Color pattern variation in Trichomycterus iheringi ( Eigenmann , 1917 ) ( Siluriformes : Trichomycteridae ) from rio Itatinga and rio Claro , São Paulo , Brazil

Color pattern is recognized as an important characteristic for diagnosing Trichomycterus species and for elucidating their relationships. An analysis based on morphological and molecular data confirms the existence of a single species of Trichomycterus in the rio Itatinga, a costal river drainage on the escarpment of the Serra do Mar and the rio Claro on the upper course of the rio Tietê. The only species found, Trichomycterus iheringi, shows two clearly distinct patterns of body pigmentation and intermediate color patterns related to body size and microhabitat preference.

Trichomycterus is a diverse assemblage with about 100 species ranging throughout Central and South America on both sides of the Andes (de Pinna & Wosiacki, 2003).Most members inhabit headwaters and small courses of cold clear water streams with stony beds and strong currents (de Pinna, 1998).The species generally have a restricted geographic distribution and display a pronounced level of endemism (Costa, 1992;de Pinna, 1992b).The diversity of the genus is still poorly known and species-level identification is problematic due to the overwhelming number of and poorly characterized species, as well as scarce information available on nominal species.The majority of them can be only diagnosed by the combination of characteristics, and many studies consider the redescription of the poorly known species essential to a better taxonomic knowledge of the genus (Arratia, 1998;Fernández, 2000).Approximately 33 species of Trichomycterus have been described for the upper rio Paraná, upper rio São Francisco, and coastal drainages of southeastern Brazil, which also contain many undescribed species (Costa, 1992;Alencar & Costa, 2004).The color pattern is recognized as a very distinctive and important characteristic for diagnosing Trichomycterus species (Alencar & Costa, 2004, 2006;Bockmann et al., 2004;Lima & Costa, 2004;Wosiacki, 2004Wosiacki, , 2005;;Wosiaki & de Pinna, 2008b;Castellanos-Morales, 2008) and also for establishing relationship among species (Barbosa & Costa, 2003;Bockmann & Sazima, 2004;Bockmann et al., 2004;Alencar & Costa, 2006;Wosiack & de Pinna, 2008a).In spite of this, Arratia et al. (1978)  Trichomycterus iheringi was recently redescribed by Wosiacki (2005) based only on the type-series.In the present study, we describe newly collected specimens from near the type locality, showing color variation not formerly described, in which two distinct color patterns and intermediate transitional stages related to ontogenetic variation and microhabitat preference are present.We also revise and discuss the variation of other morphological characters interpreted as important for diagnosing T. iheringi.

Material and Methods
The specimens examined were collected in the rio Itatinga and tributaries located on the escarpment of the Serra do Mar in the Parque das Neblinas, a Natural Patrimony Private Reserve, and in the rio Claro on the upper course of the rio Tietê in the Paulistano plateau, the first in the Bertioga municipality and the second in the Salesópolis municipality, both in São Paulo State.The specimens were collected in regions with shallow stretches, fast rapids and pools with calm waters, alternating river beds composed of sand or stone and cold water (around 15ºC) that was totally transparent.Thirty-two specimens, ranging from 14.2 to 110.9 mm SL, were collected together with field observations concerning their behavior.Additionally, we examined another twelve specimens, ranging from 39.9 to 133.9 mm SL, from the collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP).
Methodology and terminology for measurements and counts follow de Pinna (1992aPinna ( , 1992b) ) and Bockmann & Sazima (2004).The conspecificity of examined specimens was tested by sequencing the mitochondrial cytocrome c oxidase subunit I (COX1) gene (Ward et al., 2005;Steinke et al., 2009).DNA was isolated from 12 specimens with distinct length and color pattern, according to Taggart et al. (1992).The DNA integrity and concentration were evaluated using agarose gel and spectrophotometer (NanoDrop ® ND-1000).The COX1 region was amplified using the primers L6252 (5'-AAG GCG GGG AAA GCC CCG GCA-3') and H7271 ( 5'-TCC TAT GTA GCC GAA TGG TTC TTT T-3') (Cláudio Oliveira, pers.comm.) using the following conditions: a master mix of 10 pmol of each primer, 2.5 mM MgCl 2 , 2.5 mM of deoxynucleotide triphosphate, 1 unit of Taq DNA polymerase, 50-100 ng of total DNA, 2.5 µl of 10x Taq buffer and a volume of water to bring the final volume to 50µl.Cycling conditions were as follows: hot start denaturation step at 94ºC for 3 min followed by 35 cycles of denaturation at 94ºC for 1 min, annealing at 59.8ºC for 45 s and extension at 72ºC for 2 min with a final 10 min extension step at 72ºC.PCR products were visualized in 1% agarose gel and purified using the E.Z.N.A. ® System (Omega Bio-Tek, Inc.).The purified PCR products were sequenced with the BigDye TM Terminator Cycle Sequencing kit (PE Applied Biosystems) according to the protocols provided by the manufacturer.Electrophoresis of the purified samples was performed in an ABI Prism 3100 DNA Sequencer (Perkin Elmer).
The sequences were aligned with the software ClustalW (Thompson et al., 1994) and checked manually.The consensus sequences were obtained by BIOEDIT Sequence Alignment Editor 7.0.9(Hall, 1999).In silico sequences nucleotide divergence was implemented with MEGA 4.0 (Kumar et al., 2001)

