New banded Leporinus (Characiformes: Anostomidae) from the Madeira River basin, Brazil, and redescription of L. bleheri, based on integrative taxonomy

Abstract

Leporinus bleheri was described for the Guaporé-Iténez basin, in the border between Bolivia and Brazil. More recently, specimens of a similar-looking banded Leporinus were sampled in distinct rivers in the Madeira basin. Herein, we use an integrative approach combining molecular and morphological data to investigate the taxonomic status of the new samples. Morphometric data were used to perform a Principal Components Analysis (PCA). Both species specimens were cleared and double-stained for osteological description. DNA barcodes were used to investigate the genetic distance between samples and for species delimitation analyses. Molecular markers COI, CytB, 16S, Myh6, RAG1, and RAG2 were applied to estimate the phylogenetic relationships of the two species. Our results show morphological and genetic differences between samples of L. bleheri and the new species. Both species are herein (re)described and illustrated. The new species is distinguished from L. bleheri by having 12 scale rows around the caudal peduncle (vs. 16). The genetic distance between the new species and L. bleheri was of 3.93%, and species delimitation analysis recovered the samples as separated molecular units. The multi-loci analysis corroborated the sister-group relationships between both species, including them within the Leporinus fasciatus group, which was recovered as non monophyletic.

Keywords:
Anostomoidea; Amazon; Ostariophysi; Species delimitation analysis; Taxonomy

Resumo

Leporinus bleheri foi descrita da bacia do rio Guaporé-Iténez, na fronteira entre a Bolívia e o Brasil. Mais recentemente, exemplares de uma espécie barrada parecida foram amostrados em rios da drenagem do Madeira. Aqui, nós utilizamos uma abordagem integrativa combinando dados morfológicos e moleculares para investigar o status taxonômico das novas amostras. Dados morfométricos foram usados para elaboração de uma Análise de Componentes Principais (PCA). Exemplares das duas espécies foram diafanizados para descrição osteológica. DNA barcodes foram utilizados para investigar a distância genética entre as amostras, e para análises de delimitação de espécies. Marcadores moleculares COI, CytB, 16S, Myh6, RAG1 e RAG2 foram aplicados para estimar as relações filogenéticas entre as duas espécies. Nossos resultados mostraram diferença morfológica e genética entre amostras de L. bleheri e da espécie nova. As duas espécies foram aqui (re)descritas e ilustradas. A nova espécie difere de L. bleheri por ter 12 fileiras de escamas ao redor do pedúnculo caudal (vs. 16). A distância genética entre a nova espécie e L. bleheri foi de 3.93%, e as análises de delimitação de espécie recuperaram as amostras como unidades moleculares separadas. A análise multi-loci corroborou a relação de grupo-irmão das duas espécies, incluindo-as dentro do grupo Leporinus fasciatus, que foi recuperado como não monofilético.

Palavras chave:
Análises de delimitação de espécies; Anostomoidea; Amazônia; Ostariophysi; Taxonomia

INTRODUCTION

The Neotropical fish fauna is one of the most diverse in the world and still underestimated, with a hundred species being described yearly in the past decade (Reis et al., 2016; Birindelli, Sidlauskas, 2018). The systematics of Neotropical fish significantly advanced in the past years due to the combination of morphological and genetic data methods, such as DNA barcodes (Gomes et al., 2015; Fisch-Muller et al., 2018; García-Melo et al., 2019; Costa et al., 2022). Despite the efforts to describe the Neotropical diversity, the fish fauna has been suffering the impacts of environmental changes at an alarming rate, with a decline of 80% of the freshwater vertebrate populations registered in the past fifty years (Darwall et al., 2018). This decline was caused by several factors, from exacerbated use of natural resources and pollution to increased urban and agricultural areas (Garcia-Moreno et al., 2014; Darwall et al., 2018; Garcia et al., 2021). Freshwater ecosystems are probably the most endangered on the planet, and the threats have been aggravated by the impacts of human activity on climate change (Garcia-Moreno et al., 2014).

