Monophyly or Homoplasy? The Relationships of a Rare New Species of Cambeva (Siluriformes: Trichomycteridae) from the Brazilian Atlantic Forest with a Bicolored Caudal Pattern †

: The Brazilian Atlantic Forest, one the five main biodiversity hotspots in the world, still houses many organisms that are rare and unknown to science, mostly concentrated in mountain ranges. Herein, we describe a rare new species of Cambeva from the Rio Tiet ê drainage at the Serra da Mantiqueira, exhibiting a caudal fin color pattern comprising a light-yellowish–white bar posteriorly edged by a black pigmented area. A similar color pattern was described for C. castroi , C. diabola , and C. melanoptera , also endemic to the Rio Paran á basin, and considered evidence of close relationships among these three species. However, other morphological characteristics highly suggest that the new species is not closely related to C. castroi , C. diabola , and C. melanoptera ). Herein, we perform a molecular phylogenetic analysis using two mitochondrial genes and one nuclear gene, including all species with that bicolored caudal pattern. The analysis strongly supports the new species as a member of the alpha-clade, and therefore as distantly related to those three species belonging to the beta-clade, indicating that this color pattern has homoplastically arisen in Cambeva . The data reported here reinforce the urgent need for field studies to better understand the different evolutionary patterns found in the genus, since an intense process of environmental degradation is in course.


Introduction
The Atlantic Forest of eastern Brazil is one of the largest centers of biodiversity in the world [1].Despite undergoing a major process of environmental degradation, the Atlantic Forest still houses many components of this rich biota that are still unknown to science and sometimes rare, especially in the various mountain ranges that characterize the landscape of southeast and southern Brazil, where numerous endemic species are responsible for establishing this biome as one of the five main biodiversity hotspots on the planet [1].Among the most biodiverse mountain ranges is Serra da Mantiqueira, located in southeastern Brazil, with peaks that reach around 2900 m asl, which are the highest elevations in eastern South America.Serra da Mantiqueira is known for harboring a rich biodiversity [2][3][4], highlighting among the fish a large concentration of catfish species of the Trichomycterinae (hereafter trichomycterines).Over 20 trichomycterine species have been reported to be endemic to the streams flowing through Serra da Mantiqueira (e.g., [5] and included references).
Here, we first report and describe a rare species of Cambeva endemic to the Rio Tietê drainage at Serra da Mantiqueira, which was first collected by one of us (WJEMC) over 20 years ago, but due to the scarcity of available specimens, it remained undescribed until now.During the first collection on 3 March 2003, only two specimens were found, Taxonomy 2024, 4 342 although the biotope did not show signals of environmental degradation at that time.A preliminary description was prepared, but the paper was not submitted, waiting for another opportunity to collect more specimens.Between 2021 and 2022, three collecting trips were made to try to collect more specimens of this species, both at the same area sampled in 2003 and in neighboring areas of the same drainage, but no specimen was found.The first attempt was made by AMK between 10 and 12 October 2021, the second one by CRMF on 15 December 2021, and the third by CRMF between 5 and 6 November 2022.Finally, between 18 and 19 August 2023, CRMF found two specimens that completed the type series for the description presented herein.
The new species has a peculiar color pattern on the caudal fin, comprising a lightyellowish-white bar on the caudal fin base, posteriorly edged by a black pigmented area.A similar bicolored pattern of the caudal fin has been reported for three other congeners endemic to other regions of the Rio Paraná basin: Cambeva castroi (de Pinna, 1992) and Cambeva melanoptera Costa, Abilhoa, Dalcin & Katz, 2022 from the Rio Iguaçu drainage, and Cambeva diabola (Bockmann, Casatti & de Pinna, 2004) from the Rio Paranapanema drainage [6][7][8].Although the caudal fin color pattern is not identical in those species, varying in the extent and intensity of each color, it has been considered the main evidence of monophyly of the species sharing it [7,8].On the other hand, recent molecular data indicate that C. castroi and C. diabola are not sister taxa [9].In addition, the new species described here exhibits some morphological traits similar to those of Cambeva iheringi (Eigenmann, 1917), which occurs in the same river drainage as the new species but is distantly related to C. castroi, C. diabola, and C. melanoptera, thus suggesting that the caudal fin bicolored pattern has arisen independently within the genus.The objectives of the present paper are to provide a first molecular phylogenetic analysis comprising all species with the bicolored caudal pattern as terminal taxa to infer relationships among them and to describe the new species.

