Multigene analysis of the catfish genus Trichomycterus and description of a new South American trichomycterine genus (Siluriformes, Trichomycteridae)

Trichomycterus comprises about 170 valid species, but its monophyly has been challenged in the last decades. Bayesian Inference and Maximum Likelihood analyses comprehending mitochondrial genes COI and CYTB and nuclear genes GLYT, MYH6 and RAG2 from 71 Trichomycterinae terminal taxa and eight outgroups were performed. The analyses highly supports a clade containing Trichomycterus nigricans, the type species of the genus, and several other congeners endemic to eastern and northeastern Brazil, herein considered as the genus Trichomycterus, the sister clade the southern Brazil and adjacent areas clade; the latter clade comprises two subclades, one comprising species of the genus Scleronema and another comprising species previously placed in Trichomycterus, herein described as a new genus. Cambeva gen. n. is distinguished from all other trichomycterines by the presence of a bony flap on the channel of the maxillo-dentary ligament, the interopercle shorter than the opercle, a deep constriction on the basal portion of the antero-dorsal arm of the quadrate, absence of teeth in the coronoid process of the dentary, the maxilla shorter than the premaxilla, the cranial fontanel extending from the the medial posterior of frontal to the medial region of supraoccipital, and absence of the postorbital process of the sphenotic-prootic-pterosphenoid.


Introduction
Siluriformes (catfishes) is among of the most diverse vertebrate orders, with about 3700 species in 39 families (Nelson et al. 2016, Frick et al. 2018. Trichomycteridae, the second most diverse catfish family, with about 300 species (Frick et al. 2018 ), is characterized by having a modified opercular system, involving the presence of odontodes in the interopercular and opercular bones (Baskin 1973, de Pinna 1992. It occurs between southern Central America, in Costa Rica, and southern South America, in Patagonia (Baskin 1973, de Pinna 1998, exhibiting an impressive species diversity and endemism around higher elevations (de Pinna 1992). Presently, Trichomycteridae comprises eight subfamilies: Trichomycterinae Bleeker, 1858, Vandelliinae Bleeker, titude rapid stream rivers, a remarkable feature occurring among species of Trichomycterus is the ability to climb waterfalls (Eigenmann 1918, Arratia 1983 with the support of the interopercle and opercle odontodes, in an 'elbowing' behaviour (de Pinna 1992, Adriens et al. 2010. Since Baskin (1973), monophyly of Trichomycterus has been challenged by several authors (Arratia et al. 1978, de Pinna 1989, Arratia 1990a, Costa and Bockmann 1993, Datovo and Bockmann 2010, Ochoa et al. 2017), but its phylogenetic status still remains unclear. Morphological taxonomic studies could not find unique diagnostic characters for Trichomycterus (de Pinna 1989), whereas some species traditionally placed in Trichomycterus have been reallocated in other trichomycterine genera (e.g. Costa and Bockmann 1993, Arratia 1998, Henschel et al. 2017. Furthermore, recent molecular analyses have suggested that species still today placed in Trichomycterus do not form a monophyletic group (Ochoa et al. 2017).
In addition to the uncertain status of Trichomycterus, two other trichomycterine genera, Scleronema and Ituglanis, have unclear relationships. They are often considered more related to non-trichomycterine trichomycterids (Myers and Weitzman 1966, de Pinna 1989, Costa and Bockamnn 1994, DoNascimiento 2015, but more recent studies indicated that they are members of the Trichomycterinae (Datovo and Bockmann 2010, Henschel et al. 2017, Ochoa et al. 2017.
The objective of this paper is to provide the more inclusive molecular based phylogenetic analysis of Trichomycterus, assigning the phylogenetic position of its type species, Trichomycterus nigricans Valenciennes, 1832 and describing a new genus, sister to Scleronema, that was hidden under the lack of consistent information about Trichomycterus relationships.

Taxon sampling
Specimens were euthanized by sub-merging them in a buffered solution of tricaine methanesulphonate (MS-222) at a concentration of 250 mg/L, for a period of 10 min, following the guidelines of the Journal of the American Veterinary Medical Association (AVMA Guidelines) (Leary et al. 2013) and European Commission DGXI consensus for fish euthanasia (Close et al. 1996(Close et al. , 1997. Molecular data were obtained from specimens fixed and preserved in absolute ethanol. Specimens used for morphological comparisons were fixed in formalin for a period of 14 days and then transferred to 70% ethanol. All the collected material was deposited in the fish collection of Institute of Biology, Federal University of Rio de Janeiro (UFRJ).