Molecular evidence of the conspecificity of specimens with different color patterns
An average of 409 base pairs (bp) of COX1 in 12 Trichomycterus iheringi specimens from rio Itatinga and rio Claro were sequenced.The transition/transversion rate ratios were 6.402 for purines and 5.885 for pyrimidines.The overall transition/transversion bias was R = 2.983.The average nucleotide frequencies were A = 24.2%,T = 29.4%,C = 28.3% and G = 18.1%.COX1 sequences of all 13 specimens of T. iheringi (including 1 specimen from Botucatu, rio Alambari, LBP 1296) were either identical or very similar.The average Kimura-2-parameter [K2P] differences among them was 0.53% ranging from 0 to 1% which is lower than the 5.73% genetic divergence of the other recognized Trichomycterus auroguttatus Costa, 1992 and T. paolence (Eigenmann, 1917) species.The neighbor-joining (NJ) tree showed shallow intraspecimen divergences among specimens with different color patterns (Fig. 1).The mean of COX1 genetic divergence sequences between Trichomycterus specimens with different color patterns was lower than the 2% expected for different species (Hebert et al., 2003).This indicates the conspecificity of the specimens of Trichomycterus iheringi with different color patterns, including those caught in different river drainages.

Taxonomic identity of specimens examined
Trichomycterus iheringi was described by Eigenmann (1917)  paratypes CAS 64586 and FMNH 58074).The species was recently redescribed by Wosiacki (2005), who has not found uniquely derived features to diagnose the species and presented a new diagnosis based on the following combination of characters: 9 to 10 dorsal-fin branched rays; first pectoral-fin ray not prolonged as a filament; i,7 pectoralfin rays; caudal-fin margin rounded; two s6 pores paired at the interorbital space; pelvic-fin margin distance from the urogenital opening equal to half of its length; and adult specimens with uniform light tan color pattern with numerous, poorly defined small spots irregularly distributed on the body.
In the present study, the specimens examined were recognized as T. iheringi and distinguished from its congeners from south and southeastern Brazil by the same distribution of the type-specimen and by possessing the first pectoral-fin ray not prolonged as a filament; i,7 pectoral-fin rays (Table 2); caudal-fin margin rounded; two s6 pores paired at the interorbital space; and adult specimens with uniform grayish brown color, with numerous poorly defined small spots irregularly distributed on the body (Fig. 2).