Specimens
The collection process followed the ethical protocols described in our recent previous papers on trichomycterines (e.g., [10]), approved by CEUA-CCS-UFRJ (Ethics Committee for Animal Use of Federal University of Rio de Janeiro; permit numbers: 065/18 and 084/23) and with permission given by ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade; permit number: 38553-13).The specimens were analyzed in the Laboratory of Systematics and Evolution of Teleost Fishes (Instituto de Biologia, Universidade Federal do Rio de Janeiro) and deposited in its fish collection (UFRJ).The specimen prepared for osteological examination following Taylor and Van Dyke [11] and partially dissected was deposited under a separate catalogue number.In listing the specimens, we used the following abbreviations: C&S, cleared and stained specimens for osteological examination; SL, standard length.Comparative material is listed in our previous studies on the genus Cambeva (see [9,10] and included references).

Morphological Data
The methods used to describe the morphological characters are those described in our recent taxonomical study on Cambeva [9], which followed Costa [12], Bockmann et al. [7], and Costa et al. [13] for meristic and morphometric data; Costa [14] and Kubicek [15] for osteological terminology; and Arratia and Huaquin [16] and Bockmann et al. [7] for laterosensory pore terminology.The osteological characters listed in the species description are those with informative variability in Cambeva (e.g., [9,10]) or those containing useful information allowing us to phylogenetically position the new species.

Molecular Phylogenetic Analysis
The methods used for obtaining DNA sequences are those described in our recent phylogenetic studies on Cambeva (e.g., [9]), using the same markers and primers, which were first described by Villa-Verde et al. [17], Unmack et al. [18], Barros et al. [19], Ward et al. [20], Hardman and Page [21], Costa et al. [22], and Cramer et al. [23], the same PCR reaction parameters, and MEGA 11 [24] for reading and interpretating the chromatograms and editing the sequences.
The terminal taxa of the phylogenetic analysis included the 4 species with the bicolored caudal pattern, another 30 species of Cambeva representing all generic lineages, and 5 outgroup species representing other trichomycterid groups.The concatenated molecular data matrix with 2469 bp comprised sequences generated here or in our previous studies [9,10,25], as well as those taken from GenBank and first published by other authors [26][27][28].The GenBank accession numbers appear in Appendix A. The Clustal W algorithm [29] implemented in MEGA 11 was used to align each gene data set and the PartitionFinder 2.1.1 algorithm [30] was used to calculate best-fit evolutive model schemes (Appendix B) with the Corrected Akaike Information Criterion.Phylogenetic trees were generated by using Bayesian Inference (BI) in Beast 1.10.4[31] (two independent Markov Chain Monte Carlo (MCMC) runs with 3 × 10 7 generations; a sampling frequency of 1000 generations; using Tracer 1.7.2 [32] to verify the convergence of the MCMC chains and the proper burn-in value and LogCombiner v.1.10.4 [31] and Tree Annotator version 1.10.4[31] to combine and calculate the consensus tree, 25% burn-in, and Bayesian posterior probabilities) and the Maximum Likelihood (ML) approach performed in IQTREE 2.2.0 [33], with node support estimated by ultrafast bootstrap (UFBoot) [34] and the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) [35], both using 1000 replicates.

Phylogenetic Relationships and Comparative Morphology
The phylogenetic analyses generated identical trees, where the three most inclusive clades of Cambeva (i.e., alpha, beta, and gamma clades [9]) are corroborated (Figure 1).The generated tree refutes the hypothesis that species with the bicolored caudal pattern form a monophyletic group.

Molecular Phylogenetic Analysis
The methods used for obtaining DNA sequences are those described in our recent phylogenetic studies on Cambeva (e.g., [9]), using the same markers and primers, which were first described by Villa-Verde et al. [17], Unmack et al. [18], Barros et al. [19], Ward et al. [20], Hardman and Page [21], Costa et al. [22], and Cramer et al. [23], the same PCR reaction parameters, and MEGA 11 [24] for reading and interpretating the chromatograms and editing the sequences.
