A new species of Knodus (Characiformes: Characidae), with deep genetic divergence, from the Mearim and Munim river basins, Northeastern Brazil, and evidence for hidden diversity in adjacent river basins

Abstract

A new species of Knodus from the Mearim and Munim River basins, Northeastern Brazil, is herein described based on integrative taxonomy, by using different molecular based species delimitation methods and independent approaches. The new species possesses the combination of character states that usually diagnoses the genus. The new species possesses a similar colour pattern to K. victoriae, which is also morphologically similar to it. The species described herein differs from K. victoriae by possessing more total vertebrae, more branched anal-fin rays, and fewer circumpeduncular scales. We also provide a detailed discussion of the morphological diagnostic features exhibited by Knodus species from adjacent river basins.

Keywords: Cryptic species; Integrative Taxonomy; Stevardiinae

Resumo

Uma nova espécie de Knodus das bacias dos rios Mearim e Munim, Nordeste do Brasil, é descrita com base em taxonomia integrativa, utilizando diferentes métodos moleculares de delimitação de espécies e abordagens independentes. A nova espécie possui a combinação de estados de caráter que geralmente é utilizada para diagnosticar o gênero. A nova espécie possui um padrão de coloração semelhante a K. victoriae, que também é morfologicamente semelhante a ela. A espécie aqui descrita difere de K. victoriae por possuir mais vértebras totais, mais raios ramificados na nadadeira anal e menos escamas circumpedunculares. Nós também fornecemos uma discussão detalhada das características morfológicas diagnósticas exibidas por espécies de Knodus de bacias hidrográficas adjacentes.

Palavras-chave: Espécie críptica; Stevardiinae; Taxonomia Integrativa

INTRODUCTION

Knodus Eigenmann, 1911 is one of the most species-rich characid genera within the subfamily Stevardiinae (Thomaz et al., 2015; Mirande, 2019; Ferreira et al., 2021), including 35 valid species (Menezes, Marinho, 2019; Sousa et al., 2020; Fricke et al., 2022). It is distributed among the Amazon, Tocantins-Araguaia, Orinoco, Paraná-Paraguay, Parnaíba, São Francisco, and Jequitinhonha river basins (Fricke et al., 2022), with its diversity peaking in the Amazon River basin (García-Melo et al., 2019; Fricke et al., 2022).

The genus Knodus is diagnosed only by the possession of two rows of pre-maxillary teeth – the inner row with four teeth – and a scaled caudal-fin (Eigenmann, 1918; Géry, 1972, 1977). This combination traditionally used to diagnose the genus has been questioned by some authors (e.g., Schultz, 1944; Taphorn, 1992; Román-Valencia, 2000; Román-Valencia et al., 2008) who consider Knodus a synonym of the closely related genus Bryconamericus Eigenmann, 1907.

Recent phylogenetic hypotheses based on molecular and morphological data have improved our knowledge on Knodus diversity and its intrageneric relationships (e.g., Thomaz et al., 2015; Mirande, 2019; García-Melo et al., 2019). However, although some species have been described within Knodus in recent years, the alpha taxonomy of most species and related genera is still somewhat confusing (García-Melo et al., 2019; Sousa et al., 2020).

Recent sampling efforts conducted in tributaries of the Mearim and Munin river basins (Northeast Brazil) revealed the existence of a new species morphologically similar to but still distinct from Knodus victoriae (Steindachner, 1907), whose type locality is in the upper Parnaiba River basin (Steindachner, 1907). Thus, we describe a new cryptic species of Knodus sensuBickford et al., (2007), from the Mearim and Munim river basins, based on an integrative taxonomic approach.

MATERIAL AND METHODS

Taxon sampling, specimen collection, and preservation. Specimens were captured with a manual trail-net (2 m long × 1.8 m high; mesh size, 2 mm) and euthanized in a buffered solution of ethyl-3-amino-benzoat-methanesulfonate (MS–222) at a concentration of 250 mg/L until complete cessation of opercular movements, according to animal welfare laws and guidelines (Close et al., 1996; Leary et al., 2013). Specimens selected for morphological analysis were fixed in formalin for 10 days, after which they were preserved in 70% ethanol. Molecular data were obtained from specimens fixed and preserved in absolute ethanol. Specimens for morphological analysis are included in the lists of type and comparative material. Specimens for molecular approaches are listed in Tab. 1. Type material are deposited in the following ichthyological collections: Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu (LBP); Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro (UFRJ); Coleção Ictiologica do Centro de Ciências Agrárias e Ambientais da Universidade Federal do Maranhão, Chapadinha (CICCAA); and Coleção Ictiológica da Universidade Estadual do Maranhão, São Luís (CIUEMA).

Morphological analysis. Measurements and counts were made according to Fink, Weitzman, (1974), with exception of the scale rows below the lateral line, which were counted to the insertion of the pelvic fin. Vertical scale rows between the dorsal-fin origin and lateral line do not include the scale of the median predorsal series situated just anterior to the first dorsal fin ray. Counts of supraneurals, vertebrae, procurrent caudal fin rays, unbranched dorsal and anal fin rays, branchiostegal rays, gill-rakers, premaxillary, maxillary, and dentary teeth were taken only from cleared and stained paratypes (C&S), prepared according to Taylor, Van Dyke, (1985). The four modified vertebrae which constitute the Weberian apparatus were not included in the vertebral counts and the fused PU1 + U1 was considered a single element. Osteological nomenclature follows Weitzman, (1962). Comparisons with other species of Knodus were based on examined material, as well as information from literature (Zarske, Géry, 2006; Ferreira, Lima, 2006; Zarske, 2007, 2008; Ferreira, Carvajal, 2007; Román-Valencia et al., 2008; Menezes et al., 2009, 2020; Ferreira, Netto-Ferreira, 2010; Esguícero, Castro, 2014; Menezes, Marinho, 2019; Sousa et al., 2020; Deprá et al., 2021).

DNA extraction, amplification, and sequencing. DNA was extracted from fin clips using a saline buffer extraction protocol (Aljanabi, Martinez, 1997). Fragments of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) were amplified with the universal primers designed by Ward et al., (2005) (FISHF1 5´-TCAACCAACCACAAAGACATTGGCAC-3´ and FISHR1 5´-TAGACTTCTGGGTGGCCAAAGAATCA-3´), primers from Melo et al., (2011) (COI L6252-Asn 5’-AAGGCG GGGAAAGCCCCGGCA G-3’ and H7271-COXI 5’-TCC TATGTAGCCGAATGGTTC TTT T-3’) and primers developed in the present study (KNODUS-TOF 5´-GGGCGATGACCAAATCTA-3´ and KNODUS-TOR 5´-AGGGTCGAAGAATGAGGTAT-3’). Polymerase chain reactions (PCR) for samples from Knodus sp. “Mearim”, Knodus cf. savannensis, Knodus sp. “Maracaçumé”, and Knodus cf. victoriae comprised a total volume of 15 µL containing 1x polymerase buffer, 1.5 mM MgCl₂, 200 µM dNTP, 0.2 µM of each primer, 1U Taq polymerase (Invitrogen), 100 ηg DNA template, and ultrapure water. The PCR cycles were as follows: 2 min at 94ºC, followed by 35 cycles of 94ºC for 30s, 54ºC for 30s and 72ºC for 1 min and 10 min at 72ºC. Polymerase chain reactions (PCR) for samples from the Knodus sp. “Munim”, and Knodus sp. “Itapecuru” comprised a total volume of 15 µl containing 1x Polymerase buffer, 400 µM dNTP, 0,4 uM of each primer, 1U Taq Polymerase (Invitrogen), 100 ηg DNA template, and ultrapure water. The PCR cycles were as follows: 5 min at 94ºC, followed by 35 cycles of 94ºC for 45s, 48°-52ºC for 45s, and 72ºC for 1 min, and 10 min at 72ºC. Amplicons were purified using ExoSAP-IT PCR Product Clean-up (Thermo Fisher Scientific) and Gel Purification Kit (GE Healthcare Systems), and sequenced using forward and reverse primers and the BigDye Terminator 3.1 Cycle Sequencing kit in a ABI 3730 DNA Analyzer (Thermo Fisher Scientific).

