Comparative Osteology, Phylogeny and Classification of the Eastern South American Catfish Genus Trichomycterus (Siluriformes: Trichomycteridae)

Abstract

Trichomycterus has been considered for a long time to be the most problematic genus of the diverse neotropical subfamily Trichomycterinae. Recently, Trichomycterus was restricted to a clade from eastern South America supported by molecular data, but no unique morphological character state was found to distinguish it, making it difficult to allocate new species based on morphology alone. The objectives of this study were to conduct an osteological comparative analysis comprising a large sample of valid species of Trichomycterus, to conduct a total evidence phylogenetic analysis, combining osteological characters and a multigene database, and to propose an intrageneric classification based on the results of the phylogenetic analysis. Fifty-two osteological characters were combined with a multigene molecular data set of 2974 bp for 44 species of Trichomycterus, and 21 outgroups generated a well-supported phylogenetic tree, making it possible to delimit and diagnose intrageneric lineages, of which six subgenera are recognized. The high morphological diversity of osteological structures herein first reported for Trichomycterus from eastern South America is possibly related to some ecological specializations. This study shows that osteological characters combined with molecular data may be useful to consistently delimit and distinguish between trichomycterines, shedding light on the still persistent problems in trichomycterine systematics.

1. Introduction

The spectacular diversity of South American freshwater fishes has been repeatedly reported in the recent literature [1,2]. Among the most iconic fish groups in the region is the Trichomycteridae, a neotropical family of catfishes including the popularly known candiru [3] and many other taxa with uncommon morphological and ecological specializations [4,5,6,7]. The greatest species diversity among trichomycterids occurs in the Trichomycterinae, a subfamily containing about 250 valid species [8] distributed in all the main river basins of South America [9]. The Trichomycterinae, hereafter trichomycterines, are present in a large array of freshwater environments from southern Central America to southern South America, including Lake Titicaca in the central Andes, Patagonian rivers, and Amazonian lowland streams [10]. However, trichomycterine species diversity is particularly high in rivers draining the Andes in western South America and the mountain ranges of eastern South America [11]. Among the eight genera included in this subfamily, species of Cambeva Katz, Barbosa, Mattos and Costa 2018, Eremophilus Humboldt 1805, Hatcheria Eigenmann 1909, Rhizosomichthys Miles 1943, Silvinichthys Arratia 1998 and Trichomycterus Valenciennes 1832 typically inhabit aquatic mountain environments, whereas members of the genera Bullockia Arratia, Chang, Menu-Marque and Rojas 1978, Ituglanis Costa and Bockmann 1993 and Scleronema Eigenmann 1917 are found in low altitude streams [10,12,13,14]. At least one species inhabits a phreatic environment [15], whereas several others are specialized troglomorphic species [16,17].

The taxonomy of the trichomycterine genus Trichomycterus has been considered the most problematic among trichomycterids [18]. Its diagnosis was limited to a combination of features allowing to include all species lacking the derived character states diagnosing the other trichomycterine genera [13,14,18]. As a result, Trichomycterus comprised a broad artificial assemblage of trichomycterids, occupying the whole geographical range of the family. Even after some species of Trichomycterus being transferred to Ituglanis and Silvinichthys [14,19], Trichomycterus was still considered as a paraphyletic genus, with over 100 valid species, part of them endemic to the Andes and adjacent high elevation areas of northern South America, and others from eastern South America [20,21].

Concordantly to previous predictions, recent multigene analyses have strongly indicated Trichomycterus as paraphyletic [9,22]. In these analyses, trichomycterines were grouped in two major clades, a well-supported clade containing taxa endemic from eastern South America, including several species of Trichomycterus and Scleronema, and another clade, weakly supported containing the remaining trichomycterine genera and several other species of Trichomycterus from western and northern South America. Within the inclusive eastern South American clade (hereafter CST-clade), all analyses strongly supported three subclades: one comprising several species of Trichomycterus from eastern Brazil, including its type species T. nigricans Valenciennes, 1832; another one comprising species of Scleronema; and a third subclade, sister to Scleronema comprising nominal species placed in Trichomycterus [9,22]. The nomenclatural solution found by Katz et al. [9] was to restrict Trichomycterus to the molecularly well-supported subclade including T. nigricans and to describe a new genus Cambeva Katz, Barbosa, Mattos and Costa 2018 for the subclade sister to Scleronema. Presently, Cambeva comprises 29 species [23], Scleronema, eight species [24] and Trichomycterus sensu Katz et al. [9] (hereafter Trichomycterus s.s. [sensu stricto]), 60 species [25]. Trichomycterus s.s. occurs in a broad area between the Rio de Contas basin, north-eastern Brazil, at about 13°30′ S [26] and the rivers draining the Baía de Paranaguá basin, in southern Brazil, at about 48°15′ S [27].

The classification proposed by Katz et al. [9] solved an old taxonomical problem regarding monophyly and delimitation of Trichomycterus, but also imposed some practical difficulties for subsequent taxonomical studies since no unique apomorphic morphological character state was found by Katz et al. [9] for Trichomycterus s.s. The absence of a formal unambiguous morphological diagnosis for Trichomycterus and the absence of intrageneric comparative morphological studies involving a broad taxon sample makes it difficult to allocate new species on the basis of morphology alone. The objective of this study was to conduct an osteological comparative analysis comprising a large sample of valid species of Trichomycterus s.s., integrating with a multigene dataset in order to perform the most comprehensive phylogenetic study on this clade.

History of Trichomycterus Intrageneric Classification

Due to the large number of species and absence of detailed comparative studies in Trichomycterus, some taxonomical studies have tried to delimit informal intrageneric groups when diagnosing new species. Costa [13] tentatively delimited unnamed groups and subgroups of Trichomycterus s.s. from south-eastern and eastern Brazil based on some putative apomorphic character states of the external morphology, such as the relative position of supraorbital pores s6, the relative position between the urogenital orifice and the dorsal-fin base, relative position of pelvic-fin bases, barbels extent, caudal-fin shape, and color patterns. Species were then divided into two main groups, the first one including most species presently placed in Trichomycterus s.s. that were described before 1992, and a second one comprising T. reinhardti (Eigenmann 1917) and species presently placed in the T. brasiliensis group (see below) and in Ituglanis. Species presently placed in Cambeva were then considered distantly related from those two groups and placed in a separate third group.

Barbosa and Costa [28] named the T. brasiliensis species group based on derived conditions of osteological characters and color pattern. In spite of some diagnosis changes [29,30], this group has been corroborated in all molecular phylogenetic analyses [9,22,31,32,33,34], but different names have been used to this clade, including T. brasiliensis species-complex [29] and the brasiliensis clade [31]. Barbosa and Costa [35] named a group of Trichomycterus from south-eastern Brazil as the T. travassosi species complex based on coloration alone, but this group has not been corroborated by molecular data [9,32]. More recently, based on osteology and molecular evidence, Costa et al. [32,36] named the T. nigroauratus and T. nigricans groups, and Costa and Katz [25] named the T. reinhardti group, as well as Ochoa et al. [31], based on molecular evidence, named the itatiayae clade (including T. itatiayae and the T. reinhardti group in [25]) and the immaculatus clade. The use of different names for the same clades and the use of misidentified species to name clades have made the intrageneric informal classification of Trichomycterus s.s. confusing. The secondary objective of this paper was to provide an intrageneric classification well supported by both morphology and DNA evidence.