The terminal taxa of the phylogenetic analysis included the 4 species with the bicolored caudal pattern, another 30 species of Cambeva representing all generic lineages, and 5 outgroup species representing other trichomycterid groups.The concatenated molecular data matrix with 2469 bp comprised sequences generated here or in our previous studies [9,10,25], as well as those taken from GenBank and first published by other authors [26][27][28].The GenBank accession numbers appear in Appendix A. The Clustal W algorithm [29] implemented in MEGA 11 was used to align each gene data set and the Partition-Finder 2.1.1 algorithm [30] was used to calculate best-fit evolutive model schemes (Appendix B) with the Corrected Akaike Information Criterion.Phylogenetic trees were generated by using Bayesian Inference (BI) in Beast 1.10.4[31] (two independent Markov Chain Monte Carlo (MCMC) runs with 3 × 10 7 generations; a sampling frequency of 1000 generations; using Tracer 1.7.2 [32] to verify the convergence of the MCMC chains and the proper burn-in value and LogCombiner v.1.10.4 [31] and Tree Annotator version 1.10.4[31] to combine and calculate the consensus tree, 25% burn-in, and Bayesian posterior probabilities) and the Maximum Likelihood (ML) approach performed in IQTREE 2.2.0 [33], with node support estimated by ultrafast bootstrap (UFBoot) [34] and the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) [35], both using 1000 replicates.

Phylogenetic Relationships and Comparative Morphology
The phylogenetic analyses generated identical trees, where the three most inclusive clades of Cambeva (i.e., alpha, beta, and gamma clades [9]) are corroborated (Figure 1).The generated tree refutes the hypothesis that species with the bicolored caudal pattern form a monophyletic group.The analyses strongly support the new species as a member of a subclade of the Cambeva alpha-clade, here called the Cambeva iheringi group, also including Cambeva iheringi (Eigenmann, 1917), Cambeva guaraquessaba (Wosiacki, 2005), and Cambeva tupinamba (Wosiacki & Oyakawa, 2005), in which the new species appears as sister to C. iheringi (Figure 1).The species of the C. iheringi group are distinguished from the other species of the Cambeva alpha-clade (i.e., Cambeva concolor (Costa, 1992), not included in the analysis, and Cambeva variegata (Costa, 1992)) by the presence of the anterior segment of the infraorbital canal (vs.absence), the absence of a skin crest on the caudal peduncle, similar to an adipose fin (vs.presence), and jaw teeth irregularly arranged, with tooth extremities being pointed to slightly rounded (vs.arranged in rows; incisiform).The new species is supported as a sister group to C. iheringi.
The other three species with the bicolored caudal fin, C. castroi, C. diabola, and C. melanoptera, are members of the Cambeva beta-clade, but are not directly related to each other according to the tree topology.Cambeva melanoptera appears as the sister group of a strongly supported clade (i.e., the Cambeva davisi group) that includes Cambeva barbosae  The analyses strongly support the new species as a member of a subclade of the Cambeva alpha-clade, here called the Cambeva iheringi group, also including Cambeva iheringi (Eigenmann, 1917), Cambeva guaraquessaba (Wosiacki, 2005), and Cambeva tupinamba (Wosiacki & Oyakawa, 2005), in which the new species appears as sister to C. iheringi (Figure 1).The species of the C. iheringi group are distinguished from the other species of the Cambeva alpha-clade (i.e., Cambeva concolor (Costa, 1992), not included in the analysis, and Cambeva variegata (Costa, 1992)) by the presence of the anterior segment of the infraorbital canal (vs.absence), the absence of a skin crest on the caudal peduncle, similar to an adipose fin (vs.presence), and jaw teeth irregularly arranged, with tooth extremities being pointed to slightly rounded (vs.arranged in rows; incisiform).The new species is supported as a sister group to C. iheringi.