Phylogenetic analyses
In-group included seven subfamilies of Trichomycteridae lineages. Since this study was directed to searching for relationships and monophyly of Trichomycterus, the analysis included a total of 50 terminal taxa presently placed in this genus, as well as 21 terminal taxa belonging to the trichomycterine genera Bullockia, Eremophilus, Ituglanis and Scleronema. Out-group selection was directed to sample representatives of other lineages of the Trichomycteridae, comprising six terminal taxa representing each of the remaining subfamilies, as well as representatives of other families of the Loricarioidea, including the nematogenyid Nematogenys inermis Guichenot, 1848 and the loricariid Pareiorhina rudolphi (Miranda-Ribeiro, 1911), in which the analyses were rooted. A list of specimens and its respective GenBank accession numbers is provided in Suppl. material 1. Sequences were aligned and edited in Mega 7 software (Kumar et al. 2016) using the ClustalW algorithm (Chenna et al. 2003). Gaps were considered as informative characters. The data set was partitioned by gene, and the best-fit evolutionary model selection was performed under the Akaike information criteria in the software jmodeltest 2 (Darriba et al. 2012) for each partition. Bayesian Inference (BI) and Maximum Likelihood (ML) analyses were conducted using the softwares Mrbayes 3.2 (Ronquist et al. 2012) and Garli 2.0 (Zwickl 2006), re-spectively. The BI analysis was conducted with the following parameters: two independent Markov chain Monte Carlo (MCMC) runs of two chains each for 20 million generations, with a tree sampling frequency of every 100 generations. The convergence of the MCMC chains and the proper burn-in value were assessed by evaluating the stationary phase of the chains using tracer v. 1.5 (Rambaut et al. 2013). The BI final consensus tree and its Bayesian posterior probabilities were generated with the remaining tree samples after removing the first 25% samples as burnin. To test the support of the nodes in the ML analysis, 1,000 bootstrap (Felsenstein 1985) replicates were made.

Morphological comparisons
Morphological comparisons were focused on the external morphology and osteological features of cleared and stained specimens prepared according to Taylor and Van Dyke (1985). Terminology for osteological nomenclature follows Arratia (1998). Measurements and counts follow Barbosa and Costa (2003). A list of material analysed appears in Suppl. material 2.

Phylogenetic analyses
The concatenated matrix comprised 4380 bp after alignment (522 bp for COI, 857 for CYTB, 890 for GLYT, 542 for MYH6, 884 for RAG2 and 680 for SH3PX3). The bestfit evolutionary models found are shown in Suppl. material 3. The best log-likelihood score for the ML analysis was -31473.209522 and the mean for BI was -lnl -32013.626.
Both analyses exhibit similar topologies (Figs 1, 2). In all analyses, monophyly of the Trichomycterinae was high- ported in the ML analysis (Fig. 2). It includes taxa endemic to a broad geographical area, including the Andean region, Amazon, and Patagonia, formally placed in the genera Bullockia, Eremophilus, Ituglanis and Trichomycterus, which is herein graphed "Trichomycterus" by being distantly related to that clade including the type species of the genus. Relationships among basal lineages of the clade b were weakly supported (BS < 50; BI < 95).
http://zoobank.org/C26BEA49-7714-44FB-957B-678D8C6C9DCC Type species. Pygidium davisi Haseman, 1911 (Fig. 3) Diagnosis. Cambeva is similar to Scleronema and distinguished from all other genera of the Trichomycteri-ly supported, but nominal species of Trichomycterus did not form a single monophyletic group. Two major clades were found in Trichomycterinae. The first one, the trichomycterine clade a, is highly supported in both analyses (Figs 1, 2) and comprises species geographically restricted to an area encompassing eastern, south-eastern and southern Brazil, which have been traditionally placed in two genera, Scleronema and Trichomycterus. Both analyses highly corroborate three major subclades (Figs 1, 2), one comprising T. nigricans, the type species of Trichomycterus, and other congeners, therefore herein recognised as the true Trichomycterus; another corresponding to the genus Scleronema; and another clade, sister to Scleronema and comprising several nominal species of Trichomycterus, herein recognised as a new genus (see taxonomic accounts below).