2. Materials and Methods

2.1. Comparative Osteology

This study is primarily based on the examination of specimens previously cleared and stained following Taylor and Van Dyke’s protocol [37] and deposited in the ichthyological collection of the Institute of Biology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil (see list of specimens in Appendix A). A total of 53 of the 60 valid species of Trichomycterus s.s. were examined, comprising samples of paratypes or topotypes. The analysis also included 33 species of non-Trichomycterus trichomycterines; two species, Listrura tetraradiata Landim and Costa 2002 and Microcambeva ribeirae Costa, Lima and Bizerril 2004, representing the Microcambevinae, the sister group of the remaining subfamilies comprising the TSVSGM-clade, which was supported as a sister to the Trichomycterinae [7]; two species, Copionodon pecten de Pinna 1992 and Trichogenes longipinnis Britski and Ortega 1983, representing the Copionodontinae and Trichogeninae, respectively, which together form the sister group to a clade comprising all other trichomycterids [22,31]; and the nematogenyid Nematogenys inermes (Guichenot 1848), a basal loricarioid catfish taxon.

The comparative analysis focused on bone structure variation, which has been used in the last almost 50 years to infer phylogenetic relationships of trichomycterids [38], as well as to diagnose trichomycterine genera and species (e.g., [12,19,20,21,39,40,41,42]). As a consequence, the considerable amount of available data in the literature on trichomycterine osteology allows more inclusive comparisons. The comparative analysis was directed to qualitative characters potentially diagnosing distinct intrageneric clades; quantitative characters and characters with high variability within different lineages or with a high level of subjectivity were not considered in this analysis. Illustrations were made using a Zeiss Stemi SV 6 stereomicroscope with camera lucida.

Terminology for bones followed Bockmann et al. [20], except for: ‘epibranchial 5 cartilage’, here using ‘ceratobranchial 4 accessory element’ according to Carvalho et al. [43]; ‘urohyal’, here replaced by ‘parurohyal’ following Arratia and Schultze [44]; ‘pleural rib’, here substituted by ‘rib’ following Britz and Bartsch [45] that have provided evidence that there is a single rib type in teleosts. The use of the names antorbital and sesamoid supraorbital here adopted, following Bockmann et al. [20] and Adriaens et al. [46], respectively, deserves some additional explanation due to some recent controversy involving homology and terminology, as below discussed.

The anterior-most bone has been identified as lacrimal [38], tendon-bone infraorbital [19], antorbital [20], and more recently lacrimal-antorbital [47]. In trichomycterines, it consists of a small flat ossification, drop-shaped in a dorsal view, associated with a segment of the infraorbital canal; it is placed close to the base of the nasal barbel externally and the antero-lateral part of the autopalatine internally. Since this bone is placed at the same position as the antorbital bone of basal siluriforms [48], it is here called antorbital following Bockmann et al. [20] and Adriaens et al. [46]. De Pinna et al. [47] found evidence that the antorbital is a compound structure derived from the fusion between the antorbital and the lacrimal. However, evidence was based only on the examination of a single cleared and stained juvenile specimen of T. longipinnis Britski and Ortega 1983, 20.3 mm of standard length, thus needing confirmation since robust evidence of a fusion between these elements is still unavailable [49].

The posterior osseous structure here identified as sesamoid-supraorbital, resulting from the ossification of a ligament between the antorbital and the neurocranium [46], was first equivocally identified as a nasal bone [10]. Since it is not associated with a sensory canal as a true nasal bone, Baskin [38] considered this structure as neomorphic and a synapomorphy of a trichomycterid group (i.e., Trichomycterinae plus Sarcoglanidinae and Glanapteryginae), naming it the fronto-lacrimal tendon bone. Different names for this bone have been used in the subsequent literature, including supraorbital [12], antorbital [50], and tendon-bone supraorbital [19]. More recently, this bone was identified as a sesamoid supraorbital bone [46] and subsequently called the sesamoid supraorbital by several authors [21,36,51]. This rod-shaped bone is present both in the Trichomycterinae and in basal members of the TSVSGM-clade [7,38], but no similar structure is present in basal trichomycterids (i.e., Copionodontinae and Trichogeninae). Two of the three species of Trichogeninae, T. longipinnis and T. beagle de Pinna, Reis and Britski 2020 have a scale-like ossification attached to the dorsal surface of the lateral process of the lateral ethmoid [47] that is not present in the other species of Trichogenes and in any species of the Copionodontinae, the sister group of the Trichogeninae [22,31]. In fact, due to its shape and position, the presence of this bone in those two trichogenine species seems to be a unique condition among trichomycterids and other loricarioids. This scale-like ossification occurring only in trichogenines was first tentatively identified as antorbital and homologous to the rod-shaped sesamoid supraorbital of trichomycterines [50], and recently named the barbular bone [47] and again considered as homologous to the fronto-lacrimal tendon bone sense by Baskin [38] (sesamoid supraorbital). This supposed homology was justified by both bones being positioned between the antorbital and the neurocranium margin and by both not being associated with any head sensory canal. However, only in T. beagle among species of Copionodontinae and Trichogeninae, there is a ligament uniting the antorbital and the barbular bone [47]. On the other hand, these structures have a different shape (scale-like in Trichogenes vs. rod-shaped), and more importantly, they have a different position (i.e., barbular bone associated with the lateral process of the lateral ethmoid in Trichogenes, vs. sesamoid supraorbital associated with the dorsal surface of the autopalatine), suggesting that they are not homologous. Therefore, it would be premature to interpret the barbular bone present only in two species of the clade encompassing copionodontines and trichogenines as homologous to the sesamoid supraorbital of the clade comprising trichomycterines and the TSVSGM-clade, without developmental studies supporting such proposal.

Terminology for minor osteological structures such as processes, bone outgrowths, and articulations, mostly followed Baskin [38], Arratia [41], and Adriaens et al. [46], as described in the text, and for cephalic latero-sensory system following Arratia and Huaquin [48].