Anostomidae, with over 150 valid species in 16 genera, is the second most diverse family of Characiformes (Fricke et al., 2023). Anostomids are distinguishable from other fishes by having three or four large incisiform teeth in each premaxillary and dentary (Garavelo, Britski, 2003; Sidlauskas, Birindelli, 2017). As for most Neotropical fish groups, the use of molecular markers greatly expanded our knowledge of the family in the past decade. The general phylogeny of Anostomidae, including the paraphyly of Leporinus Agassiz, 1829, is well documented (Ramirez et al., 2016, 2017a, 2020; Sidlauskas et al., 2022). Similarly, the DNA barcodes has been essential to show hidden diversity in Anostomidae (Ramirez et al., 2017b, 2020), and analysis combining molecular and morphological data has led to a recognition of cryptic species, as defined by Bickford et al. (2007) (i.e., different species described as a single one due to morphological similarities), or “pseudo-cryptic” species (i.e., species that have been morphologically recognized as such only after other methods have unveiled their existence). Examples of the latter are Leporinus desmotes Fowler, 1914, L. enyae Burns, Chatfield, Birindelli & Sidlauskas, 2017, L. villasboasorum Burns, Chatfield, Birindelli & Sidlauskas, 2017, and L. jatuncochi Ovchynnyk, 1971; the four were previously recognized as a single species (L. desmotes).

Leporinus has the greatest diversity in Anostomidae, with around 80 valid species (Fricke et al., 2023), occurring in all of the main basins of South America (Sidlauskas, Birindelli, 2017). The genus is characterized by its terminal or subterminal mouth and the possession of four teeth on the dentary, three or four teeth on premaxillary, all unicuspid and incisiform (Sidlauskas, Birindelli, 2017). While Leporinus is not monophyletic (Ramirez et al., 2016), some species form monophyletic groups, like the L. fasciatus (Bloch, 1794) clade, composed by L. affinis Günther, 1864, L. altipinnis Borodin, 1929, L. bleheri Géry, 1999, L. desmotes, L. enyae, L. fasciatus, L. jatuncochi, L. pearsoni Fowler, 1940, L. tigrinus Borodin 1929, L. villasboasorum, and L. y-ophorus Eigenmann, 1922. The group is distinguishable from other Leporinus by having dark vertical bars on the body of adult individuals and nine branched pelvic-fin rays (Sidlauskas, Vari, 2008; Burns et al., 2017).

Leporinus bleheri was described in 1999 by Géry based on specimens collected at the border between Bolivia and Brazil, in the Verde River, Guaporé-Iténez basin. In the original description, Géry (1999) considered that the species possessed only three teeth on each dentary and premaxillary bones. Nevertheless, Géry noted the possibility of a fourth dentary tooth, which was difficult to see without damaging the specimen. At the time, the species was not found elsewhere, and because of that, it was considered endemic to the Guaporé-Iténez basin, which is part of the Madeira River basin. Since then, the species was only reported less than a handful of times: for the upper Negro River (Britski, Birindelli, 2016), Jamari (Oliveira et al., 2020), and Eastern Amazon (Dagosta, Pinna, 2019). Two decades later, we sampled the Guaporé River and tributaries close to the border between Brazil and Bolivia and collected additional specimens of L. bleheri. In addition, specimens from the Machado and Aripuanã rivers (also in the Madeira River basin), were remarkably similar to those of L. bleheri, but showed significant morphological and meristic differences. The present contribution aims to redescribe L. bleheri, provide osteological and molecular data for the first time, and investigate the co-specificity of the samples from Machado and Aripuanã rivers. Given the results from the molecular and morphological analyses that we uncovered, we describe the samples from the Machado and Aripuanã rivers as a new species.

MATERIAL AND METHODS

Morphological analyses. Meristic and morphometric data were obtained following Birindelli, Britski (2013). Counts and measurements were taken on the left side of specimens, with a digital caliper with 0.01 accuracy. Measurements related to the body and fins are presented in percentages of standard length (SL), and measurements related to the head are given in percentages of head length (HL). Samples were defined according to their collection data and preliminary analyses of meristic data. Morphometric data was compared using mean, minimum–maximum, and standard deviation values. A Principal Components Analysis (PCA) was performed using logarithmized measures on program PAST v. 4.10 (Hammer et al., 2001), where analyzed as a variance-covariance matrix, and with graphic results showed with convex hulls plotted in PC2 vs. PC3, as PC1 is known to be influenced by body size. A regression analysis was done in R v. 4.2.2 with the sma formula of the package SMATR to compare the size of the snout length with the standard length, all values logarithmized (Warton et al., 2012). All specimens are alcohol-preserved unless indicated. Institutional abbreviations follow Sabaj (2020). One specimen from each sample was cleared and double stained (CS) according to the protocol of Taylor, Van Dyke (1985) and dissected according to the Ridewood protocol (Bemis et al., 2004) for the osteological description. Osteological elements and complexes were photographed using a DFC295 digital camera attached to a Leica M205A stereomicroscope. Osteological terminology followed Weitzman (1962) with modifications used by Sidlauskas, Vari (2008).

