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

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 Trichomycterus 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.


Introduction
Colouration has been an important source of characters for systematists diagnosing species and inferring phylogenetic 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 consider colouration as valid evidence to diagnose species, us-ing primarily morphometric data for reviewing trichomycterines 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 Andean 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 trichomycterids is highly unusual and is useful to diagnose species.
In the Atlantic Forest of south-eastern Brazil, catfishes of the genus Trichomycterus Valenciennes, 1832 represent the fish group with the greatest number of species adapted to life in mountain rivers, with about 60 valid species (Costa et al. 2020a;Costa and Katz 2021).Amongst these species is Trichomycterus albinotatus Costa, 1992, endemic to the upper section of the Rio Preto drainage, Rio Paraíba do Sul Basin (Costa 1992(Costa , 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 T. albinotatus are diurnal, being easily observed actively swimming near the river bottom during daylight (Costa 1992(Costa , 2021), but specimens above 65 mm of standard length (SL) are never seen swimming during daylight period, thus presumably considered nocturnal.
Data on distribution and colouration of T. albinotatus are still restricted to its original description (Costa 1992).At that time, T. albinotatus was recorded for a short area of the main course of the upper Rio Preto and adjacent tributaries (Córrego Véu de Noiva and Ribeirão Santa 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 collected in this area, always in altitudes above about 1200 m above sea level (a.s.l.).More recently, however, specimens similar to T. albinotatus topotypes, but exhibiting different flank colouration were found in separate areas of the same drainage, in altitudes above about 1150 m a.s.l.Specimens from these geographically disjunct areas had other morphological characters identical to those exhibited 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 T. albinotatus was found in slightly lower altitude areas between these sub-drainages, at about 1100 m a.s.l. or less.The primary objective of this study is to first describe the different geographically disjunct colour morphs and ontogenetic colouration changes in T. albinotatus, providing data on their respective geographical distribution and habitats.Since species diversity is often high in mountain regions (Hoorn et al. 2013) and the presence of closely-related species inhabiting 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 genetic divergence amongst geographically isolated colour morphs thorough a phylogenetic analysis using a segment of the mitochondrial gene cytochrome b (1098 bp) for 23 specimens from 11 collecting sites, representing all colour morphs.

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 Conservação da Biodiversidade; permit number: 38553-7) and INEA (Instituto 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 T. 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 × 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 are according to the regional popular use in Portuguese (e.g.Córrego, Ribeirão, 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 tissue 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 fragment of the mitochondrial encoded gene cytochrome b (CYTB), which have been successfully used to delimit trichomycterine species (Costa and Katz 2021).Amplification 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 μl reactions with reagents at the following concentrations: 5× GreenGo-Taq Reaction Buffer (Promega), 1 mM MgCl 2 , 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 thermocycling 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 contaminations.The PCR products were then purified using the Wizard SV Gel and PCR Clean-Up System (Promega).Sanger Sequencing reactions were made by Mac-rogen Inc. (South Korea).Sequencing chromatograms and sequences were assessed using MEGA 7 (Kumar et al. 2016).The generated DNA sequences were translated 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 is available in Online Resource 2.  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.

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 midline; two horizontal rows of rounded to horizontally elongated 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 vermiculate pattern; light marks paler.
Distribution and habitat: Areas above 1150 m a.s.l. of the upper course of the Rio Preto, the Ribeirão Santa Clara and Córrego 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 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 presence of large rocks favours formation of small pools below waterfalls where most specimens were concentrated (Fig. 3C).Bottom predominantly with light grey rocks of different sizes.
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 valley, riverine environment highly exposed to sunlight.Water clear, yellowish hyaline.Rocks small, rare small waterfalls where most specimens are concentrated (Fig. 3E).Bottom predominantly with small light grey rocks, light grey to light yellow gravel and sand.
Distribution and habitat: Upper section of the Córrego do Alcantilado (CS7-10; Fig. 1), at altitudes between 1155 and 1320 m a.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 waterfalls (Fig. 3D); riverine environment poorly exposed to sunlight.Water clear, yellowish hyaline.Bottom predominantly composed of dark grey rocks.

Phylogeny
The phylogenetic analysis highly supported T. 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 lineages.Genetic distances amongst haplotypes were low (0.1-0.4%).The eleven haplotypes found amongst the 23 sequenced individuals were scattered in different localities.H1 was the most common haplotype, present in seven analysed specimens collected in seven separate localities and exhibited all the four colour morphs.

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.Arratia 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 T. albinotatus exhibiting different colour morphs in disjunct areas of the upper Rio Preto drainage, with slightly different ecological 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 sunlight, had a colour pattern consisting of alternating white and brown to black spots (CM1, Fig. 2A-C).Contrastingly, 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 elongated 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 being available.

Ontogenetic colouration and habitat change
In all colour morphs of T. albinotatus, white marks on the flank were less conspicuous or had completely disappeared, 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 example, in T. auroguttatus, a typical diurnal psammophilic species sympatric to T. 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 T. vitalbrazili, the sister group of T. albinotatus (Vilardo et al. 2020).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 T. 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 andCosta 2008, 2010b;da Silva et al. 2010;Ferrer and Malabarba 2013;Reis et al. 2020;Costa et al. 2021Costa et al. , 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 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 T. 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 different species.Although the occurrence of geographically disjunct colour morphs is possibly uncommon amongst trichomycterids, data on colouration polymorphism provided by Arratia & Menu-Marque (1981) and Arratia (1983a) suggest that colouration may be vary according to local environmental conditions in different trichomycterid 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 T. itatiayae Miranda Ribeiro, 1906 andT. 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 T. 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 pattern characters to diagnose species and trichomycterine clades.For example, even with the occurrence of chromatic polymorphism herein reported for T. albinotatus, the presence of white marks on the flank of juveniles uniquely shared by T. albinotatus and T. vitalbrazili amongst trichomycterines, clearly corroborated monophyly of this clade formally named as subgenus Humboldtglanis Costa, 2021, which is highly supported in a phylogenetic study integrating morphology and DNA sequences (Costa 2021).Similarly, apomorphic colour patterns are shared by trichomycterine clades corroborated by both osteological and molecular data (Costa 2021;  Costa and Katz 2021), thus demonstrating that colour patterns may be effectively used for diagnosing trichomycterine species and groups.These data in fact show that colouration should not be discarded a priori as evidence of species limits or phylogenetic relationships in trichomycterines, as well as colouration alone should not be used to infer species limits, being necessary to reinforce species delimitation hypotheses using additional morphological evidence (e.

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

Table 2 .
List of specimens used in the molecular analysis, with respective voucher number in the ichthyological collection of the Biology Institute, Federal University of Rio de Janeiro (UFRJ) and GenBank accession numbers.In specimens of T. albinotatus, CS means collecting site (see Table1); CM, colour morph; H, haplotype.