2.2. DNA Sequences

The molecular dataset comprised partial sequences of two nuclear genes, myosin heavy chain 6 (MYH6, 543 bp) and recombination activating 2 (RAG2, 821 bp), and two mitochondrial genes, cytochrome c oxidase subunit I (COI, 522 bp) and cytochrome b (CYTB, 1088 bp). Sequences were first obtained in previous studies focusing on trichomycterine phylogeny [9,25,32,33,34], in which sequenced specimens were, whenever possible, collected in the species type locality. Amplification of the target DNA fragments was made through the polymerase chain reaction (PCR) method, using the primers MHRAG2-F1 and MHRAG2-R1 [52]; RAG2 TRICHO F and RAG2 TRICHO R [7]; RAG2 MCF and RAG2 MCR [53]; myh6_F459 and myh6_R1322 [54]; MYH6 TRICHO F and MYH6 TRICHO R [7]; Cytb Siluri F and Cytb Siluri R [55]; L5698-ASN and H7271-COI [55]; and FISHF1 and FISHR1 [56]. The analyses were complemented with sequences obtained from GenBank; a complete list of species and GenBank accession numbers appears in Appendix B. Alignment was conducted for each gene set using Clustal W algorithm [57] implemented in MEGA 7.0 [58]; no gap was found in the analyzed alignments.

2.3. Phylogenetic Analyses

The phylogenetic analyses comprised species that were examined in the osteological comparative analysis and had available DNA data, including 44 species of Trichomycterus s.s. and 21 outgroups (see Apenddix B for a list of species used in the phylogenetic analyses). Fifty-two characters rsulted from the osteological comparative analysis (see above); a list of characters, formulated according to Sereno [59], is provided in the Appendix C, and the distribution of character states among taxa appears in the data matrix of the Appendix D. Osteological characters were combined to a set of gene fragments more commonly used in phylogenetic studies on trichomycterines (see DNA sequences above), a total of 2974 bp. Molecular data were partitioned according to the codon position for each gene, as well as morphological data were placed in a separate partition. The best-fitting models of molecular evolution for each partition was found using the Bayesian information criterion (BIC) of ModelFinder [60], implemented in IQ-TREE 1.6.11 [61], with partition models described by Chernomor et al. [62]; the list of partitions and their respective models of nucleotide substitution are provided in the Appendix E. The concatenated dataset, a total of 3026 characters for 65 taxa, was first analyzed using IQ-TREE for Maximum Likelihood (ML), applying three methods for assessing the reliability of internal branches: the Shimodaira-Hasegawa-like procedure support (SH-aLRT) [63], the Bayesian-like transformation of SH-aLRT support (aBayes) [64] and the ultrafast bootstrap support (UFBoot) [65,66], using 1000 replicates and default parameters as implemented in IQ-TREE. The concatenated dataset was also analyzed using Maximum Parsimony (MP) methods performed with TNT 1.1 [67]. The phylogenetic analysis was rooted in the nematogenyid N. inermis. The search for most parsimonious trees was conducted using the ‘traditional’ search algorithm and setting random taxon-addition replicates to ten, tree bisection–reconnection branch swapping, multitrees in effect, collapsing branches of zero length, characters equally weighted, and a maximum of 1000 trees saved in each replicate. Character states were treated as unordered. Branch support was assessed by bootstrap analysis [68], using a heuristic search with 1000 replicates and the same settings used in the MP search. Unambiguous diagnostic character states for the main lineages of Trichomycterus were obtained by character state optimization of the combined analysis tree using TNT 1.1.

2.4. Taxonomic Accounts

Subgenera appeared following a phylogenetic order, according to Figure 1. Diagnoses comprised osteological apomorphic features supporting the taxon, followed by combinations of external morphological features additionally distinguishing the taxon from other taxa; the abbreviation ‘chast’ means character state, that is numbered according to Appendix C.

3. Results

3.1. Comparative Osteology and Phylogeny

Fifty-two osteological characters with informative variability were obtained from the comparative analysis (see Appendix C for list of characters and Appendix D for distribution of character states among terminal taxa). The ML analysis generated a phylogenetic tree with most nodes reaching high support values (Figure 1). The single most parsimonious tree generated by the MP analysis was similar to the ML tree, but with some nodes being weakly supported (see list of support values for each node of Figure 1 in

Table 1. Node support in phylogenetic analyses (Figure 1), comprising Shimodaira-Hasegawa-like procedure support (SH-aLRT) with values above 70%, the Bayesian-like transformation of SH-aLRT support (aBayes) with values above 0.7, and the ultrafast bootstrap support (UFBoot) with values above 70%, for the Maximum Likelihood (ML) analysis, and bootstrap values above 50% for the Maximum Parsimony (MP) analysis.

Node	ML			MP
	SH-aLRT	aBayes	UFBoot	Bootstrap
1	-	-	-	100
2	97.4	1	100	97
3	100	1	100	99
4 (Microcambevinae)	100	1	100	99
5 (Trichomycterinae)	98.4	1	100	98
6	84.5	1	74	-
7	85.8	1	76	-
8	99.6	1	100	78
9	100	1	100	100
10 (CST-clade)	99.8	1	100	95
11 (Cambeva + Scleronema)	99.9	1	100	99
12 (Scleronema)	100	1	100	100
13	99.9	1	100	100
14	98.6	1	100	100
15 (Cambeva)	89.0	1	95	84
16	66.7	-	79	-
17	96.6	1	95	-
18	99.1	1	100	83
19	99.4	1	100	100
20	73.3	0.8	92	-
21 (Trichomycterus)	85.1	1	92	89
22	78.6	0.8	82	-
23 (Paracambeva)	100	1	100	100
24 (T. reinhardti group)	97.3	1	99	99
25	98.8	1	100	97
26	96.9	1	97	92
27	-	1	-	-
28	100	1	100	100
29	100	1	100	99
30	83.7	0.9	97	99
31	83.1	1	91	-
32 (sg. Trichomycterus)	100	1	100	100
33 (T. nigroauratus group)	100	1	100	100
34 (T. nigricans group)	86.2	1	96	72
35	98.3	1	100	94
36	97.6	1	100	100
37	91	1	93	-
38 (Psammocambeva)	98.9	1	100	89
39 (T. pradensis group)	50.6	0.7	80	-
40	87.1	1	96	-
41 (T. travassosi group)	100	1	100	94
42	-	-	-	-
43	92.3	1	98	-
44	85.9	1	87	-
45	92.1	1	97	-
46	-	-	85	-
47	99.2	1	100	95
48 (Humboldtglanis)	99.4	1	100	100
49 (Cryptocambeva)	100	1	100	100
50	92.4	1	99	55
51	96.2	1	99	58
52	96	1	99	92
53	89.6	1	97	97
54	73	-	95	-
55	100	1	100	100
56	63.6	-	94	92
57	92.6	1	99	-

). In both analyses, the monophyly of Trichomycterus s.s. was corroborated (SH-aLRT support of 85.1%, aBayes support of 0.96, ultrafast bootstrap support of 92% for the ML analysis, and a bootstrap of 89% for the MP analysis).

Like in previous studies [9,32], T. giganteus Lima and Costa 2004, here placed in a separate subgenus, appeared as sister to a clade containing all other congeners, besides exhibiting ten unique osteological apomorphic character states, first described here (see taxonomic accounts below). The clade comprising all species of Trichomycterus s.s., except T. giganteus, contained five main subclades supported by high values in all analyses (Figure 1, Table 1), which were below recognized as subgenera.

