
44°33'32"W, about 1295 m asl; W. J. E. M. Costa et al.,
26 Jul. 2017. — UFRJ 11663, 1 (DNA); Corrego do Al-
cantilado, close to cave about 150 m from Cachoeira do
Alcantilado, 22°17'36"S, 44°33'39"W, about 1320 m asl;
W. J. E. M. Costa et al., 26 Jul. 2017. - UFRJ 11658, 4
(DNA); idem; W. J. E. M. Costa et al., 25 Sep. 2018. —
UFRJ 11964, 3 (DNA); Cachoeira da Muralha, Corrego
do Alcantilado, 22°17'38"S, 44°33'26"W, about 1170
m asl; W. J. E. M. Costa et al., 25 Sep. 2018. — UFRJ
11963, 4 (DNA); Corrego do Alcantilado, 22°17'48"S,
44°33'19"W, about 1155 m asl; W. J. E. M. Costa et al.,
25 Sep. 2018.

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