Data partitioning, evolutionary models, and alignment. The dataset included the COI sequences (401 base pairs, bp) of individuals from 10 Knodus species, including the species here described, specimens that we were not able to identify at the species level, as well as two Bryconamericus species. Many sequences were newly generated for this project, while others originated from the Barcode of Life Database (BOLD) and the National Center for Biotechnology Information (NCBI) (Tab. 1). Sequences were aligned using ClustalW (Chenna et al., 2003) and translated into amino acid residues using the program MEGA 7 (Kumar et al., 2016) to test if the sequences came from NUMTs (nuclear mitochondrial DNA sequences), in which case premature stop codons or indels would be expected. The best-fit evolutionary model (GTR+I+G) was selected using the Akaike Information Criterion (AIC) and the Corrected Akaike Information Criterion (AICc) by jModelTest 2.1.7 (Darriba et al., 2012), and used in all analyses, except for ABGD (Automatic Barcode Gap Discovery) which is a model-free approach based only on genetic distances.

Phylogenetic analysis. A Bayesian inference phylogenetic (BI) tree was estimated in MrBayes 3.2 (Ronquist et al., 2012) to reconstruct the evolutionary relationships among terminals using the General Time Reversible (GTR+I+G) evolutionary model. The BI analysis was conducted with the following parameters: two independent Markov chain Monte Carlo (MCMC) runs of two chains each for 10 million generations, with a tree sampling frequency at every 1,000 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. 1.6 (Rambaut et al., 2014). The final consensus tree and its posterior probabilities were generated with the remaining tree samples after removing the first 25% samples (burn-in). We used as outgroups sequences of Bryconamericus exodon Eigenmann, 1907 and B. iheringii (Boulenger, 1887). The remaining haplotypes were used as ingroups.

Species delimitation and molecular diagnoses. We implemented five distinct and independent single locus species delimitation methods based on molecular data, each of which rely on different operational criteria for species delimitation. DBC, DNA barcoding (hereafter Traditional DNA barcoding) was initially proposed by Hebert et al., (2003a,b). Since then, it has improved and gained supporters due to its practicality and efficiency (e.g., Hajibabaei et al., 2007; Coissac et al., 2016; DeSalle, Goldstein, 2019). The premise of the method consists of standardized sequencing of a specific gene for each species, followed by the organization of the sequences in virtual reference libraries. Once a species is added to the library, any individual of that species, at any ontogenetic stage, or even fragments, can be identified by simple comparison, simply by sequencing the standard gene (Hebert et al., 2003a,b). For fish and other animals, the gene used is the mitochondrial protein cytochrome c oxidase I (COI) and its effectiveness has been frequently demonstrated (e.g., Costa-Silva et al., 2018; García-Melo et al., 2019). The methodology suggests that the maximum genetic distance between individuals of the same species, based on the COI sequences, can be defined for each taxonomic group. Any difference higher than this cut-off value would represent discontinuity between species (Hebert et al., 2003a,b). The other approaches were CBB, a character-based DNA barcoding method (DeSalle et al., 2005) adapted by Ottoni et al., (2019) and Guimarães et al., (2020b); GMYC, the General Mixed Yule Coalescent method, single-threshold version (Fujisawa, Barraclough, 2013); bPTP, the Bayesian implementation of the Poisson tree processes (Zhang et al., 2013); and ABGD, Automatic Barcode Gap Discovery (Puillandre et al., 2012).

Traditional DNA barcoding (DBC). We used the Kimura-2-parameter model (K2P) (Kimura, 1980) to estimate the pairwise genetic distances between species in MEGA 7 software (Kumar et al., 2016). We considered a cutoff of 2% as sufficient to discriminate species, since this threshold is commonly inferred by species delimitations among freshwater Neotropical fish species based on COI (Jacobina et al., 2018).

Molecular diagnosis (CBB). The molecular diagnosis approach delimited the new species by the presence of a unique combination of nucleotides at particular sites. In addition, the new species was diagnosed by nucleotide substitutions following Costa et al., (2014), Ottoni et al., (2019), and Guimarães et al., (2020b). Nucleotide substitutions among lineages were optimized on the Bayesian inference topology using PAUP version 4 (Swofford, 2002). Each nucleotide substitution is represented by its relative numeric position determined through sequence alignment with the complete mitochondrial COI gene of Psalidodon paranae (Eigenmann, 1914) (KX609386.1:5503-7062), followed by the specific nucleotide substitution in parentheses. Unique nucleotide substitutions in our analysis are marked with asterisk.

General Mixed Yule Coalescent (GMYC). The GMYC is a single locus coalescent-based species delimitation approach that relies on branch lengths to establish a threshold between speciation and coalescent processes (Fujisawa, Barraclough, 2013). Here we applied the single-threshold version of the method, which usually outperforms the multiple-threshold version (Fujisawa, Barraclough, 2013). A new reduced dataset was created for this analysis using DAMBE5 (Xia, 2013), including only unique haplotypes following the requirements of this method.

The ultrametric phylogenetic tree needed for input was inferred in BEAST version 1.8.4 (Drummond et al., 2012), with the following parameters: an uncorrelated relaxed clock with lognormal distribution, a Yule Process as tree prior with 10 million generations and sampling frequency of 1,000. We used as outgroups sequences of Bryconamericus exodon and B. iheringii. The remaining haplotypes were used as ingroups. The GMYC analysis was performed on the Exelixis Lab’s server https://species.h-its.org/gmyc.

Bayesian implementation of the Poisson tree processes (bPTP). The bPTP is another single locus coalescent-based species delimitation method that differs from other similar approaches, such as GMYC, by not requiring an ultrametric tree and thus not relying on branch lengths to delimit species (Zhang et al., 2013). The method assumes that more molecular variability (number of nucleotide substitutions) is expected between haplotypes from different species than within a species (Zhang et al., 2013), establishing a threshold between speciation and coalescent processes. The reduced dataset for performing the bPTP was the same at that used in GMYC, following the requirements of this method. The input phylogenetic tree was estimated in software Mrbayes 3.2 (Ronquist et al., 2012) to reconstruct the evolutionary relationships among terminals using the General Time Reversible (GTR+I+G) evolutionary model. The BI analysis was conducted with the following parameters: two independent Markov chain Monte Carlo (MCMC) runs of two chains each for 10 million generations, with a tree sampling frequency at every 1,000 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 version 1.6 (Rambaut et al., 2014). The final consensus tree and its posterior probabilities were generated with the remaining tree samples after removing the first 25% samples (burn-in). The remaining haplotypes were used as ingroups. The bPTP analysis was performed on the Exelixis Lab’s web server http://species.h-its.org/ptp/, following the default parameters except for designation of a 20% burn-in. Bryconamericus exodon was chosen as the outgroup.