3.2. Taxonomic Accounts

3.2.1. Megacambeva, New Subgenus

LSID: urn:lsid:zoobank.org:act:DD5D2BBB-A0E2-4D6F-96F1-229A811ADCE7

Type species. Trichomycterus giganteus Lima and Costa 2004.

Diagnosis. Megacambeva differs from all other subgenera of Trichomycterus s.s. in possessing the following apomorphic conditions: presence of a short osseus ridge on the dorsal surface of the autopalatine, between the lateral margin and the middle portion of the bone (chast.2.1, Figure 2A; vs. absence of a similar ridge, Figure 2B–F); a posteriorly directed latero-posterior process of the autopalatine (chast.4.2, Figure 2A; vs. laterally or latero-posteriorly directed, Figure 2B–F); presence of a prominent ridge on the dorsal surface of the shell-like articular facet for the lateral ethmoid, terminating in a process latero-dorsally directed and connected by ligament to a robust sesamoid supraorbital (chast.8.1, Figure 2A; vs. never a similar structure, Figure 2B–F); presence of a deep concavity in the hyomandibular outgrowth reaching the ventral portion of the bone outgrowth (chast.13.3, Figure 3A; vs. absence, Figure 3B–F); strongly curved distal portion of largest internal-most opercular and interopercular odontodes, with their tips forming an angle of about 90° with its main axis (chast.15.1, Figure 3A; vs. nearly straight or only slightly curved, Figure 3B–F); a prominent trapezoidal process on the lateral margin of the lateral ethmoid, immediately posterior to the articular zone with the autopalatine (chast.30.1, Figure 2A; vs. process absent, Figure 2B–F); a slender Weberian capsule, subtly narrowing laterally to form a long lateral extremity (chast.44.1; Figure 4A; vs. broad, pear-like, abruptly narrowing laterally to form a short lateral extremity, Figure 4B–D); a hypertrophied tree-shaped supralateral process on the scapulo-coracoid (chast.45.1; Figure 5A; vs. needle shaped and small, Figure 5B); and three anterior processes in the pelvic bone (chast.51.1, Figure 6A; vs. two, Figure 6B–G). In addition, the only species included in Megacambeva, T. giganteus is unique among congeners by reaching a larger size, surpassing 200 mm SL (vs. usually reaching about 50–75 mm SL as maximum adult size, rarely reaching about 100–120 mm SL), and by having a unique dorsal fin morphology, that is deeper than long (vs. longer than deep, Figure 1). Megacambeva is also distinguished from all other subgenera by the following combination of character states: nine or ten pectoral-fin rays (vs. six to eight in Cryptocambeva, Humboldtglanis, and Paracambeva); anal-fin origin posterior to dorsal-fin base (vs. anal-fin origin in vertical through posterior half of dorsal-fin base in Cryptocambeva, Humboldtglanis, Paracambeva, and Psammocambeva); long maxillary barbel, its tip reaching the anterior third of the pectoral fin (vs. reaching anterior margin of pectoral-fin base or shorter in Cryptocambeva, Humboldtglanis, Paracambeva, and Trichomycterus); basal portion of maxillary barbel not distinctively widened (vs. widened in Psammocambeva); and jaw teeth always sharply pointed (vs. at least larger adults with incisiform teeth in Trichomycterus).

Included taxa. A single species, T. giganteus.

Etymology. From the Greek mega (large), and cambeva, a popular name for trichomycterids in south-eastern Brazil, referring to the large size of the only included species. Gender feminine.

Distribution and ecological notes. The only included species, T. giganteus, is known from a single locality in the Rio Guandu-Mirim, Rio Guandu basin, an isolated small coastal basin in south-eastern Brazil. Adult specimens were found where the river form small lakes below waterfalls, close to the river bottom, and below large rocks, about 200 cm deep or slightly more. Specimens were eventually seen swimming between bottom rocks during daylight, suggesting some diurnal activity.

3.2.2. Paracambeva, New Subgenus

LSID: urn:lsid:zoobank.org:act:F5037571-D95D-4091-880B-9E598AF9ED9F

Type species. Trichomycterus itatiayae Miranda Ribeiro 1906.

Diagnosis. Paracambeva is supported by two apomorphic conditions: the dorsal process of the interopercle being close to the anterior margin of the odontode patch (chast.17.1, Figure 3B; vs. dorsal process more posterior placed, Figure 3A,C–F; independently occurring in Cambeva and Scleronema according to the analysis) and a slender parapophysis of the second free vertebrae, since its proximal portion to the distal portion (chast.38.1, Figure 7C; vs. robust, Figure 7D–F). Species of Paracambeva also differ from all other species the Trichomycterus s.s. by having a unique color pattern consisting of a continuous black stripe between the snout and the caudal-fin base, becoming dispersed and spotted in larger specimens (Figure 1) [25] (Figure 1 and Figure 2). Paracambeva is also distinguished from all other subgenera of Trichomycterus s.s. by the following combination of character states: six or seven pectoral-fin rays (vs. eight in Humboldtglanis, eight or nine in Psammocambeva and Trichomycterus, and nine or ten in Megacambeva); anal-fin origin in vertical through the middle or posterior portion of the dorsal-fin base (vs. posterior to end of the dorsal-fin base in Megacambeva); basal portion of maxillary barbel not distinctively widened (vs. widened in Psammocambeva); distal tip of the maxillary barbel not reaching the pectoral-fin base (vs. reaching first pectoral-fin base third in Megacambeva); eye small, iris with an indistinct or small light area (vs. iris light area broad in Psammocambeva).

Included taxa. Paracambeva comprises two main lineages. One lineage, containing only T. itatiayae, is characterized by the following apomorphic character states: a rudimentary and curved latero-posterior process of the autopalatine (chast.3.1, Figure 2B; vs. well-developed, about straight, Figure 2A,C–F); a deep concavity on the metapterygoid accommodating an expansion of the antero-dorsal process of the quadrate (chast. 11.1, Figure 3B; vs. never a similar morphology, Figure 3A,C–F); a deep anterior outgrowth of the hyomandibula, with the dorsal margin nearly horizontal anteriorly and a distinctive U-shaped concavity posteriorly (chast. 13.1, Figure 3B; vs. never a similar morphology, Figure 3A,C–F); a medially directed antero-dorsal extremity of the lateral ethmoid (chast.27.2, Figure 2B; vs. anteriorly or laterally directed, Figure 2A,C–F); and the presence of a small process close to the cartilage supporting fin rays on the postero-medial portion of the pelvic bone (chast.52.2, Figure 6C; vs. absence, Figure 6A,B,D–G). The other lineage, the T. reinhardti species group, contains ten species: T. anaisae Katz and Costa 2021, T. funebris Katz and Costa 2021, T. humboldti Costa and Katz 2021, T. ingaiensis Katz and Costa 2021, T. luetkeni Katz and Costa 2021, T. pauciradiatus Alencar and Costa 2006, T. piratymbara Katz, Barbosa and Costa 2013, T. reinhardti (Eigenmann 1918), T. sainthilairei Katz and Costa 2021, and T. septemradiatus Katz, Barbosa and Costa 2013. The T. reinhardti species group is supported by the following apomorphic character states: a lengthened ventral flap of the autopalatine articular facet for the lateral ethmoid (chast.7.1, Figure 2I; vs. short or rudimentary, Figure 2J); infraorbital canal not attached to antorbital (chast.23.1, Figure 2G; vs. attached, Figure 2H); and a rudimentary pelvic girdle, with a narrow proximal region, without vestiges of the antero-medial and posterior processes (chast.47.1, Figure 6B; vs. pelvic girdle well-developed, Figure 6A,C–G). Species of the T. reinhardti group are also distinguished from T. itatiayae by having pointed jaw teeth (vs. incisiform).