The other three species with the bicolored caudal fin, C. castroi, C. diabola, and C. melanoptera, are members of the Cambeva beta-clade, but are not directly related to each other according to the tree topology.Cambeva melanoptera appears as the sister group of a strongly supported clade (i.e., the Cambeva davisi group) that includes Cambeva barbosae   1.
barbosae and C. davisi (three evolutionary steps) or the independent appearance in each of the three species with that color pattern (three evolutionary steps).barbosae and C. davisi (three evolutionary steps) or the independent appearance in each of the three species with that color pattern (three evolutionary steps).Diagnosis.Cambeva difficilis differs from all other species of Cambeva by having small sub-incisiform odontodes on the basal portion of the opercular and interopercular patches (vs.odontodes pointed).Cambeva difficilis is also distinguished from all other congeners of the Cambeva alpha-clade by the presence of a unique color pattern of the caudal fin, comprising a yellowish-white zone on the basal portion of the fin and posteriorly edged by a black zone, more conspicuous immediately after collection.Cambeva difficilis also differs from other species of the C. iheringi group by having short mesethmoid cornua, distally terminating in sharply pointed tips (Figure 2A; vs. longer, with rounded tips, Figure 2D), a relatively short interopercle, its largest length about half of the longitudinal length of the hyomandibula (Figure 2B; vs. longer, about two thirds of that length, Figure 2E), and a compact parurohyal, with a relatively short lateral process, a broad anterior head with an indistinct antero-lateral paired process, and without basal constriction (Figure 2C; vs. elongated lateral process, anterior head slender with a distinct antero-lateral paired process, and basal constriction, Figure 2F).Cambeva difficilis also differs from C. guaraquessaba by having shorter barbels, with the nasal barbel reaching an area just posterior to the orbit (vs.reaching the opercular patch of odontodes), the maxillary barbel reaching the posterior portion of the interopercular patch of odontodes (vs.reaching the area between the interopercular patch of the odontodes and the pectoral fin base), and the rictal barbel reaching the anterior portion of the interopercular patch of odontodes (vs.posterior portion), and by having pelvic fin bases medially separated by a broad interspace, about half of the pelvic fin base length (vs. in close proximity); it also differs from C. tupinamba by having the anal fin origin posterior to the dorsal fin base (vs.vertically through the posterior portion of the dorsal fin base).
Description.External morphology (morphometric data in Table 1): The body is relatively slender, deeper in the midway area in terms of the distance between the pectoral and pelvic fin insertions.The anterior portion of the trunk is subcylindrical, compressed on the caudal peduncle.The dorsal profile of the trunk is convex in its anterior portion and nearly straight on the caudal peduncle; the ventral profile is approximately straight in all of its extension.The anus is vertically through the middle of the dorsal fin base.The urogenital opening is minute, immediately posterior to the anus.The lateral line canal of the trunk is short, with two pores just posterior to the opercular patch of the odontodes.
The head is about two times wider than it is deep, sub-trapezoidal from a dorsal view, while the anterior profile of the snout is straight to slightly convex.The mouth is subterminal, with a large lateral fleshy lobe, and its longest length is about half of the length of the lower jaw, excluding the lateral lobes.The jaw teeth are irregularly arranged.The jaw dentition includes smaller pointed teeth on the internal part of the jaw and larger teeth with a near-rounded extremity on the external part.In total, there are 46 premaxillary teeth and 60 dentary teeth in the C&S specimen.The barbels are moderately short; the tip of the nasal barbel posteriorly reaches the area immediately posterior to the orbit, the tip of the maxillary barbel reaches the posterior portion of the interopercular patch of the odontodes, and the rictal barbel reaches the anterior portion of the interopercular patch of the odontodes.The eyes are moderately large, positioned on the dorsal surface of the head, approximately equidistant from the middle of the snout anterior edge and the anterior margin of the opercular patch of the odontodes.The distance between the anterior and posterior nostrils is about half of the distance between the posterior nostril and the orbit.The dorsal and ventral surfaces of the head are covered by minute skin papillae.The branchial membrane is attached to the isthmus at its anterior-most point.There are eight branchiostegal rays.