Automatic Barcode Gap Discovery (ABGD). The ABGD is a barcode species delimitation method that aims to establish a minimum gap that probably corresponds to the threshold between interspecific and intraspecific processes (Puillandre et al., 2012). The major advantage of ABGD when compared to the other barcode species delimitation methods is that the inference of the limit between interspecific and intraspecific processes (gap detection) is recursively applied to previously obtained groups to get finer partitions until there is no further partitioning, allowing a more refined search. Basically, the ABGD analysis indicates the number of groups (species) estimated relative to a large spectrum of p values (prior intraspecific values). A value of 0.1 indicates maximum intraspecific variability with all sequences belonging to a single species, whereas a 0.001 value indicates a small intraspecific variability with each distinct haplotype representing a different species. The reduced dataset for performing the ABGD was the same as that used in GMYC, following the requirements of this method. We ran ABGD on the ABGD server website https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html with default parameters, except for the X value (we used an X value of 1.0 as recommended by the server for our analysis). After running the ABGD, additional molecular, morphological, or ecological characters are needed to infer the correct number of species if the analysis will follow an integrative taxonomy paradigm.

RESULTS

Knodus guajajara Aguiar, Brito, Ottoni & Guimarães, new species

urn:lsid:zoobank.org:act:4EEDDC6E-4088-4B60-828D-AF79EF84DC26

(Figs. 1–3; Tabs. 2–3)

Knodus victoriae [non Knodus victoriae (Steindachner, 1907)]. ―Guimarães et al., (2020a:90). ―Oliveira et al., (2020:6–7).

FIGURE 1 |
Knodus guajajara, holotype, CICCAA 4883, 31.4 mm SL, Alto Alegre do Pindaré municipality, Igarapé Arapapá, Pindaré River drainage, Mearim River basin.

TABLE 1 |
Specimens and DNA sequence information included in the study. Sequences made available by this study follow with asteristic.

Nº	Species	Depositories of vouchers			Genbank ID
		Collection	Tissue code	Voucher
1	Bryconamericus exodon	LBP	26191	5118	MH002960
2	Bryconamericus iheringii	LBP	60645	14473	MH002991
3	Knodus alpha	STRI	9564	531	MH003217
4	Knodus alpha	LBP	9561	663	MH003216
5	Knodus borki	LBP	53820	12472	MH003218
6	Knodus borki	LBP	–	53821	KT248111
7	Knodus caquetae	MPUJ	637	11073A	MH003221
8	Knodus caquetae	MPUJ	639	11073B	MH003222
9	Knodus heteresthes	LBP	57371	13870	MH003232
10	Knodus heteresthes	LBP	37317	7959	MH003233
11	Knodus megalops	LBP	54223	12566B	MH003234
12	Knodus meridae	LBP	15818	7569	MH003235
13	Knodus tiquiensis	LBP	–	33217	KT248096
14	Knodus tiquiensis	LBP	–	33218	KT248097
15	Knodus tiquiensis	LBP	–	33216	KT248098
16	Knodus victoriae	LBP	–	27366	KT248128
17	Knodus victoriae	LBP		27370	KT248129
18	Knodus victoriae	LBP	27342	5607	KT248130
19	Knodus victoriae	LBP	–	27254	KT248131
20	Knodus victoriae	LBP	–	27368	KT248132
21	Knodus victoriae	LBP	–	27295	KT248133
22	Knodus victoriae	LBP		27369	KT248134
23	Knodus victoriae	LBP	–	27253	KT248135
24	Knodus victoriae	LBP	–	27255	KT248136
25	Knodus victoriae	LBP	–	27367	KT248137
26	Knodus aff. victoriae (Balsas)*	CICCAA	4817.1	4817	MW556675
27	Knodus aff. victoriae (Balsas)*	CICCAA	4817.2	4817	MW556676
28	Knodus aff. victoriae (Balsas)*	CICCAA	4817.3	4817	MW556677
29	Knodus aff. victoriae (Balsas)*	CICCAA	4818.1	4818	MW556678
30	Knodus aff. victoriae (Balsas)*	CICCAA	4818.2	4818	MW556679
31	Knodus aff. victoriae (Balsas)*	CICCAA	4818.3	4818	MW556680
32	Knodus aff. victoriae (Itapecuru)*	CICCAA	2064.1	2064	MW556681
33	Knodus aff. victoriae (Itapecuru)*	CICCAA	2064.2	2064	MW556682
34	Knodus cf. savannensis	LBP	–	66360	KT248199
35	Knodus cf. savannensis	LBP	–	66339	KT248200
36	Knodus cf. savannensis	LBP	–	66340	KT248201
37	Knodus cf. savannensis	LBP	–	66356	KT248202
38	Knodus cf. savannensis	LBP	–	66362	KT248203
39	Knodus cf. savannensis	LBP	–	66363	KT248204
40	Knodus cf. savannensis	LBP	–	66363	KT248218
41	Knodus cf. savannensis	LBP	–	62500	KT248219
42	Knodus cf. savannensis (Carolina-MA, Tocantins River basin)*	CICCAA	3609.1	3609	MW556695
43	Knodus sp.“Guamá”	LBP	43070	9141	KT248223
44	Knodus sp.“Guamá”	LBP	–	43718	KT248225
45	Knodus sp.“Guamá”	LBP	–	43067	KT248226
46	Knodus sp.“Guamá”	LBP	–	43068	KT248227
47	Knodus sp.“Tapajós”	LBP	57048	13750	KT248123
48	Knodus sp.“Tapajós”	LBP	–	57049	KT248124
49	Knodus sp.“Xingu”	LHGP	–	66314	KT248184
50	Knodus sp.“Xingu”	LHGP	–	66315	KT248185
51	Knodus sp.(Marabá)*	CICCAA	2087.1	2087	MW556692
52	Knodus sp.(Marabá)*	CICCAA	2087.2	2087	MW556693
53	Knodus sp.(Marabá)*	CICCAA	2087.3	2087	MW556694
54	Knodus sp.(Maracaçumé)*	CICCAA	2390.1	2390	MW556683
55	Knodus sp.(Maracaçumé)*	CICCAA	2390.2	2390	MW556684
56	Knodus sp.(Maracaçumé)*	CICCAA	2390.5	2390	MW556685
57	Knodus guajajara*	CICCAA	2052.2	2052	MW556688
58	Knodus guajajara*	CICCAA	2052.3	2052	MW556689
59	Knodus guajajara*	CICCAA	2052.4	2052	MW556690
60	Knodus guajajara*	CICCAA	2052.5	2052	MW556691
61	Knodus guajajara*	CICCAA	2391.1	2391	MW556686
62	Knodus guajajara*	CICCAA	2391.2	2391	MW556687

Holotype. CICCAA 4883, 31.4 mm SL, Alto Alegre do Pindaré municipality, Igarapé Arapapá, Pindaré River drainage, Mearim River basin, 03°42’26”S 46°00’25”W, Nov 2016. E. C. Guimarães & P. S. Brito.