Distribution and ecological notes. Trichomycterus itatiayae is endemic to the upper Rio Paraíba do Sul basin, south-eastern Brazil; it was found associated with leaf litter, occasionally swimming above it during daylight [69]. The T. reinhardti group occurs in a wide geographical range, including the upper Rio Paraná, upper Rio São Francisco, upper Rio Doce, and middle Rio Paraíba do Sul basin, south-eastern Brazil. During daylight field works, species of Paracambeva are found hidden within roots of marginal vegetation in small shallow streams, indicating to have nocturnal habits [25].

Etymology. The name Paracambeva is derived from the Greek prefix para, meaning resembling, and cambeva, a popular name for trichomycterids in south-eastern and southern Brazil and a nominal trichomycterine genus, in reference to the superficial resemblance with some species of Cambeva (e.g., Cambeva poikilos (Ferrer and Malabarba 2013)). Gender feminine.

3.2.3. Subgenus Trichomycterus Valenciennes 1832

Type species. Trichomycterus nigricans Valenciennes 1832.

Diagnosis. The subgenus Trichomycterus is supported by three apomorphic character states: the presence of a posterior expansion in the metapterygoid (chast.10.1, Figure 3D; vs. absence, Figure 3A–C,E,F); a long lateral process of the sphenotic-prootic-pterosphenoid complex (chast.32.1, Figure 8A; vs. short, Figure 8B); and the presence of an expansion on the antero-distal portion of the second free vertebra parapophysis, overlapping the adjacent rib that is widened in its sub-proximal portion (chast.39.1, Figure 7D; vs. absence of expansion, rib not widened in its sub-proximal portion, Figure 7C,E–G). The subgenus Trichomycterus is distinguished from all other subgenera of Trichomycterus s.s. by having the opercular odontode patch more posteriorly placed, with its posterior limit reaching a vertical through the pectoral-fin base (vs. anterior to pectoral-fin base). The subgenus Trichomycterus also differs from all other subgenera of Trichomycterus s.s. by the following combination of character states: eight or nine pectoral-fin rays (vs. six or seven in Cryptocambeva and Paracambeva); opercular odontodes transversely arranged (vs. diagonally in Cryptocambeva and Humboldtglanis); dorsal fin longer than deep (vs. deeper than long in Megacambeva); basal portion of maxillary barbel not distinctively widened (vs. widened in Psammocambeva).

Included taxa. Eight valid species in two groups: the T. nigricans group, including T. caipora Lima, Lazzarotto, and Costa 2008, T. immaculatus (Eigenmann and Eigenmann 1889), T. nigricans, and T. santaeritae (Eigenmann 1918), supported by the presence of an enlarged posterior process of the maxilla (chast.22.1, Figure 2C; vs. not enlarged, Figure 2A,B,D–G); and the T. nigroauratus group, including T. maculosus Barbosa and Costa 2010, T. nigroauratus Barbosa and Costa 2008, and T. quintus Costa 2020, supported by the presence of a folded laminar expansion of the lateral ethmoid border, just above the articular facet for the autopalatine (chast.26.1, Figure 2D; vs. absence, Figure 2A–C,E–G); and an elongate pelvic bone, with a narrow and straight posterior process (chast.48.1, Figure 6D; vs. never a similar shape, Figure 6A–C,E–G). Species of the T. nigricans group are also distinguished from species of the T. nigroauratus group by having eight pectoral-fin rays (vs. nine) and posterior margin of the caudal fin straight to slightly convex (vs. concave at least in juveniles).

Distribution and ecological notes. Species of the subgenus Trichomycterus are endemic to an area comprising the Rio Paraíba do Sul basin and the neighboring smaller coastal basins. Most species have cryptical habits during the day, suggesting that they are nocturnal, but T. santaeritae is a specialized diurnal psammophilic species [32,36].

3.2.4. Psammocambeva, New Subgenus

LSID: urn:lsid:zoobank.org:act:4ED967A8-D2DB-43F1-9918-6A3384652165

Type species. Trichomycterus travassosi Miranda Ribeiro, 1949.

Diagnosis. Psammocambeva is supported by the presence of a long maxilla, that is longer than the premaxilla (chast.20.1, Figure 2F; vs. shorter, Figure 2A–E,G,H), a derived condition independently acquired in a subclade of the subgenus Trichomycterus. Psammocambeva is distinguished from other subgenera of Trichomycterus s.s. by the presence of a widened basal portion of the maxillary and rictal barbels (Figure 9B; vs. not widened, Figure 9A,C,D) and a broad ventral expansion of the light portion of the iris (Figure 9B; vs. light portion of the iris not ventrally widened, Figure 9A,C,D). Distinguished from Paracambeva also by the anterior segment of the infraorbital latero-sensory canal being always present and close to the nostrils (vs. separated by an interspace from the nostrils when present); from Cryptocambeva and Paracambeva by the presence of eight or nine pectoral-fin rays (vs. six or seven); from Cryptocambeva and Humboldtglanis by the opercular odontodes being transversely arranged, vs. diagonally); from Humboldtglanis by the absence of white spots on the flank (vs. presence); from Trichomycterus by the opercular odontode patch not extending posteriorly above the pectoral-fin base (vs. extending); and from Megacambeva by the dorsal fin being longer than deep (vs. deeper than long).

T. melanopygius Reis, dos Santos, Britto, Volpi, and de Pinna 2020, T. pradensis Sarmento-Soares, Martins-Pinheiro, Aranda, and Chamon 2005, and T. tete Barbosa and Costa 2010, is characterized by two apomorphic conditions: a shallow anterior hyomandibular outgrowth (chast.12.1, Figure 3C; vs. deep, Figure 3A,B,D–G); and an expanded ventral preopercular flap (chast.19.1, Figure 3C; vs. short, Figure 3A,B,D–G). Three species not available for the present study, showing great external morphological similarity, are probably members of this clade: T. itacambirussu Triques and Vono 2004, T. jequitinhonhae Triques and Vono 2004, T. landinga Triques and Vono 2004, and T. trefauti Wosiacki 2004. The T. travassosi group, comprising T. auroguttatus Costa 1992, T. cf. alternatus (Eigenmann 1917), T. caudofasciatus Alencar and Costa 2004, T. gasparini Barbosa 2013, T. goeldii Boulenger 1896, T. jacupiranga Wosiacki and Oyakawa 2005, T. longibarbatus Costa 1992, T. macrophthalmus Barbosa and Costa 2012, T. mimosensis Barbosa 2013, T. pantherinus Alencar and Costa 2004, T. puriventris Barbosa and Costa 2012, and T. travassosi Miranda Ribeiro 1949, is supported by: a widened lateral margin of the lateral ethmoid, (chast.31.1, Figure 2F; vs. not widened, Figure 2A–E).