Variation of body pigmentation
Trichomycterus iheringi specimens show two very distinct well-defined patterns of body pigmentation, and intermediate color patterns clearly related to size and microhabitat preference.The first color pattern is observed in smaller juvenile specimens, between 14.2 to 51.2 mm SL (Fig. 3a, b), invariably captured partially or totally buried in clear sand, with same tonality as trunk background color of specimens.The color pattern is characterized by head uniform gray dorsally, gradually lighter laterally, and latero-ventrally with same ground color as trunk.Nasal barbel dark, with same color as dorsal portion of head, maxillary and rictal barbels unpigmented from base to half of their length and slightly pigmented on distal half.Ground color of trunk and caudal peduncle light cream to pale yellow (likely the color of river bottom), with two regular longitudinal rows, each with 8 to 13 well-defined dark brown blotches, larger than eye-diameter, formed of densely grouped chromatophores, one along dorsal region and other, most conspicuous, along midlateral region.Sometimes with poorly defined small spots loosely distributed between these two regular longitudinal rows.Pectoral and pelvic fins unpigmented, dorsal and anal fins unpigmented from base to half of their length, with few small spots on   distal half, caudal fin with small spots from base to tip.
A second, very distinct color pattern was observed in larger adult specimens, between 52.4 to 133.9 mm SL (Figs. 2 and 3f), always captured in areas with river bottom formed by with variously sized dark stones.The pattern is characterized by head dark dorsally, gradually lighter ventrally; nasal barbel dark as dorsal portion of head, maxillary and rictal barbels slightly pigmented, with same coloration as trunk.Trunk ground color uniformly grayish brown, with numerous small spots, as large as eye-diameter or smaller, irregularly distributed on the dorsal and lateral surface of head, trunk and caudal peduncle, gradually less numerous ventrally.Pectoral and pelvic fins unpigmented, dorsal and caudal fins with same coloration as trunk and anal fin relatively lighter, with few spots.
Specimens with intermediate sizes, between 36.7 to 68.9 mm SL (Fig. 3c, d, e), with transitional phases of body coloration were captured in the same areas as the larger adults, generally associated with those.Well-defined dark brown large blotches, larger than eye-diameter along dorsal region of trunk are absent, but those along midlateral region are always present but variable.Blotches in some specimens are quite conspicuous and formed by densely grouped chromatophores (Fig. 3c), in other specimens are less conspicuous, formed by loosely arranged chromatophores (Fig. 3d, e).These specimens with intermediate sizes begin to show small spots, as large as eye-diameter or smaller, irregularly distributed on dorsal and lateral surfaces of head, trunk and caudal peduncle, which are also quite variable, ranging from few and inconspicuous spots along lateral region of trunk (Fig. 3c, e), to quite numerous and conspicuous (Fig. 3d).Head, trunk, caudal peduncle, nasal, maxillary and rectal barbels, and fins with same ground color as described for juvenile specimens.
Our results have shown that this is not the case for T. iheringi.Wosiacki (2005) redescribed T. iheringi based only on adult specimens, while variations exhibited by juvenile specimens led Serra et al. (2007) to consider the Trichomycterus from the rio Itatinga as a species with undefined taxonomic status and possibly being an undescribed species, and not juveniles of T. iheringi as proposed here based on analysis of a larger series.Other comparative material examined (see in comparative material examined, MZUSP 44536, MZUSP 47596, and MZUSP 86988) also show similar color pattern variation.
Besides color pattern variation, some other diagnostic characteristics proposed by Wosiacki (2005) for T. iheringi were also highly variable when large series of variously sized specimens were examined, and therefore not useful for diagnosis.A pelvic-fin margin distance to the urogenital opening equal to half of the fin length was observed in specimens between 41.6 to 133.9 mm SL (19 specimens), whereas those between 14.2 to 58.0 mm SL (25 specimens) have the pelvic-fin margin contacting the urogenital opening.Wosiacki (2005) only examined the type-specimens with 117.6 to 139.6 mm SL, with pelvic-fin margin distance to the urogenital opening equal to half of the fin length.Dorsal-fin rays also show a greater variation, with ii,7 (1 specimen), ii,8 (29 specimens), or ii,9 (13 specimens; Table 2), contrarily to the range ii-iii,9-10 described by Wosiacki (2005).