Molecular analyses. DNA extraction, amplification, and sequencing. Total DNA was extracted from tissues (fins and muscle) of the two specimens of the new species (MZUEL 21726) and one of L. bleheri (MZUEL 21725), using the Wizard Kit (Promega), following the manufacturer’s instructions. The integrity of the DNA was evaluated by electrophoresis in 8% agarose gel, and the quantification was performed using the Quantus Fluorometer (Promega) device following the manufacturer’s instructions. All samples were diluted to 10ng. For DNA barcodes analyses, a fragment of approximately 650 bp of the Cytochrome c Oxidase subunit I (COI) was amplified by PCR (Polymerase Chain Reaction) using the primers FishF1 and FishR1 (Ward et al., 2005). PCR was performed using GoTaq Green Master Mix (Promega), following the manufacturer’s instructions. The DNA barcodes sequences of the three specimens were compared to species currently recognized in the Leporinus fasciatus group, sensu Burns et al., 2017 (L. affinis, L. altipinnis, L. bleheri, L. desmotes, L. enyae, L. fasciatus, L. jatuncochi, L. pearsoni, L. tigrinus, and L. villasboasorum) obtained from the GenBank. Additionally, we included samples of Leporinus multimaculatus Birindelli, Teixeira & Britski, 2016 and L. octomaculatus Britski & Garavello, 1993, as they share approximately 89% similarity with the new species, and L. friderici. This common and widespread congener was used as a tree root, for which only sequences were included of samples collected in French Guiana and Suriname (the latter is the type-locality of L. friderici). For phylogenetic reconstruction, we sequenced additional partial sequences of Cytochrome B (CytB), using primers AnosCytBF and AnosCytBR (Ramirez, Galetti Jr., 2015), and two nuclear markers (RAG1 and RAG2 - Oliveira et al., 2011). For the 16S and Myh6 markers, only sequences from Genbank were included. We included specimens in the phylogenetics analyses with genetic sequences available for three or more markers. Nuclear markers were amplified according to Oliveira et al. (2011) and sequenced for both strands using an automatic ABI 3500 (Applied Biosystems) sequencer. Sequences were aligned in Mega, v. 11 (Tamura et al., 2021) using the Muscle algorithm (Edgar, 2004) and trimmed their tips. Contigs were assembled and edited using BioEdit (Hall, 1999). GenBank accession numbers are given in Tabs. 1–2.

TABLE 1 |
Accession numbers of COI sequences from GenBank used for the Leporinus species delimitation analysis, and additional information of each voucher specimen.