Trichomycterus astromycterus Reis, de Pinna, and Pessali 2020, not available to the present study, probably is a member of the T. travassosi group. Although considered to be possibly closely related to the Chilean monotypic genus Bullockia [70], T. astromycterus exhibits the derived character states supporting Psammocambeva and the T. travassosi group, as well as three synapomorphies supporting a subclade comprising T. cf. alternatus, T. auroguttatus, and T. goeldii: a long autopalatine postero-lateral process, its length nearly equal or slightly longer than the autopalatine longitudinal length excluding the postero-lateral process (chast.5.1, Figure 2F; vs. distinctively shorter, Figure 2A–E); a slightly folded maxilla (chast.21.1, Figure 2F; vs. curved or straight, Figure 2A–E); and the anterior cranial fontanel represented by a minute aperture (chast.35.1, Figure 8B; vs. broad aperture, Figure 8A), besides all having a protruded orbit (Figure 9B).

Etymology. From the Greek, psammos (sand), and cambeva, a popular name for trichomycterids in south-eastern and southern Brazil and a nominal trichomycterine genus, an allusion to the psammophilic habits of the type species and other included species. Gender feminine.

Distribution and ecological notes. Psammocambeva occupies the entire geographical distribution of Trichomycterus s.s., from the Rio de Contas basin in north-eastern Brazil to the rivers associated with the Baía de Paranaguá in southern Brazil. Field studies indicate that species of Psammocambeva are always found associated with gravel or sand substrate [35], but at least some species (e.g., T. auroguttatus and T. cf. alternatus) are specialized psammophilic species, often found completely buried in sand stretches on the middle of river bottom [13]. At least T. pradensis has migrating behavior, which is supported by field studies conducted in February 2014, when hundreds of juvenile specimens of this species, about 20 mm of total length, were seen forming a continuous upstream flow in the lower Rio Jucuruçu (16°23′34” S 39°17′09” W), eastern Brazil (person. observ.).

3.2.5. Humboldtglanis, New Subgenus

LSID: urn:lsid:zoobank.org:act:09886ED2-C1BC-4A84-8824-07E6EEA8B510

Type species. Trichomycterus albinotatus Costa 1992.

Diagnosis. Species of Humboldtglanis are distinguished from all other congeners by the following apomorphic features: a slightly convex medial margin of the autopalatine in larger individuals (above about 45 mm SL; chast.6.1, Figure 2H; vs. concave), steep anterior and posterior portions of the dorsal margin of the hyomandibula anterior outgrowth, converging to a pronounced V-shaped notch (chast.13.1, Figure 3E; vs. outgrowth never with similar morphology, Figure 3A–D,F), and a sharp process in the latero-posterior margin of the epioccipital (chast.34.1, Figure 4E; vs. process absent, Figure 4A–C). Humboldtglanis is also distinguished from other subgenera, except Cryptocambeva, by the following apomorphic features: presence of a long and narrow ridge on the dorsal surface of the autopalatine, close to its lateral margin (chast.1.1, Figure 2E,H; vs. absence, Figure 2A–D,F,G); an oblique arrangement of odontodes of the basal portion of the opercular odontode patch (chast.14.1, Figure 3E; vs. vertical, Figure 3A–D,F); ventral surface of the lateral margin of the mesethmoid ventrally folded around the vomer margin (chast.25.1, Figure 2J; vs. not folded, Figure 2I); lateral ethmoid medially separated from its symmetrical homologous (chast.28.1, Figure 2E; vs. in contact, Figure 2A–D,F); presence of a lateral expansion of the lateral ethmoid margin close to the sesamoid supraorbital (chast.29.1, Figure 2E; vs. absence, Figure 2A–D,F); the presence of a small notch in the posterior margin of the parapophysis of the second free vertebra, in an area close to the proximal extremity of the adjacent rib (chast.40.1, Figure 7E; vs. absence, Figure 7A–D,F); and a rudimentary posterior process of the pelvic bone (chast.49.1, Figure 6E,F; vs. well-developed, Figure 6A,C,D,G). Species of Humboldtglanis are also distinguished from other congeners by the presence of rows of small white spots along the flank in specimens about 50 mm SL or less (Figure 1; vs. white spots absent). Humboldtglanis is also distinguishable from other subgenera of Trichomycterus s.s. by the following combination of characters: the anal-fin origin is placed in a vertical through the posterior portion of the dorsal-fin base (vs. anal-fin origin posterior to the dorsal-fin base in Megalebias), eight pectoral-fin rays (vs. six or seven in Cryptocambeva and Paracambeva, nine or ten in Megacambeva), dorsal fin longer than deep (vs. deeper than long in Megacambeva, Figure 1); presence of an anterior infraorbital canal attached to the antorbital, externally close to nostrils (vs. anterior infraorbital canal when present is not attached to the antorbital and is externally separated from nostrils by an interspace in Paracambeva); never having a continuous black stripe between the snout and the caudal-fin base, becoming disperse and spotted in larger specimens (vs. always present in Paracambeva); orbital diameter about 12–16% of the head length in specimens between about 40 and 60 mm SL (vs. about 7–11% of the head length in Cryptocambeva), by having the basal portion of maxillary barbel not distinctively widened (vs. widened in Psammocambeva); the light portion of iris not extending ventrally (vs. broad, extending ventrally in Psammocambeva, Figure 9); and the posterior margin of the opercular patch of odontodes placed in a vertical anterior to pectoral-fin base (vs. in vertical through the pectoral-fin base in Trichomycterus).

Included taxa. Two species, T. albinotatus and T. vitalbrazili Vilardo, Katz, and Costa 2020.

Etymology. The name Humboldtglanis was used in honor of the Prussian naturalist Friedrich Wilhelm Heinrich Alexander von Humboldt (1769–1859), for his valuable and pioneering contribution in studies on distribution, ecology, and conservation of mountain organisms; the name glanis, is from the ancient Greek and is commonly used for generic catfish names. The name is also an allusion to several species of Humboldtglanis only inhabiting mountain rivers 1000 m above sea level. Gender masculine.

Distribution and ecological notes. Humboldtglanis is endemic to the Rio Paraíba do Sul basin, south-eastern Brazil, occurring in areas about 1100 m above sea level. Field observations indicate that species are diurnal, swimming actively during daylight a few centimeters above gravel substrate and small rocks, but larger specimens about 60 mm SL or more, are mostly found hidden among stones, only sporadically swimming around this habitat [13,33].