Paratypes. All from Brazil, Maranhão State: CICCAA 1535, 1, 29.2 mm SL, collected with holotype. CICCAA 1585, 3, 28.0–34.7 mm SL, Alto Alegre do Pindaré municipality, igarapé Arapapá, Pindaré River drainage, Mearim River basin, 03°42’26”S 46°00’25”W, Nov 2015, E. C. Guimarães & P. S. Brito. CICCAA 4860, 2 C&S, 29.9–31.6 mm SL, Alto Alegre do Pindaré municipality, igarapé Arapapá, Pindaré River drainage, Mearim River basin, 03°42’26”S 46°00’25”W, Nov 2015, E. C. Guimarães & P. S. Brito. LBP 31041, 16, 24.5–31.4 mm SL, Alto Alegre do Pindaré municipality, Igarapé Igarapá, Pindaré River drainage, Mearim River basin, 03°45’51”S 46°08’15”W, Nov 2015, E. C. Guimarães & P. S. Brito. CICCAA 4858, 5 C&S, 22.9–27.0 mm SL, Alto Alegre do Pindaré municipality, Igarapé Igarapá, Pindaré River drainage, Mearim River basin, 03°45’51”S 46° 08’15”W, Nov 2015, E. C. Guimarães & P. S. Brito. CICCAA 1517, 2, 21.8–24.4 mm SL, Alto Alegre do Pindaré municipality, igarapé Caititu, Pindaré River drainage, Mearim River basin, 03°42’30”S 46°01’19”W, Jul 2017, E. C. Guimarães & P. S. Brito. CICCAA 4859, 2 C&S, 23.5–27.6 mm SL, Alto Alegre do Pindaré municipality, igarapé Caititu, Pindaré River drainage, Mearim River basin, 03°42’30”S 46°01’19”W, Jul 2017, E. C. Guimarães & P. S. Brito. CICCAA 1536, 2, 28.8–29.8 mm SL, Buriticupu municipality, Buritizinho River, Pindaré River drainage, Mearim River basin, 04°11’53”S 46°28’41”W, Nov 2016, E. C. Guimarães & P. S. Brito. CICCAA 4861, 1 C&S, 31.9 mm SL, Buriticupu municipality, Buritizinho River, Pindaré River drainage, Mearim River basin, 04°11’53”S 46°28’41”W, Nov 2016, E. C. Guimarães & P. S. Brito. CICCAA 1227, 1, 29.6 mm SL, Buriticupu municipality, Buritizinho River, Pindaré River drainage, Mearim River basin, 04°19’45”S 46°29’46”W, 27 Jan 2017, E. C. Guimarães & P. S. Brito. UFRJ 7023, 10, 22.8–29.3 mm SL, Alto Alegre do Pindaré municipality, igarapé Jenipapo, Pindaré River drainage, Mearim River basin, 03°51’20”S 46°11’09”W, Jul 2017, E. C. Guimarães & P. S. Brito. CICCAA 4862, 5 C&S, 23.9–29.3 mm SL, Brazil, Maranhão State, Alto Alegre do Pindaré municipality, igarapé Jenipapo, Pindaré River drainage, Mearim River basin, 03°51’20”S 46°11’09”W, Jul 2017, E. C. Guimarães & P. S. Brito. CICCAA 1321, 39, 19.4–29.2 mm SL, Miranda do Norte municipality, Mearim River basin, 04°19’45”S 46°29’46”W, Nov 2016, E. C. Guimarães & P. S. Brito. CICCAA 4863, 11 C&S, 19.7–24.4 mm SL, Miranda do Norte municipality, Mearim River basin, 04°19’45”S 46°29’46”W, Nov 2016, E. C. Guimarães & P. S. Brito. CIUEMA 1021, 8, 21.2–26.9 mm SL, Alto Alegre do Pindaré municipality, Igarapé Timbira, Pindaré River drainage, Mearim River basin, 03°32’57”S 44°39’38”W, Nov 2015, E. C. Guimarães & P. S. Brito. CICCAA 3423, 42, 19.5–30.21 mm SL, CICCAA 3424, 57, 22.35–26.9 mm SL,, Chapadinha municipality, Riacho da Raiz, RESEX Chapada Limpa, Munim River basin, 03°53’45”S 43°29’21”W, 11 Aug 2019, J. Reis, L. Oliveira, F. Ottoni, R. Fernandes & A. Silva. Chapadinha municipality, Bandeira River, Povoado Mata do Jeroca, RESEX Chapada Limpa, Munim River basin, 03°59’40”S 43°29’24”W, 11 Aug 2019, J. Reis, L. Oliveira, F. Ottoni, R. Fernandes & A. Silva. CICCAA 3425, 22, 20.22–26.71 mm SL, CICCAA 3426, 6, 28.87–32.6 mm SL, Chapadinha municipality, stream at Bairro Aldeia, Munim River basin, 03°44’53”S 43°21’32”W, 28 Jan 2019, F. Carvalho, H. Silva, L. Oliveira & I. Gôuvea. CICCAA 3467, 2, 21.22–21.43 mm SL, Anapurus municipality, stream on the road at Povoado de Paços, Munim River basin, 03°33’44”S 43°03’52”W, Sep 2019, D. Campos, J. Reis & F. Ottoni. CICCAA 4808, 3, 25.19–34.2 mm SL, CICCAA 4822, 1, 36.89 mm SL, Chapadinha municipality, stream at balneário Repouso do Guerreiro, Bairro Independência, Munim River basin, 03°44’57”S 43°20’26”W, Nov 2019, B. Furtado, M. Paiva, A. Bezerra, M. Coelho & I. Gouvêa. CICCAA 4490, 5, 24.73–29.11 mm SL, Chapadinha municipality, Riacho Fundo, Munim River basin, 03°42’20”S 43°31’46”W, 23 Sep 2018, L. Sousa, L. Oliveira & I. Gouvêa, CICCAA 4491, 3 C&S 23.26–28.19 mm SL, Chapadinha municipality, Riacho Fundo, Munim River basin, 03°42’20”S 43°31’46”W, 23 Sep 2018, L. Sousa, L. Oliveira & I. Gôuvea.

Morphological diagnosis.Knodus guajajara differs from K. borki Zarske, 2008 and K. delta Géry, 1972 by having a complete lateral line (vs. incomplete lateral line) and from K. cupariensis de Sousa, Silva-Oliveira, Canto & Ribeiro, 2020 and K. geryi Lima, Britski & Machado, 2004 by having caudal fin lobes with sparse chromatophores and lacking basal blotches (vs. a dark basal blotch on each caudal fin lobe, Sousa et al., 2020; fig. 1); from Knodus borki, K. diaphanus (Cope, 1878), K. victoriae, K. heteresthes (Eigenmann, 1908), K. deuterodonoides (Eigenmann, 1914), K. longus Zarske & Géry, 2006, K. septentrionalis Géry, 1972, K. figueiredoi Esguícero & Castro, 2014, K. geryi, K. meridae Eigenmann, 1911, K. nuptialis Menezes & Marinho, 2019, K. orteguasae (Fowler, 1943), K. tiquiensis Ferreira & Lima, 2006, K. angustus Menezes, Ferreira & Netto-Ferreira, 2020, K. rufford Deprá, Ota, Vitorino-Júnior & Ferreira, 2021 and K. obolus Deprá, Ota, Vitorino-Júnior & Ferreira, 2021 by having 20–25 branched rays in the anal-fin (mode 23) (vs. 12–19, combined); and from K. tiquiensis by having a single humeral spot (vs. two). Knodus guajajara is distinguished from K. breviceps (Eigenmann, 1908) and K. savannensis Géry, 1961 by having a conspicuous round humeral blotch (vs. inconspicuous and vertically elongate); from K. dorsomaculatus Ferreira & Netto-Ferreira, 2010 by having a hyaline dorsal-fin (vs. dark blotch on the base of the first five branched dorsal fin rays); from K. alpha (Eigenmann, 1914), K. chapadae (Fowler, 1906), K. geryi, K. hypopterus (Fowler, 1943), K. mizquae (Fowler, 1943) and K. shinahota Ferreira & Carvajal, 2007 by having 4 or 5 rows of scales between the lateral line and the dorsal-fin origin (vs. 6 rows of scales); from K. cinarucoensis (Román-Valencia et al., 2008), K. gamma Géry, 1972 and K. longus Zarske & Géry, 2006 by having 12 or 13 scales in the median series between the tip of the supraoccipital spine and the dorsal-fin origin (vs. 10 or 11 rows of scales in K. gamma and 17 to 18 in K. longus); from K. jacunda (Fowler, 1913), K. moenkhausii (Eigenmann & Kennedy, 1903), Knodus cismontanus (Eigenmann, 1914), Knodus caquetae Fowler, 1945, K. tanaothoros (Weitzman, Menezes, Evers & Burns, 2005), K. weitzmani (Menezes, Netto-Ferreira & Ferreira, 2009) and by having 3 to 5 maxillary teeth (vs. absence in K. jacunda, one in K. caquetae, and 2 in K. moenkhausii, K. tanaothoros, K. weitzmani and K. cismontanus); from K. megalops Myers, 1929 by having 3 or 4 tricuspid teeth in the premaxillary outer row (vs. 5); from K. jacunda by having 3 to 5 maxillary teeth (vs. absence); from K. smithi (Fowler, 1913) by having 3 to 5 cusps on the teeth of the inner row of the premaxilla (vs. 7); and from K. figueredoi, K. heteresthes, K. meridae, K. mizquae, K. moenkhausii, K. victoriae, K. pasco Zarske, 2007 by having 12 circumpeduncular scales (vs. 13–14 combined). Furthermore, Knodus guajajara differs from K. victoriae by having more total vertebrae 33–35, mode 34 (vs. 30–33, mode 32).