Species	Drainage, Country	Voucher	COI	Reference
L. affinis	Tocantins, Brazil	L101	KF568975	Ramirez et al. (2017)
L. affinis	Araguaia, Brazil	LBP12699-43695	KY524537	Burns et al. (2017)
L. affinis	Tocantins, Brazil	TO640	OP782390	Nascimento et al. (2023)
L. affinis	Tocantins, Brazil	TO661	OP782391	Nascimento et al. (2023)
L. affinis	Tocantins, Brazil	TO603	OP782392	Nascimento et al. (2023)
L. affinis	Tocantins, Brazil	TO634	OP782393	Nascimento et al. (2023)
L. affinis	Tocantins, Brazil	TO636	OP782394	Nascimento et al. (2023)
L. affinis	Tocantins, Brazil	TO637	OP782395	Nascimento et al. (2023)
L. affinis	Tocantins, Brazil	TO638	OP782396	Nascimento et al. (2023)
L. altipinnis	Aquarium specimen	MZUEL7250	unregistred	previously unpublished
L. bleheri	Guaporé, Brazil	MZUEL21725	unregistred	previously unpublished
L. bleheri	Guaporé, Brazil	MZUSP113988-L079	KF568976	Ramirez et al. (2016); Ramirez et al. (2020)
L. desmotes	Essequibo, Guyana	ANSP179650-2068	KY524534	Melo et al. (2016); Burns et al. (2017)
L. enyae	Orinoco, Venezuela	AUM43700-V5274	KX086798	Burns et al. (2017)
L. enyae	Orinoco, Venezuela	AUM43700-V5274	KY524545	Burns et al. (2017)
L. fasciatus	Madeira, Brazil	UFRO-I9367	KF568981	Ramirez et al. (2016)
L. fasciatus	Nickerie, Suriname	MHNGuncat-157_2	KY524532	Burns et al. (2017)
L. fasciatus	Amazon, Brazil	LBP4248-22701	KY524536	Burns et al. (2017)
L. fasciatus	Nannay, Peru	ANSP180321-NS61	KY524540	Burns et al. (2017)
L. fasciatus	Orinoco, Venezuela	AUM43300	KY524541	Burns et al. (2017)
L. fasciatus	Maroni, French Guiana	GF15-106	MZ050948	Papa et al. (2021)
L. fasciatus	Marowijne, Suriname	SU08-342	MZ050971	Papa et al. (2021)
L. fasciatus	Marouini, French Guiana	GFSU14-178	MZ051056	Papa et al. (2021)
L. fasciatus	Tampok, French Guiana	GF15-219	MZ051126	Papa et al. (2021)
L. fasciatus	Maroni, French Guiana	GF10-007	MZ051274	Papa et al. (2021)
L. fasciatus	Maroni, French Guiana	GF15-105	MZ051400	Papa et al. (2021)
L. fasciatus	Maroni, French Guiana	GF15-328	MZ051452	Papa et al. (2021)
L. fasciatus	Marouini, French Guiana	GFSU14-393	MZ051560	Papa et al. (2021)
L. fasciatus	Marouini, French Guiana	GFSU14-148	MZ051598	Papa et al. (2021)
L. fasciatus	Marouini, French Guiana	GFSU14-372	MZ051768	Papa et al. (2021)
L. friderici	Maroni, French Guiana	GFSU14-084	MZ050815	Papa et al. (2021)
L. friderici	Maroni, French Guiana	PAG-0094	MZ050836	Papa et al. (2021)
L. friderici	Maroni, French Guiana	GF15-280	MZ051019	Papa et al. (2021)
L. friderici	Marowijne, Suriname	SU08-655	MZ051034	Papa et al. (2021)
L. friderici	Maroni, French Guiana	GF15-012	MZ051078	Papa et al. (2021)
L. friderici	Marouini, French Guiana	GFSU14-083	MZ051153	Papa et al. (2021)
L. friderici	Tampok, French Guiana	PAG3639	MZ051168	Papa et al. (2021)
L. friderici	Maroni, French Guiana	PAG-0105	MZ051214	Papa et al. (2021)
L. friderici	Maroni, French Guiana	PAG-0670	MZ051421	Papa et al. (2021)