3.2.6. Cryptocambeva, New Subgenus

LSID: urn:lsid:zoobank.org:act:E0BE3FF5-ABAE-4881-9875-5FA5C23FE2C4

Type species. Trichomycterus mirissumba Costa 1992.

Diagnosis. Species of Cryptocambeva are distinguished from all other congeners by the following apomorphic features: a rounded, narrow, and elongated extremity of the pterotic lateral process (chast.33.2, Figure 4C; vs. pterotic lateral process truncate and not elongated, Figure 4A,B,D), a small posttemporo-supracleithrum, resulting in a broad interspace between the posttemporo-supracleithrum and adjacent bones, and a short postero-medial process attached on the distal portion of the Weberian capsule (chast.46.1, Figure 4C; vs. interspace narrow, postero-medial process attaching on the proximal portion of the Weberian capsule, Figure 4A,B,D). Species of Cryptocambeva also differs from all other congeners by having a distinctively narrowed area adjacent to the base of the anterior processes of the pelvic bone (chast.50.1, Figure 6F), but this character state is not applicable to diagnose one species T. candidus (Miranda Ribeiro 1949), in which pelvic fin and girdle are absent. Cryptocambeva is also distinguished from other subgenera, except Humboldtglanis, by the following apomorphic features: presence of a long and narrow ridge on the dorsal surface of the autopalatine, close to its lateral margin (chast.1.1, Figure 2E,H; vs. absence, Figure 2A–D,F,G); an oblique arrangement of odontodes of the basal portion of the opercular odontode patch (chast.14.1, Figure 3E; vs. vertical, Figure 3A–D,F); ventral surface of the lateral margin of the mesethmoid ventrally folded around the vomer margin (chast.25.1, Figure 2J; vs. not folded, Figure 2I); lateral ethmoid medially separated from its symmetrical homologous (chast.28.1, Figure 2E; vs. in contact, Figure 2A–D,F); presence of a lateral expansion of the lateral ethmoid margin close to the sesamoid supraorbital (chast.29.1, Figure 2E; vs. absence, Figure 2A–D,F); the presence of a small notch in the posterior margin of the parapophysis of the second free vertebra, in an area close to the proximal extremity of the adjacent rib (chast.40.1, Figure 7E; vs. absence, Figure 7A–D,F); and a rudimentary posterior process of the pelvic bone (chast.49.1, Figure 6E,F; vs. well-developed, Figure 6A,C,D,G). Species of Cryptocambeva are distinguished from all other congeners by a color pattern consisting of minute dark brown dots scattered over all the body (Figure 1; vs. never a similar color pattern). Species of Cryptocambeva are also distinguishable from species of all other subgenera by the following combination of character states: six or seven pectoral-fin rays (vs. nine or ten in Megacambeva, eight in Humboldtglanis, eight or nine in Psammocambeva and Trichomycterus), the anal-fin origin in a vertical through the posterior portion of the dorsal-fin base (vs. anal-fin origin posterior to the dorsal-fin base in Megacambeva), dorsal fin longer than deep (vs. deeper than long in Megacambeva); never having a continuous black stripe between the snout and the caudal-fin base, becoming disperse and spotted in larger specimens (vs. always present in Paracambeva); jaw teeth always sharply pointed (vs. at least larger adults with incisiform teeth in Trichomycterus); the basal portion of maxillary barbel not distinctively widened (vs. widened in Psammocambeva); and an inconspicuous light portion of the iris (vs. broad in Psammocambeva).

Included taxa. Cryptocambeva comprises fifteen valid species: T. brasiliensis Lütken 1874, T. brunoi Barbosa and Costa 2010, T. candidus, T. claudiae Barbosa and Costa 2010, T. fuliginosus Barbosa and Costa 2010, T. maracaya Bockmann and Sazima 2004, T. mariamole Barbosa and Costa 2010, T. mimonha Costa 1992, T. mirissumba, T. macrotrichopterus Barbosa and Costa 2010, T. novalimensis Barbosa and Costa 2010, T. pirabitira Barbosa and Azevedo-Santos 2012, T. potschi Barbosa and Costa 2003, T. rubiginosus Barbosa and Costa 2010, T. vermiculatus (Eigenmann 1917), and at least three undescribed species. Trichomycterus argos Lezama and Triques e Santos 2012, not available for the present study, was considered to belong to the T. brasiliensis group by Lezama et al. [71], but the morphology of the neurocranium and adjacent bones schematically illustrated in its original description [71] (Figure 3) suggests that is not a member of this group. In this illustration, the two derived conditions relative to the pterotic and posttemporo-supracleithrum seem to be not present.

Etymology. From the Greek cryptos (hidden) and cambeva, a popular name for trichomycterids in south-eastern and southern Brazil and a nominal trichomycterine genus, referring to the cryptic habits of species of this group during daylight collections (see below). Gender feminine.

Distribution and ecological notes. Species of Cryptocambeva occur in a broad area of south-eastern Brazil, encompassing the upper courses of the Rio São Francisco, Rio Itabapoana, Rio Paraíba do Sul, and Rio Paraná basins, besides small isolated coastal basins emptying in the Baía de Sepetiba. Field studies indicate that species of the T. brasiliensis group are nocturnal, with adults being found deeply buried in river banks and juveniles in leaf litter deposits during the day [13,28].

This section may be divided into subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.

4. Discussion

4.1. Phylogeny

The phylogenetic tree obtained in the present analysis was similar to those generated when using only molecular data [32,33,34], supporting the same intrageneric clades here recognized as subgenera and species groups. The only exception was the T. pradensis group, here supported as monophyletic, but with relatively low support values, whereas two lineages formed a paraphyletic lineage in previous studies, but also with weakly supported nodes. These discrepant results are due to the inclusion of unique osteological character states herein assigned to the T. pradensis group (see taxonomical accounts above).

4.2. Morphological Diversity of Osteological Structures

A high morphological diversity of osteological structures in different lineages of Trichomycterus s.s. was herein first recorded and described. This diversity, mainly concentrated in bones of the mesethmoidal region and jaw suspensorium, is comparable or superior to that found among closely related genera of other trichomycterid groups. For example, the morphological divergence among the subgenera of Trichomycterus s.s. and included groups is conspicuously greater than that reported to distinguish the trichomycterine genera Bullockia and Hatcheria [12], and the copionodontine genera Copionodon de Pinna 1992 from Glaphyropoma de Pinna 1992 [72].