FIGURE 2 |
Knodus guajajara, paratypes. A. CICCAA 1518, 22.8 mm SL, Brazil, Maranhão, Alto Alegre do Pindaré municipality, igarapé Jenipapo, Pindaré River drainage, Mearim River basin. B. CICCAA 2696, 40.2 mm SL, Brazil, Maranhão, Chapadinha municipality, stream in riparian forest on the road BR–222, Munim River basin.

Description. Morphometric data presented in Tabs. 2–3. Body comparatively small, with largest specimen examined measuring 37.7 mm SL. Greatest body depth at dorsal-fin origin. Dorsal profile of head convex from upper lip to vertical through middle portion of eye; slightly concave from this point to tip of supraoccipital spine; straight to slightly convex from posterior tip of supraoccipital spine to dorsal-fin origin; dorsal-fin base straight; slightly convex to straight from end of dorsal-fin base to adipose fin and concave from latter point to anterior dorsal-procurrent ray. Ventral profile of body convex from lower lip to anal-fin origin; straight, posterodorsally inclined along anal-fin base. Dorsal and ventral profile of caudal peduncle slightly concave.

Mouth sub-terminal; jaws isognathous. Posterior terminus of maxilla extending beyond anterior margin of orbit. Premaxillary teeth in two rows; outer row with 3(18) and 4(8) tricuspid teeth, inner row with 4(26) tri-to pentacuspid teeth. Maxilla with 3(11), 4(13), 5(2) tri-to pentacuspid teeth. Dentary with three anteriormost teeth large, tri and pentacuspid teeth, followed by 4(2), 5(2), 6(4), 7(13) and 8(5) smaller, uni, tricuspid or pentacuspid teeth (Fig. 3).

FIGURE 3 |
Knodus guajajara, CICCAA 4861, paratype, 31.9 mm SL, jaw suspensorium. A. Premaxillary. B. Maxilla. C. Dentary. Scale bar = 1 mm.

Scales cycloid, moderately large, with 6–8 well-marked radii; circuli only present proximally. Lateral line complete, slightly curved ventrally along its anterior third, with 36(8), 37(61), 38*(137), 39(13), or 40(7) perforated scales. Longitudinal series of scales between dorsal-fin origin and lateral line 4(30) or 5*(202). Longitudinal series of scales between lateral line and pelvic-fin origin 4*(63), or 5(184). Predorsal scales 11(3), 12(106) or 13*(122). Circumpeduncular scales 12*(220). Single series of scales on anal-fin base.

Dorsal fin rays ii,7(45) or 8*(191). Dorsal-fin origin slightly posterior to middle of body, and slightly posterior to vertical through pelvic-fin origin. First unbranched ray approximately half length of second unbranched ray. Pectoral-fin rays i,9(9), 10(142) or 11*(94); tip of pectoral fin reaching or exceeds pelvic fin origin when adpressed. Pelvic-fin rays i,5,i(41) or i,6,i*(189); tip of pelvic fin usually reaching first anal fin rays when adpressed. Anal-fin rays iv, 20(2), 21(8), 22(40), 23*(140), 24(37) or 25(15). Anal-fin origin located at vertical through middle of dorsal fin. Anal-fin rays decreasing gradually in length. Caudal fin forked, lobes equal in size. Principal caudal-fin rays i,9,8,i(219). Dorsal procurrent caudal-fin rays 8(1), 9(1), 10(18), 11(3) or 12(3) and ventral procurrent caudal-fin rays 9(3), 10(2), 11(15), 12(3) or 13(3). Adipose-fin origin approximately at vertical through base of 19th to 21st branched anal-fin rays. Gill rakers on first gill arch 15(2), 16(4) or 17(2): hypobranchial 2(4) or 3 (4), ceratobranchial 7(8), cartilage between epibranchial and ceratobranchial 1(8), epibranchial 5(4) or 6(4). Gill rakers setiform. Branchiostegal rays 4(8), 3(8) on anterior ceratohyal and 1(8) on posterior ceratohyal. Total vertebrae 33(3), 34(18) or 35(8). First dorsal fin pterygiophore inserted between 10th and 12th vertebrae. First anal fin pterygiophore inserted between 16th and 17th vertebrae. Supraneurals 4(2), 5(12) or 6(9).

TABLE 2 |
Morphometric data (N = 110) of the holotype and paratypes of Knodus guajajara from the Mearim River basin. SD = Standard deviation.

	Holotype	Paratypes	Mean	SD
Standard length	31.4	19.4‒34.7	25.3	‒
Percents of standard length
Depth at dorsal-fin origin (body depth)	29.8	20.1‒30.2	24.7	3.3
Snout to dorsal-fin origin	55.3	47.8‒57.1	52.2	5.5
Snout to pectoral-fin origin	27.1	21.7‒29.32	24.9	3.0
Snout to pelvic-fin origin	45.7	37.6‒49.3	42.2	4.9
Snout to anal-fin origin	60.2	44.0‒61.6	53.9	6.1
Caudal peduncle depth	10.5	7.6‒11.7	9.3	1.2
Caudal peduncle length	15.3	7.1‒15.2	11.1	1.9
Pectoral-fin length	20.5	14.8‒23.8	18.7	2.6
Pelvic-fin length	13.5	9.8‒15.4	12.6	1.7
Dorsal-fin base length	9.1	8.7‒15.2	11.5	1.7
Dorsal-fin height	26.9	17.7‒27.0	21.3	2.8
Anal-fin base length	29.4	25.5‒36.3	30.5	3.8
Anal-fin lobe length	13.0	9.8‒20.3	15.3	2.4
Eye to dorsal-fin origin	42.5	33.6‒43.8	38.9	4.5
Dorsal-fin origin to caudal-fin base	52.5	39.4‒56.7	46.9	5.8
Percents of head length
Head length	23.9	21.3‒26.5	23.3	2.5
Horizontal eye diameter	41.3	32.6‒45.6	39.5	4.8
Snout length	24.9	15.8‒29.6	21.8	3.3
Least interorbital width	35.1	23.7‒36.8	32.3	4.0
Upper jaw length	47.5	31.5‒48.8	38.3	5.3

TABLE 3 |
Morphometric data (N = 136) for the paratypes of Knodus guajajara from the Munim River basin. SD = Standard deviation.