The opercular patch of the odontodes is rounded, with broad skin folds around its margins, separated from the interopercular patch by a large interspace that is larger than the vertical length of the opercular patch.The interopercular patch of the odontodes is small, slightly longer than the opercular patch.The odontodes are arranged in transverse rows in the opercle and in irregular longitudinal rows in the interopercle.The larger odontodes are conical and slightly curved, while the smaller odontodes are incisiform.There are 19 to 21 opercular odontodes and 29 interopercular odontodes.The canals of the cephalic latero-sensory system are well developed, including having a short anterior segment of the infraorbital canal, with two paired pores (i1 and i3) attached to the lacrimal bone and separated from the other canals.The supraorbital canal is continuous, with three paired pores (s1, s3, and s6), connected to the posterior infraorbital section, with two pores (i10 and i11), and connected to the postorbital section, with two pores (po1 and po2).The s6 pore is roughly equidistant from the orbit and its symmetrical homologous pore.
The fins are well developed, thickened at the basal portion, and thin at the distal one.The dorsal and anal fins are subtriangular.The dorsal fin origin is nearer to the caudal fin base than to the posterior margin of the head.The anal fin origin is vertically immediately posterior to the dorsal fin base.In total, there are 12 (ii + II + 8) dorsal fin rays and 9 (ii + II + 5) anal fin rays.The caudal fin truncate has 13 (I + 11 + I) principal caudal fin rays, 12 (xi + I) dorsal procurrent rays, and 8 (vii + I) ventral procurrent rays.The pectoral fin is rounded, with 8 (I + 7) rays.The first pectoral fin is shorter than the second one, not forming a filament in its tip.The pelvic fin subtruncate has a rounded posterior margin, with 5 (I + 4) rays.The pelvic fin bases are medially separated by an interspace about half of the length of the pelvic fin base.The pelvic fin insertion is vertically anterior to the dorsal fin origin.The pelvic fin has an extremity reaching vertically through the posterior half of the dorsal fin base, but not reaching the anus.
Osteology: The bones of the mesethmoidal region are longitudinally elongated (Figure 2A).The mesethmoid is T-shaped, robust, and without lateral expansions.The anterior margin of the mesethmoid is weakly concave, and the mesethmoid cornu is short, terminating in a sharp tip.The lateral margin of the lateral ethmoid is straight, without projections.The premaxilla is sub-rectangular, slightly tapering laterally.The maxilla is boomerang-shaped, slightly bent, and longer than the premaxilla.The lacrimal and sesamoid supraorbital bones are flat, thin, without distinctive projections, and well ossified.The lacrimal bone is long and sub-elliptical.The sesamoid supraorbital bone is long, longer than the autopalatine excluding the postero-lateral process, and in close proximity to the lacrimal bone.
The autopalatine is relatively elongated, with its smallest width being about half of the autopalatine length, and sub-rectangular from a dorsal view when excluding its posterolateral process, with a roughly straight lateral margin and a weakly concave medial margin (Figure 2A).The postero-lateral process of the autopalatine is triangular, with its length being about two-thirds of the autopalatine length.The metapterygoid is derate in size and longer than it is deep, with a slightly pointed dorsal extremity, its five facets forming an irregular pentagon, and its dorsal and posterior margins being slightly sinuous (Figure 2B).The quadrate is slender, with deep anterior constriction just above its main longitudinal arm.The dorsoposterior quadrate outgrown, is in contact with the anterior margin of the hyomandibula outgrowth.The hyomandibula is long, occupying about three-fourths of the jaw suspensorium length.
The opercle is robust, with a broad and weakly curved dorsal process (Figure 2B).The opercular odontode patch has a depth slightly smaller than the length of the dorsal hyomandibula articular facet.The opercular articular facet for the hyomandibula has a prominent, rounded rim projecting over the hyomandibula.The articular facet for the preopercle is small and rounded.The interopercle is short, with its longest length being about half of the longitudinal length of the hyomandibula.The preopercle is slender, without distinctive projections.
The parurohyal is compact, with a short, subtriangular, and slightly curved lateral process (Figure 2C).The parurohyal head is broad, without distinct posterior constriction and an indistinct antero-lateral paired process.The middle parurohyal foramen is broad.The posterior process is short, about half of the length of the distance between the anterior margin of the parurohyal and the anterior insertion of the posterior process.