Although field reports on trichomycterines from eastern Brazil are sparse and limited to a few notes in taxonomical papers, some data suggest that the high morphological diversity here reported for Trichomycterus s.s. is possibly related to some ecological specializations found in different lineages. Trichomycterus giganteus, the only species of Megacambeva and supported as sister to the clade comprising all other congeners (Figure 1), highly differs from other taxa of the CST-clade by unique osteological character states (see results above). However, T. giganteus also differs from other congeners by its larger size and habitat. This species reaches about 200 mm of standard length (SL) [73], a size never recorded for any member of the CST-clade, which rarely reach about 120 mm SL. Trichomycterus giganteus is found in small lake-like areas formed between river waterfalls, where larger specimens may be seen swimming about 200 cm below the water surface (person. observ.). Other congeners are typically found in fast-flowing, shallow streams, in depths about 100 cm or less. Interestingly, a few other trichomycterine taxa reaching large size are found in Andean lakes, such as Eremophilus mutisii Humboldt 1805 reaching about 300 mm SL and Trichomycterus rivulatus Valenciennes 1846 reaching about 350 mm SL [11], suggesting that life in lacustrine habitats may favor the development of larger sizes.

Some sister lineages seem to exhibit divergent ecological specializations. Within Paracambeva, T. itatiayae has diurnal habits and is occasionally observed swimming on the river bottom during daylight, close to leaf litter [69], thus differing from species of the sister group, the T. reinhardti species group, that are only found buried in the marginal vegetation of small shallow streams during daylight collections [25]. Similarly, species of Cryptocambeva are typical nocturnal catfishes, with adult specimens found deeply buried in riverbanks during daylight field studies, whereas juveniles are often found buried in leaf litter [13,28,30]. This behavior highly contrasts with the diurnal activity recorded for juveniles and smaller adults of species of Humboldtglanis [13,33], the sister group of Cryptocambeva. In the upper Rio Preto drainage, where populations of five species of Trichomycterus have been observed in the field by the author since 1979, including monthly field observations in the period between 2017 and 2020, T. albinotatus (subgenus Humboldtglanis) was easily seen actively swimming just above the gravel substrate during daylight. On the other hand, the sympatric species T. mirissumba (subgenus Cryptocambeva) was found hidden in holes below the marginal vegetation during daylight but commonly fished at night by the local people in any part of the river bottom, making obvious its nocturnal habits. These observations are consistent with sporadic field observations involving other species of both subgenera [28,30,33]. Interestingly, in species of Cryptocambeva, the eye is smaller and darker, without a conspicuous light iris (Figure 9D), contrasting with the larger eye with light iris area around pupil in Humboldtglanis (Figure 9C), conditions probably related to their nocturnal and diurnal habits, respectively. The other sympatric congeners are also equally specialized, with T. auroguttatus, a psammophilic species of Psammocambeva showing high diurnal activity (Figure 9B), and T. quintus, a typically nocturnal species of the subgenus Trichomycterus, only found buried into the bottom leaf litter among rocks during the daytime, in places with strong water flow (Figure 9A) [32].

Species of the subgenera Psammocambeva and Trichomycterus apparently have more variability regarding habitat preferences. Species of the latter subgenus typically have predominant overall dark brown to black coloration, which is probably related to their cryptic habits during daylight [32]. Species of the T. nigroauratus group have been reported to be associated with the bottom leaf-litter [32,69], and most species of the T. nigricans group are commonly found below large rocks during day light [74], but T. santaeritae, a species of the T. nigricans group, highly differs by having lighter coloration (i.e., light yellow with dark brown spots), is diurnal and psammophilic [36]. Similarly, field studies have indicated different habitats preferences for species of Psammocambeva, with many having diurnal habits [13,35,75] and some others nocturnal [76]. Most species of this clade have been found associated with gravel substrate [35,75], but some species such as T. auroguttatus are highly associated to sandy bottom [13], exhibiting a relatively large eye that is slightly dorsally protruded (Figure 6B). Field studies have shown that T. auroguttatus has an intense diurnal activity, being usually found swimming close to sandy stretches on the river bottom, in parts with intense waterflow; when disturbed, specimens quickly bury into the sand, about 3–5 cm below sand surface (person. observ. 2017–2020). However, detailed field studies on other species of Psammocambeva are necessary to make some inferences about possible ecological specializations.

4.3. CST-Clade and Trichomycterus s.s. Diagnosis

Using criteria traditionally accepted for genus delimitation, such as monophyly, morphological gaps, and ability to discriminate, it would be justifiable to divide Trichomycterus s.s. in different genera. However, in order to preserve the nomenclatural stability of included species that are consistently placed in this genus for a long time, the option here adopted was to divide Trichomycterus s.s. in subgenera. The morphological gaps found between Trichomycterus s.s. subclades was compatible with the relatively long genetic distances found among these subclades [32] (Figure 1). However, no unique morphological character state was found to be synapomorphic for Trichomycterus s.s. On the other hand, the comparative osteological analysis provided some evidence relative to vertebra morphology supporting both the CST-clade and the clade comprising Cambeva and Scleronema.

All taxa belonging to the CST-clade have the parapophysis main axis of the second free vertebra postero-laterally directed (chast.37.1; Figure 7B–F), instead of being more laterally directed as in other trichomycterines here examined (Figure 7A). However, a supposedly similar postero-laterally directed parapophysis may be convergently present in other trichomycterines not examined in this study since reconstruction of the Colombian trichomycterine Rhizosomichthys totae (Miles 1942) [42] (Figure 2C) showed a similar morphology.

Species of Cambeva and Scleronema differ from species of Trichomycterus s.s. by having a branched parapophysis of the first free vertebra, with a dorsal branch in contact with the dorsal surface of the articular zone of the first rib, and a ventral branch ventrally supporting the proximal extremity of the rib (chast.36.1; Figure 7B), instead of being unbranched as in Trichomycterus s.s. (Figure 7C–F) and other trichomycterids (Figure 7A), and by the morphology of the parapophysis of the third free vertebra that is posteriorly directed (chast.42.1; Figure 7B), instead of being postero-laterally directed as in other trichomycterines (Figure 7A,C–F). It is important to note that the latter condition, in which the whole parapophysis is posteriorly directed with its main axis reaching a position nearly parallel to the vertebral column axis, differs from that occurring in some species of Ituglanis, in which the parapophysis is curved, and only its extremity is posteriorly directed [77] (Figure 2B). Therefore, the combination of an apomorphic postero-laterally directed parapophysis main axis of the second free vertebra and the plesiomorphic unbranched parapophysis of the first free vertebra and a postero-laterally directed parapophysis of the third free vertebra would tentatively characterize Trichomycterus s.s.

5. Conclusions

The Trichomycterinae has been considered a problematic taxon (see introduction above), but recent studies are quickly changing this scenario. Major advances have resulted from studies on phylogenetic relationships [9,22,31,32], but some problems were not still addressed. The present study provides morphological evidence supporting and making it possible to distinguish between Trichomycterus lineages previously supported only by molecular data, thus allowing future unequivocal placement of new species without molecular data. Furthermore, this study has shown that osteological characters combined with molecular data may be useful to consistently delimit and distinguish between trichomycterine groups that cannot be properly distinguished using only external morphological features. More importantly, this integrative approach sheds light upon the way of solving still persistent problems in trichomycterine systematics, including a formal generic placement for the numerous Andean and Guianan incertae sedis taxa [9,31,78].