	Range	Mean	SD
Standard length	19.5‒37.7	25.3	‒
Percents of standard length
Depth at dorsal-fin origin (body depth)	20.4‒37.2	24.7	2.06
Snout to dorsal-fin origin	47.4‒53.4	50.3	1.13
Snout to pectoral-fin origin	21.9‒27.1	24.4	0.79
Snout to pelvic-fin origin	39.4‒45.7	42.5	1.24
Snout to anal-fin origin	53.1‒59.8	56.2	1.38
Caudal peduncle depth	8.3‒10.4	9.3	0.41
Caudal peduncle length	9.2‒12.8	10.4	0.75
Pectoral-fin length	18.5‒23.4	20.9	0.74
Pelvic-fin length	12.0‒15.7	13.8	0.68
Dorsal-fin base length	8.4‒12.6	10.1	0.76
Dorsal-fin height	20.1‒24.6	22.1	0.86
Anal-fin base length	22.8‒28.7	26.4	1.17
Anal-fin lobe length	16.1‒20.2	18.1	0.82
Eye to dorsal-fin origin	33.1‒39.2	36.1	1.12
Dorsal-fin origin to caudal-fin base	47.4‒54.3	49.5	1.14
Percents of head length
Head length	21.5‒25.7	23.4	0.82
Horizontal eye diameter	33.8‒42.6	37.4	1.56
Snout length	20.1‒25.9	22.5	1.34
Least interorbital width	27.1‒35.3	31.7	1.77
Upper jaw length	34.5‒41.2	37.9	1.47

Color in alcohol. Ground of body and head light brown, with dorsal region of body (especially dorsal profile), dorsal portion of head (just above eyes), and upper snout area (upper jaw and adjacent areas) darker. Ventral region of trunk and head with light brown coloration, especially on scales adjacent to anal-fin base. Dark colored chromatophores concentrated on scales of dorsal region (from both body and head), on opercle, and lateral band of flank (especially on posterior region, near caudal peduncle and caudal-fin base). Conspicuous vertically elongated humeral spot, with some dark scattered chromatophores above and below this blotch. Lateral band formed by chromatophores located above lateral line, extending gradually from humeral blotch to middle rays of caudal fin, where they become thicker and more concentrated. All fins including adipose hyaline, with chromatophores on inter-radial membranes of dorsal fin. Anal and caudal fins with posterior margins dark grey.

Color in life. Yellowish coloration in dorsolateral region and silver coloration in ventrolateral region of body (Fig. 2). Gray lateral band located in middle portion of trunk, with bright pigment extending from operculum to caudal-fin base. Dorsal portion of eye yellowish-orange and lower portion silver; mouth and dorsolateral portion of head yellowish with concentrated chromatophores; operculum with silver coloration. Fins hyaline with chromatophores present and especially concentrated in medial region of dorsal fin, distal region of anal and caudal fins, on first pectoral-fin ray, and just posterior to medial lateral band on caudal fin. Adipose fin with yellowish-orange pigmentation; yellowish-orange pigments also present in middle region of caudal fin.

Sexual dimorphism. Hooks present on pelvic fins and anal fin of sexually mature males. Pelvic fins with 5 to 14 hooks per branched ray; anal fin with 2 to 8 hooks per ray. Hooks concentrated on anterior anal fin rays (Fig. 4).

FIGURE 4 |
Knodus guajajara, CICCAA 4861, paratype, male, 31.9 mm SL Maranhão, Mearim River basin. A. Hooks on pelvic fin. B. Hooks on anal fin. (Photographed by F. P. Ottoni).

Geographical distribution.Knodus guajajara occurs in the rivers, streams, and lagoons of the Mearim and Munim river basins, in the State of Maranhão, Northeast Brazil (Fig. 5).

Etymology. The specific epithet honors the Guajajara indigenous tribe, which is one of the most numerous indigenous peoples in Brazil. They inhabit more than 10 Indigenous Lands on the eastern margin of the Amazon, all located in Maranhão. A noun in apposition the language spoken by them is the Teneteara, from the Tupi-Guarani linguistic family.

Conservation status.Knodus guajajara occurs in two distinct river basins of Maranhão, the Mearim and Munim river basins. It was found in several collecting sites along these basins, and it is always abundant. Therefore, according to the guidelines given in the International Union for Conservation of Nature (IUCN) categories and criteria (IUCN, 2019), we recommend that Knodus guajajara should be categorized as Least Concern (LC).

FIGURE 5 |
Geographical distribution of Knodus guajajara.

Molecular species delimitation

Molecular diagnosis.Knodus guajajara is diagnosed molecularly by a combination of 19 nucleotide substitutions, four of them unique* (listed below):

COI 279 (C→A), COI 282 (C→T), COI 306 (C→T), COI 327 (C→T), COI 357 (A→G*), COI 372 (A→G*), COI 378 (A→G), COI 414 (T→C), COI 450 (T→C), COI 459 (C→T), COI 510 (T→C), COI 528 (C→T), COI 537 (C→T), COI 565 (C→T), COI 585 (T→C), COI 589 (C→T), COI 597 (C→T), COI 627 (A→G*), COI 645 (T→A*).

Traditional DNA Barcoding. COI sequences support the existence of a new species of Knodus inhabiting the Mearim and Munim River basins. After trimming, the final alignment yielded 401 base pairs with 132 variable sites and 62 haplotypes. Knodus guajajara is 14% divergent, on average, from the other taxa, with a minimum genetic distance of 11% to K. borki, K. caquetae and Knodus sp. “Maracaçumé”, and a maximum genetic distance of 16% to Bryconamericus exodon Eigenmann,1907 and Knodus tiquiensis. The genetic distance between the new species and K. victoriae is 13% (Tab. 4).

TABLE 4 |
Kimura-2-parameters pairwise genetic distances among species.