There are 38 free vertebrae and 13 ribs.The base of first dorsal fin ray is vertically through the centrum of the 17th vertebra, while the base of the first anal fin ray is vertically through the centrum of the 23rd vertebra.The posterior portion of the caudal skeleton has two dorsal plates, corresponding to hypurals 3 + 4 and 5, and a single ventral plate corresponding to hypurals 1 + 2 and a parhypural.
The coloration of the live species is shown (Figure 4).The flank, head side, and dorsum are pale-brownish-yellow, with black dots that are larger along the longitudinal midline of the flank and the dorsal and posterior portion of the caudal peduncle.The nasal barbel is dark gray, while the maxillary and rictal barbels are light gray.The venter is pinkish-white.The dorsal, pelvic, and pectoral fins are hyaline, with sparse minute dark gray dots.The anterior half of the caudal fin has a yellowish-white zone on the basal portion, posteriorly edged by a black zone; the posterior half is hyaline, with a great concentration of minute black dots.The pelvic fin is hyaline.
The coloration of the species in alcohol is shown (Figure 3).The coloration is similar to its coloration in life, but the dark marks are paler and the basal portion of the caudal fin is light gray.
Etymology.From the Latin, the name difficilis (difficult) is an allusion to the great difficulty it takes to collect this new species (see Introduction).
Distribution (Figure 5).Cambeva difficilis is known only from nearby localities in the Rio da Cachoeira and a close tributary, which is a tributary of the Rio Atibaia, itself a tributary of the Rio Piracicaba that belongs to the Rio Tietê drainage, the upper Rio Paraná basin, southeastern Brazil, at altitudes about 870-890 m asl.

Discussion
Among species of Cambeva, the bicolored caudal pattern described here (i.e., a lightyellowish-white bar on the caudal fin base, posteriorly followed by a black area) is present in the C. iheringi group of the Cambeva alpha-clade and in the C. davisi group of the Cambeva beta-clade.A caudal fin color pattern comprising a broad light area contrasting with a black posterior margin occurs in Scleronema Eigenmann, 1917, the sister group of Cambeva [25]; a bicolored caudal fin is present in the S. operculatum group of the subgenus Scleronema [36], but does not occur in its sister group, the S. minutum group, and it is absent in the subgenus Plesioscleronema Costa, Sampaio, Giongo, Almeida, Azevedo-Santos & Katz, 2022.Therefore, the occurrence of this bicolored caudal fin pattern in isolated distant lineages indicates that this pattern is an apomorphic condition that has arisen at least twice in Cambeva.
The independent occurrence of similar apomorphic characteristics in different lineages (i.e., homoplasies) is common in several organisms and may be explained by different evolutionary processes such as convergence, parallel evolution, and reversals, although distinction among them may be unclear [37].The term parallel evolution has been commonly applied in cases where the emergence of apomorphic characteristics occurs independently in different lineages of the same group as an adaptive response to similar environmental pressures (e.g., [38]).Cases of parallel evolution have been reported from studies on fish (e.g., [39]) to studies even on human populations (e.g., [40]).Our analysis indicating that the bicolored caudal pattern arises independently at least twice in the same genus, Cambeva, once in the alpha-clade and at least one time in the beta-clade (Figure 1), suggests this to be a case of parallel evolution.The species of Cambeva live in similar environments (e.g., fast-flowing streams with bottoms typically composed of gravel and sand), in which several microhabitats are available.However, we do not have detailed field data about the ecological preferences and behavior of Cambeva allowing us to infer the existence of environmental factors or specific behaviors favoring the homoplastic appearance of the bicolored caudal pattern, which would support an interpretation of the parallel evolution process.