Included species. Cambeva davisi
Distribution. Species of Cambeva gen. n. occur in the Paraná, São Francisco, Ribeira de Iguape, and Uruguay river basins, as well as in smaller isolated coastal river basins of south-eastern and southern Brazil.
Etymology. Cambeva, probably derived from the Tupi-Guarani, is a popular name for trichomycterid fishes in southern and south-eastern Brazil. Gender: feminine.

Monophyly of Trichomycterinae
Since the first objective phylogenetic analysis of the Trichomycteridae by Baskin (1973), monophyly of the Trichomycterinae has been challenged by several authors. Besides not finding unique diagnostic features for the subfamily, supposed derived traits shared by some trichomycterines and species of other trichomycterid subfamilies made uncertain the position of some trichomycterine members. de Pinna (1989) suggested that the trichomycterine genus Scleronema is more closely related to the subfamily Sarcoglanidinae than to other trichomycterines, as already discussed by Myers & Weitzman (1966), due to the common possession of an enlarged maxilla; Arratia (1990) also reported a maxilla enlargement for the trichomycterine genera Bullockia and Hatcheria, besides species of 'Trichomycterus'. Our results clearly indicate that taxa having long maxilla (e.g., sarcoglanidines, Bullockia, Scleronema) do not form a monophyletic group. Considering the psammophilic habits exhibited by species of Sarcoglaninidae and Scleronema, with specimens digging into sand or gravel (Zuanon andSazima 2004, Schaefer et al. 2005, AM Katz and WJEM Costa pers. obs.), the long maxilla exhibited by these taxa may be interpreted as an adaptive convergence generating homoplasies in both groups. Costa and Bockmann (1993) considered Ituglanis and Scleronema more closely related to the TSVSG clade, comprising the Tridentinae, Sarcoglanidinae, Vandelliinae, Stegophilinae and Glanapteryginae (Costa and Bockmann 1994), than to other trichomycterids based on a thin tip of the lateral process of the urohyal and a slightly reduced interopercular patch of odontodes. However, as noted by subsequent authors (e.g. Fernández and de Pinna 2005; Datovo and Bockmann 2010; the present study) other trichomycterines also exhibit in some extent those character states. More recently, Datovo and Bockmann (2010), in their myological analysis of trichomycterids, found evidence indicating that Ituglanis and Scleronema are more closely related to other Trichomycterinae than to the TSVSG clade. A similar result has been proposed in molecular phylogenies (e.g. Henschel et al. 2017, Ochoa et al. 2017. In our multigene analysis, using a broader genetic sample, the phylogenetic position of Ituglanis and Scleronema within a monophyletic Trichomycterinae is highly corroborated (Figs 1, 2). Ituglanis, with species distributed in the main tropical and subtropical Cis-Andean river basins of South America, did not appear as closely related to Scleronema, but sister to a clade comprising Andean, Patagonian and Amazon species of 'Trichomycterus' (Figs 1, 2).

Monophyly of Cambeva
All species here included in Cambeva were previously placed in Trichomycterus, instead of being considered as more closely related to Scleronema (Eigenmann 1918, Bizerril 1994, Wosiacki and Garavello 2004. Datovo and Bockmann (2010) examined myological characters of several trichomycterids, among which were two species of Cambeva, C. davisi and , which according those authors share a unique apomorphic condition consisting of the extensor tentaculi originating exclusively from the ethmoidal region of the neurocranium. We have searched this derived condition in several species of Trichomycterus and Cambeva herein examined (see Suppl. material 2), but it was only found in species endemic to some coastal rivers of southern Brazil, and to the Iguaçu, Ribeira do Iguape and Parapanema river basins (C. castroi, C. cubataonis, C. davisi, C. naipi and C. zonata), suggesting that this derived condition is synapomorphic for a subclade of Cambeva.
Cambeva is here supported by high values of bootstrap and posterior probability as monophyletic and sister to Scleronema (Figs 1, 2). However, in spite of the clade comprising Cambeva and Scleronema being diagnose by morphological synamoporphies, we did not find unique character states for Cambeva, which is only distinguishable from other trichomycterine genera by a combination of derived and primitive morphological character states (see Diagnosis above).