	Species	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18
1	Bryconamericus exodon
2	Bryconamericus iheringii	0.12
3	Knodus heteresthes	0.15	0.16
4	Knodus cf. savanensis (Clade 1)	0.13	0.17	0.09
5	Knodus cf. savanensis (Clade 2)	0.13	0.16	0.08	0.06
6	Knodus sp. “Tapajós”	0.13	0.18	0.08	0.10	0.09
7	Knodus sp. “Xingu”	0.15	0.16	0.05	0.09	0.10	0.09
8	Knodus tiquiensis	0.14	0.18	0.13	0.11	0.11	0.12	0.14
9	Knodus borki	0.11	0.15	0.12	0.12	0.11	0.11	0.12	0.15
10	Knodus megalops	0.13	0.14	0.14	0.14	0.14	0.12	0.13	0.15	0.08
11	Knodus caquetae	0.11	0.14	0.14	0.14	0.13	0.12	0.14	0.15	0.07	0.04
12	Knodus meridae	0.16	0.16	0.12	0.11	0.10	0.12	0.13	0.15	0.11	0.13	0.13
13	Knodus alpha	0.14	0.18	0.10	0.06	0.09	0.13	0.09	0.15	0.14	0.14	0.15	0.15
14	Knodus sp. “Marabá”	0.14	0.16	0.16	0.13	0.12	0.14	0.15	0.14	0.15	0.15	0.15	0.17	0.15
15	Knodus sp. “Maracaçumé”	0.12	0.14	0.12	0.13	0.11	0.12	0.13	0.11	0.11	0.12	0.10	0.12	0.15	0.14
16	Knodus victoriae	0.15	0.18	0.07	0.08	0.08	0.10	0.08	0.13	0.11	0.15	0.14	0.12	0.10	0.16	0.12
17	Knodus aff. victoriae (Balsas)	0.13	0.19	0.09	0.09	0.09	0.10	0.10	0.13	0.12	0.13	0.15	0.10	0.10	0.14	0.14	0.05
18	Knodus aff. victoriae (Itapecuru)	0.14	0.15	0.10	0.09	0.09	0.10	0.11	0.13	0.12	0.11	0.14	0.12	0.09	0.14	0.12	0.08	0.06
19	Knodus guajajara	0.16	0.13	0.15	0.13	0.13	0.14	0.14	0.16	0.11	0.13	0.11	0.13	0.13	0.13	0.11	0.13	0.15	0.13

Phylogenetic analysis. The BI phylogenetic analysis delimited 19 lineages: two of them are outgroups (Bryconamericus species) and the remaining correspond to Knodus species (Fig. 6). One of the 17 lineages of Knodus represents the new species described herein (Knodus guajajara) with haplotypes from the Mearim and Munim river basins forming an exclusive clade supported by the highest possible support value (PP = 1) (Fig. 6). The BI phylogenetic analysis also delimited new lineages of Knodus inhabiting the Tocantins River (Knodus sp. “Marabá”) and the Maracaçumé River (Knodus sp. “Maracaçumé”). In addition, two well supported clades were recovered within Knodus cf. savannensis: the first one containing Knodus cf. savannensis (Tocantins River, Carolina, MA), Knodus “Guamá” and Knodus cf. savannensis (KT248218 and KT248219) (posterior probability = 0.96). The second clade of Knodus cf. savannensis corresponds to six haplotypes (KT248199, KT248200, KT248201, KT248202, KT248203, and KT248204) of Knodus cf. savannensis (PP = 1). Two other lineages closely related to K. victoriae were detected: Knodus cf. victoriae (Balsas River drainage) and Knodus cf. victoriae (Itapecuru River basin) (PP = 1).

GMYC, bPTP and ABGD. The results of the three species delimitation methods (GMYC, bPTP, and ABGD) were congruent (Fig. 6), and all three delimited the same 19 lineages. Two of them are outgroups (Bryconamericus species), and 17 are Knodus lineages (species) (Fig. 6). The new species herein described (K. guajajara) was recovered in all three species delimitation methods. Furthermore, some potential undescribed species were detected: Knodus “Marabá”, Knodus “Maracaçumé”, Knodus cf. victoriae (Itapecuru River basin) and Knodus cf. victoriae (Balsas River drainage). Knodus victoriae (upper Parnaiba River basin) was also recovered, as well as two K. cf. savannensis clades.

FIGURE 6 |
Bayesian Inference phylogenetic tree generated by this study. The results of GMYC, bPTP and ABGD are included. Numbers above and below branches are posterior probability values. The gray bars correspond to the congruent results of the different species delimitation methods (Con.).

DISCUSSION

Knodus guajajara is the first species of the genus described using a combination of morphological and genetic data. The new species has the traditional morphological characteristics that define Knodus, especially the small scales covering the procurrent rays of the caudal fin (Eigenman, 1918; Géry, 1972, 1977; Román-Valencia et al., 2008).

There are some congeners that occur geographically close to the new species described here: Knodus cupariensis, K. dorsomaculatus and K. heteresthes (from the Tapajós River basin); K. nuptialis and K. weitzmani (from the Xingu River basin); K. savannensis (from the lower/middle Tocantins River basin); and K. victoriae (from the upper Parnaiba River basin) (Aguiar et al., 2021; Fricke et al., 2022; and this study). Furthermore, Knodus septentrionalis from Ecuador’s upper Pastaza River basin was previously considered by Géry, (1972) as a subspecies of Knodus victoriae, but was elevated to the species category by Chang, Ortega, (1995). The new species is morphologically distinguished from these aforementioned species, as well as all the other congeners, by a unique combination of character states (see diagnosis). Of the geographically proximate species to Knodus guajajara, K. savannensis and K. victoriae are the closest ones (lower/middle Tocantins River and Parnaiba River basins, respectively). However, Knodus guajajara is distinguished from K. savannensis in the form of its conspicuous and slightly rounded humeral patch, which is inconspicuous and vertically elongated in K. savannensis. It is morphologically most similar to K. victoriae, having been previously misidentified as that species in the Munim and Mearim river basins (Oliveira et al., 2020; Guimarães et al., 2018a, 2020a). However, some morphological characteristics differ between these two species, such as the presence of fewer circumpeduncular scales, more branched rays in the anal fin and more vertebrae in Knodus guajajara (see diagnosis).

Accurate discussions regarding phylogenetic relationships, as well as the biogeography of the species in the studied area, would be speculative due to the lack of available data, poor taxonomic resolution of some species and knowledge gaps for Knodus species in this region, as well as for the coastal river systems of Maranhão.

Using only morphological characteristics to delimit species in Knodus has caused identification errors in taxonomic studies (García-Melo et al., 2019). For this reason, García-Melo et al., (2019) did not solely consider the presence of scales at the base of the tail fin as a convincing feature to delimit the genus, but rather emphasized the use of DNA sequences to delimit Knodus species more precisely.

In accordance with that recommendation, in addition to morphological evidence, the description of Knodus guajajara is also supported by molecular data (see Fig. 6 and Tab. 4) from five different and independent single locus species delimitation methods. It has a high genetic distance value (13%) (Tab. 4) relative to K. victoriae, the most morphologically similar species, and appears to be phylogenetically distant (Fig. 6). Knodus guajajara is the sister group to a clade comprising K. borki, K. caquetae and K. megalops, possessing 11%, 11% and 13% of genetic distance, respectively, to these species. It is important to emphasize that K. guajajara is more genetically similar to these three species than to several geographically closer congeners (see Tab. 4). Knodus borki is known from near Iquitos, in Peru, K. caquetae occurs in the Amazon and Caquetá River basins of Colombia, and K. megalops is distributed in the Upper Amazon River basin, in Peru and Ecuador (Fricke et al., 2022). All three of these regions are far away from the known geographical distribution of Knodus guajajara. This pattern may be an artifact of the gap of knowledge for Knodus or of the scarcity of genetic data for the genus.

In addition to the new species described here, our results reveal the presence of even more potentially undescribed species for the genus Knodus (Fig. 6) than García-Melo et al., (2019), indicating that a knowledge gap for the group still likely exists and that more undescribed species await discovery. Studies that incorporate both molecular and morphological data, as well as, different operational criteria (integrative taxonomy) have shown success in delimiting and describing species and complexes of cryptic or hidden species (e.g., Mazetti et al., 2012; Guimarães et al., 2018b, 2019, 2020b; Brito et al., 2019, 2021; Ottoni et al., 2019; Faria et al., 2020). The same result is observed for the present study, in which the different species delimitation methods based on molecular data demonstrate congruence with each other. All these methods supported the validity of the new species, the hidden diversity in K. victoriae and the possible presence of new species of Knodus in the Maracaçumé and Tocantins river basins (Fig. 6).