We recently recorded a case of repeated pelvic fin loss in Cambeva, involving five species without any trace of the fin and pelvic girdle [10], a rare event among trichomycterines from eastern South America.Molecular analysis indicated that losses occurred at least three or four times independently, once or twice in the beta-clade and twice in the

Discussion
Among species of Cambeva, the bicolored caudal pattern described here (i.e., a lightyellowish-white bar on the caudal fin base, posteriorly followed by a black area) is present in the C. iheringi group of the Cambeva alpha-clade and in the C. davisi group of the Cambeva beta-clade.A caudal fin color pattern comprising a broad light area contrasting with a black posterior margin occurs in Scleronema Eigenmann, 1917, the sister group of Cambeva [25]; a bicolored caudal fin is present in the S. operculatum group of the subgenus Scleronema [36], but does not occur in its sister group, the S. minutum group, and it is absent in the subgenus Plesioscleronema Costa, Sampaio, Giongo, Almeida, Azevedo-Santos & Katz, 2022.Therefore, the occurrence of this bicolored caudal fin pattern in isolated distant lineages indicates that this pattern is an apomorphic condition that has arisen at least twice in Cambeva.
The independent occurrence of similar apomorphic characteristics in different lineages (i.e., homoplasies) is common in several organisms and may be explained by different evolutionary processes such as convergence, parallel evolution, and reversals, although distinction among them may be unclear [37].The term parallel evolution has been commonly applied in cases where the emergence of apomorphic characteristics occurs independently in different lineages of the same group as an adaptive response to similar environmental pressures (e.g., [38]).Cases of parallel evolution have been reported from studies on fish (e.g., [39]) to studies even on human populations (e.g., [40]).Our analysis indicating that the bicolored caudal pattern arises independently at least twice in the same genus, Cambeva, once in the alpha-clade and at least one time in the beta-clade (Figure 1), suggests this to be a case of parallel evolution.The species of Cambeva live in similar environments (e.g., fast-flowing streams with bottoms typically composed of gravel and sand), in which several microhabitats are available.However, we do not have detailed field data about the ecological preferences and behavior of Cambeva allowing us to infer the existence of environmental factors or specific behaviors favoring the homoplastic appearance of the bicolored caudal pattern, which would support an interpretation of the parallel evolution process.
We recently recorded a case of repeated pelvic fin loss in Cambeva, involving five species without any trace of the fin and pelvic girdle [10], a rare event among trichomycterines from eastern South America.Molecular analysis indicated that losses occurred at least three or four times independently, once or twice in the beta-clade and twice in the gamma-clade, whereas in closely related genera, it occurred only once [10].Although the repeated independent occurrence of identical and complex new morphological features during the evolution of the same group is unlikely to happen by chance, therefore being admissible evidence of parallel adaptation [41], the lack of methodologically robust field observations prevents us from assuming both the case of repeated pelvic fin loss and the case of independent acquisition of the bicolored caudal pattern to be cases of parallel evolution.

Conclusions
The genus Cambeva possesses a great diversity of species only recently revealed (e.g., [9,10]).Recent studies have shown us that species of this genus may possess uncommon or unique morphological traits among trichomycterines, but some evidence casually obtained in field studies also suggests unusual behavioral and ecological characteristics (e.g., [10,[42][43][44]).In view of the intense process of environmental degradation throughout the distribution area of the genus, these data demonstrate the urgent need for field studies to better understand these preliminary data on the evolution and ecology of the group.
Costa, Feltrin & Katz, 2021, C. castroi, Cambeva davisi (Haseman, 1911), and C. diabola, among which the last species is supported as the sister group of C. barbosae, and C. castroi as the sister to the clade comprising C. barbosae plus C. diabola.The distribution of the bicolored caudal pattern among the taxa of the Cambeva beta-clade offers us some equally parsimonious interpretations about the characteristic state evolution, such as a single appearance at the base of the C. davisi group, with the subsequent disappearance in C.
Costa, Feltrin & Katz, 2021, C. castroi, Cambeva davisi (Haseman, 1911), and C. diabola, among which the last species is supported as the sister group of C. barbosae, and C. castroi as the sister to the clade comprising C. barbosae plus C. diabola.The distribution of the bicolored caudal pattern among the taxa of the Cambeva beta-clade offers us some equally parsimonious interpretations about the characteristic state evolution, such as a single appearance at the base of the C. davisi group, with the subsequent disappearance in C. barbosae and C. davisi (three evolutionary steps) or the independent appearance in each of the three species with that color pattern (three evolutionary steps).