L. friderici	Maroni, French Guiana	PAG-0671	MZ051423	Papa et al. (2021)
L. friderici	Marouini, French Guiana	GFSU14-008	MZ051433	Papa et al. (2021)
L. friderici	Maroni, French Guiana	PAG-0082	MZ051463	Papa et al. (2021)
L. friderici	Maroni, French Guiana	GF15-269	MZ051557	Papa et al. (2021)
L. friderici	Paloemeu, Suriname	SU08-202	MZ051564	Papa et al. (2021)
L. friderici	Maroni, French Guiana	PAG-0480	MZ051659	Papa et al. (2021)
L. friderici	Paloemeu, Suriname	SU08-656	MZ051752	Papa et al. (2021)
L. friderici	Marouini, French Guiana	GFSU14-007	MZ051774	Papa et al. (2021)
L. friderici	Maroni, French Guiana	GF15-013	MZ051963	Papa et al. (2021)
L. jatuncochi	Upper Amazon, Peru	OS18325-PE10-164	KY524542	Burns et al. (2017)
L. jatuncochi	Upper Amazon, Peru	OS18325-PE10-165	KY524543	Burns et al. (2017)
L. jatuncochi	Upper Amazon, Peru	OS18325-PE10-166	MF285873	Burns et al. (2017); Sidlauskas et al. (2021)
L. multimaculatus	Tocantins, Brazil	MZUEL20500-23	OR497019	Ito et al. (2023)
L. multimaculatus	Tocantins, Brazil	MZUEL20500-24	OR497020	Ito et al. (2023)
L. multimaculatus	Tocantins, Brazil	MZUEL20500-25	OR497021	Ito et al. (2023)
L. multimaculatus	Tocantins, Brazil	MZUEL20500-26	OR497022	Ito et al. (2023)
L. multimaculatus	Tocantins, Brazil	MZUEL20500-27	OR497023	Ito et al. (2023)
L. octomaculatus	Upper Tapajós, Brazil	MZUEL69386	OR497016	Ito et al. (2023)
L. octomaculatus	Upper Tapajós, Brazil	MZUEL7917-38	OR497017	Ito et al. (2023)
L. octomaculatus	Upper Tapajós, Brazil	MZUEL7918-35	OR497018	Ito et al. (2023)
L. octomaculatus	Upper Tapajós, Brazil	MZUSP113992	KF568989	Ramirez et al. (2016)
L. pearsoni	Upper Amazon, Peru	ANSP180841-4124	MF285874	Burns et al. (2017)
L. tigrinus	Xingu, Brasil	LIAM2877	PP506335	Boaretto et al. (2024)
L. tigrinus	Xingu, Brasil	LIAM4639	PP506336	Boaretto et al. (2024)
L. tigrinus	Araguaia, Brazil	MZUEL20673-6	PP506331	Boaretto et al. (2024)
L. tigrinus	Araguaia, Brazil	MZUEL20673-9	PP506332	Boaretto et al. (2024)
L. tigrinus	Araguaia, Brazil	MZUEL20673-60	PP506333	Boaretto et al. (2024)
L. tigrinus	Araguaia, Brazil	MZUEL20673-61	PP506334	Boaretto et al. (2024)
L. tigrinus	Upper Paraná, Brazil	GTCrvE001	MN186394	Boaretto et al. (2024)
L. tigrinus	Upper Paraná, Brazil	GTCrvE002	MN186395	Boaretto et al. (2024)
L. tigrinus	Upper Paraná, Brazil	GTCrvE003	MN186396	Boaretto et al. (2024)
L. tigrinus	Upper Paraná, Brazil	GTCrvE004	MN186397	Boaretto et al. (2024)
L. tigrinus	Upper Paraná, Brazil	GTCrvE005	MN186398	Boaretto et al. (2024)
L. tigrinus	Araguaia, Brazil	GEPEMA5514	KF568997	Ramirez et al. (2016, 2020); Sidlauskas et al. (2021)
L. villasboasorum	Xingu	INPA40521-T8731	KY524538	Burns et al. (2017)
L. villasboasorum	Xingu	ANSP-195954-T10752	KY524539	Burns et al. (2017)
Leporinus sp. n. (described below)	Machado, Brazil	MZUEL21726-A3	unregistred	This work