Knodus savannensis is distributed throughout the Tocantins basin and has two possible type-localities according to Lima, Géry, (2001): “Brazil, state of Tocantins, municipality of Itacajá, between the Manoel Alves Pequeno river and the Vermelho river, Tocantins river basin approx. 8°19’S, 47°25W”, or “Brazil, Tocantins river basin, Javaés river, Bananal Island, approx. 11°S, 51°W”. A more comprehensive study focusing on K. savannensis is necessary to solve the taxonomy complexity of this species and its type locality.

Our results point to another lineage (Knodus sp. “Marabá”) in the Tocantins River that is apparently not closely related to the Knodus savannensis complex, suggesting two distinct colonization or speciation events in the basin. The occurrence of geographically close species in the genus was recently recorded by Deprá et al., (2021), in which two new species (K. rufford and K. obolus) share the same type locality in the upper Tocantins river basin, thus demonstrating another case of hidden diversity within Knodus.

Still another case of cryptic diversity occurs in the Maracaçumé river basin (Maranhão State). In this river a divergent lineage occurs (Knodus sp. “Maracaçumé”, see Tab. 4), but despite its geographic proximity, it possesses a high genetic distance (11%) to Knodus guajajara, suggesting that is a new species according to the species delimitation methods conducted. The occurrence of new characids has been demonstrated for the Maracaçumé River basin, including cryptic species (e.g., Brito et al., 2019; Guimarães et al., 2020b). The description of new fish species for this basin is expected since according to Abreu et al. (2019, 2020), biogeographic processes favored the isolation of drainages in Maranhão, providing opportunities for the emergence of new species.

We also identified cryptic diversity occurring in Knodus victoriae with the detection of two additional lineages: one from the Balsas River drainage of the upper Parnaiba River basin [Knodus aff. victoriae (Balsas)]; and the other from the upper Itapecuru River basin [Knodus aff. victoriae (Itapecuru)] (Fig 6; Tab. 4), totaling three lineages under the name Knodus victoriae. A noteworthy aspect is that two of these lineages, Knodus aff. victoriae (Balsas) and Knodus victoriae, occur in upper sections of the Parnaiba River basin (Aguiar et al., 2021; this study). It is likely that the presence of these sister lineages in the upper Parnaiba and upper Itapecuru river basins is due to past headwater captures between these river systems.

In addition to contributing to the description of a new species (Knodus guajarara), the results of our present work revealed that a hidden diversity of species of the genus Knodus inhabits the hydrographic basins of Maranhão and adjacent systems. The use of different data sources (morphology, molecular, geographic distribution, etc.) can help to identify genetically distinct groups with high morphological similarity, thereby avoiding identification errors and helping to solving problems of validity and taxonomic placement within Characidae.

Comparative material examined.Knodus borki: Peru. MTDF 31357, 1, 33.99 mm SL, holotype, Umgebung von Iquitos. Knodus longus: Bolivia. MTDF 28853, 1, 46.1 mm SL, holotype, río Warinilla (oder Harinilla) nahe Choiro. Knodus pasco: Peru. MTDF 30634, 1, 55.4 mm SL, holotype, Departamento Pasco, erste Quebrada bei San Antonio de Cacazú (eigentlich nach dem Vorort Baja Cacazú) auf der Straße, nach Puerto Bermúdez. Knodus savannensis: Brazil. USNM 196088, 1, 30.4 mm SL, holotype, Tocantins, Tocantins basin. Knodus savannensis (Clade 1): Brazil. CICCAA 2397, 108, 36.83–48.67 mm SL; CICCAA 2399, 73, 31.46–38.52 mm SL; CICCAA 2400, 27, 38.37–61.08 mm SL, Maranhão, Carolina. Knodus sp. “Marabá”: Brazil. CICCAA 1522, 2, 29.16–33.92 mm SL; CICCAA 1306, 14, 22.92–36.22 mm SL; CICCAA 1503, 3, 34.36–38.24 mm SL, Pará, Marabá. CICCAA 1525, 9, 24.4–37.74 mm SL, Pará, Curinópolis. CICCAA 1436, 20, 17.74–27.51 mm SL, Pará, Paragominas. CICCAA 1409, 6, 30.7–38.9 mm SL; CICCAA 1521, 4, 29.85–37.67 mm SL; CICCAA 1516, 1, 29.62 mm SL; CICCAA 1524, 5, 20.68–20.82 mm SL;CICCAA 1509, 3, 34.86–38.24 mm SL, Maranhão, São Pedro da Água Branca. CICCAA 1295, 27, 20.36–28.27 mm SL; CICCAA 1523, 8, 17.82–22.58 mm SL, Maranhão, Vila Nova dos Martírios. Knodus victoriae: Brazil. NMW 57823, 16 syntypes; NMW 57824, 13 syntypes; NMW 57825, 18 syntypes of Tetragonopterus victoriae, Maranhão, Parnaíba basin; CICCAA 2708, 18; CICCAA 2709, 17; CICCAA 2710, 7 C&S, Maranhão, Alto Parnaíba municipality, Parnaíba river basin; Knodus aff. victoriae: Brazil. CICCAA 2842, 1, 29.5 mm SL, Maranhão, Itapecuru River basin. CICCAA 4809, 9, 15.57–28.47 mm SL; CICCAA 4810, 31, 20.73–43.48 mm SL; CICCAA 4811, 29, 17.18–42.22 mm SL; CICCAA 4812, 34, 17.32–42.18 mm SL; CICCAA 4892, 8 C&S, CICCAA 4893, 2 C&S, Maranhão, Parnaíba basin. Knodus cf. victoriae: Brazil. CICCAA 2395, 6, 25.6–36.08 mm SL; CICCAA 2391, 5 C&S, 27.51–31.4 mm SL, Maranhão, Itapecuru River basin.

ACKNOWLEDGEMENTS

We thank Axel Zarske, Ingo Schindler and Ronald Fricke for providing useful literature; Vale S.A, Amplo Engenharia and Instituto Maranhense de Estudos Socioeconômicos e Cartográficos (IMESC) for sharing part of the data analyzed in this study; Mark Sabaj Pérez (CAS), James Maclaine (FMNH), Riedel Bettina (NMW), Sandra Raredon (Smithsonian Institution) for providing photographs, x-ray images, and information on the type material of some species; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES – Finance Code 001) and Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA) for providing the scholarship under the process 88882.445737/2019–01 to RGA, BD–05604/19 to ECG, 88887.159561/2017–00 to PSB; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant 307974/2021–9 to FPO) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ; grant E–26/202.005/2020 to AMK). This study was supported by the project “PROCESSO UNIVERSAL – 00724/17”, from FAPEMA.

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ADDITIONAL NOTES

• HOW TO CITE THIS ARTICLE
  Aguiar RG, Guimarães EC, Brito PS, Santos JP, Katz AM, Dias LJBS, Carvalho-Costa LF, Ottoni FP. A new species of Knodus (Characiformes: Characidae), with deep genetic divergence, from the Mearim and Munim river basins, Northeastern Brazil, and evidence for hidden diversity in adjacent river basins. Neotrop Ichthyol. 2022; 20(2):e210173. https://doi.org/10.1590/1982-0224-2021-0173

• Edited-by
  Brian Sidlauskas

Publication Dates

• Publication in this collection
  03 June 2022
• Date of issue
  2022

History

• Received
  26 Jan 2022
• Accepted
  8 Apr 2022