DNA barcodes analysis. Genetic distances between and within species were calculated in Mega using K2P+G as a model for nucleotide evolution and considering both transversions and transitions, using 1,000 bootstrap pseudoreplicates. Overall mean genetic distance was also calculated using the same parameters. The minimum interspecific distance was used as a threshold to obtain molecular operational taxonomic units (MOTUs) (Blaxter et al., 2005). Two species delimitation analyses were performed: ASAP (Assemble Species by Automatic Partitioning), which uses a pairwise genetic distance matrix to infer species partitions (Puillandre et al., 2020), and PTP (Poisson Tree-Processes), which infer species partition based on a maximum-likelihood based tree (Zhang et al., 2013).

TABLE 2 |
Accession numbers of COI, Cytb, 16S, Myh6, RAG1, and RAG2 sequences from GenBank used for the phylogenetic analysis, and additional information for each voucher specimen.

Species of Leporinus	Drainage	Voucher	16S	COI	Cytb	RAG1	RAG2	Myh6	Reference
L. affinis	Araguaia, Brazil	LBP12699-43695	KY524562	KY524537	KY524551	KY524510	KY524523	KY524496	Burns et al. (2017)
L. affinis	Tocantins, Brazil	L101		KF568975	KF569018	KF569061	KF569104	KF569147	Ramirez et al. (2017)
L. altipinnis	Aquarium specimen	MZUEL7052		unregistred	unregistred	unregistred	unregistred		Previously unpublished
L. altipinnis	Negro, Brazil	LBP24381	MF285868		MF285876	MF285883	MF285887	MF285879	Burns et al. (2017)
L. bleheri	Guaporé, Brazil	MZUSP113988-L079		KF568976	KF569019	KF569062	KF569105	KF569148	Ramirez et al. (2016, 2020)
L. desmotes	Essequibo, Guyana	ANSP179650-2068	KY524572	KY524534	KY524534	KY524507	KY524520	KY524493	Burns et al. (2017)
L. enyae	Orinoco, Venezuela	AUM43700-V5274	KY524570	KY524545	KY524559	KY524518	KY524531	KY524504	Burns et al. (2017)
L. fasciatus	Nickerie, Suriname	MHNGuncat-157-2	KY524490	KY524532	KY524546	KY524505	KY524519	KY524491	Burns et al. (2017)
L. fasciatus	Tapanahony, Suriname	MHNGuncat-157-8	KY524571	KY524533	KY524547			KY524492	Burns et al. (2017)
L. fasciatus	Amazon, Brazil	LBP4248-22701	KY524561	KY524536	KY524550	KY524509	KY524522	KY524495	Burns et al. (2017)
L. altipinnis	Negro, Brazil	LBP4459-24831	HQ171321			HQ289223	HQ289417	HQ289030	Oliveira et al. (2011)
L. fasciatus	Madeira, Brazil	UFROI3343-L496		KF568981	KF569024	KF569067	KF569110	KF569153	Ramirez et al. (2016)
L. fasciatus	Orinoco, Venezuela	AUM43300	KY524566	KY524541	KY524555	KY524514		KY524500	Burns et al. (2017)
L. fasciatus	Upper Amazon, Peru	ANSP180321-NS61	KY524565	KY524540	KY524554	KY524513	KY524526	KY524499	Burns et al. (2017)
L. friderici	Marowijne, Guyana	ANSP189264	KX087039	KX086747	KX086812	KX086958	KX086985	KX086872	Frable et al. (2016); Melo et al. (2016)
L. jatuncochi	Upper Amazon, Peru	OS18323-164	KY524567	KY524542	KY524556	KY524515	KY524528		Burns et al. (2017)
L. jatuncochi	Upper Amazon, Peru	OS18324-165	KY524568	KY524543	KY524557		KY524529		Burns et al. (2017)
L. jatuncochi	Upper Amazon, Peru	OS18325-166	MF285869	MF285873				MF285880	Burns et al. (2017); Sidlauskas et al. (2021)
L. octomaculatus	Tapajós, Brazil	MZUSP113992-L091		KF568989	KF569032	KF569075	KF569118	KF569161	Ramirez et al. (2016)
L. pearsoni	Upper Amazon, Peru	ANSP180841-4124	MF285867	MF285874	MF285877	MF285882	MF285886	MF285878	Burns et al. (2017)
L. tigrinus	Araguaia, Brazil	GEPEMA5514-L176		KF568997	KF569040	KF569083	KF569126	KF569169	Ramirez et al. (2016, 2020); Sidlauskas et al. (2021)
L. villasboasorum	Xingu, Brazil	INPA40521	KY524563	KY524538	KY524552	KY524511	KY524524	KY524497	Burns et al. (2017)
L. villasboasorum	Xingu, Brazil	ANSP195954-T10752	KY524564	KY524539	KY524553	KY524512	KY524525	KY524498	Burns et al. (2017)
Leporinus sp. n. (described below)	Machado, Brazil	MZUEL21726-A3		unregistred	unregistred	unregistred	unregistred		This work

Phylogenetic analysis. A phylogenetic hypothesis was generated through a Bayesian Inference in BEAST, v. 2.7.6 (Bouckaert et al., 2014). Models of nucleotide evolution were estimated independently for each gene via the bModelTest package with an estimated mutation rate, and the model was set to namedExtended using empirical frequencies (Bouckaert, Drummond, 2017). We used a relaxed Log-normal clock modelwhich was linked to mitochondrial genes (COI, Cytb, 16S, Myh6), and also to nuclear markers (RAG1, RAG2), but not to all markers at the same time. We used Birth-Death model as a tree prior and all other parameters were set as default. For calibration, we applied a most recent common ancestor constraint on the node, including all terminal taxa. We based the age of that clade on Sidlauskas et al. (2022) hypothesis that estimated that the most recent common ancestor of Leporinus friderici and L. fasciatus dated 16.99 million years. For that node, we applied a log-normal constraint with an offset of 14.3, M of 1, and Sigma of 0.9, with a median of 17.0. The analysis included two runs of 100.000.000 generations, sampled every 10.000 generations whose results were later combined in LogCombiner v. 2.7.6. Tracer v. 1.7.1 (Rambaut et al., 2018) was used to check if all parameters´ effective sample size (ESS) values exceeded 200. A maximum clade credibility tree was constructed in TreeAnnotator, v. 2.7.6, using a burn-in of 10%.

RESULTS

Molecular analyses. A total of 587 base pairs of 78 samples were recovered, including 177 variable sites and 144 parsimony-informative. The lowest genetic distance between species was 0.69%, obtained by L. desmotes and L. jatuncochi (Tab. 3). All other species distances ranged from 3.93, between Leporinus sp. n. and L. bleheri, to 19.98%, between L. altipinnis and L. jatuncochi. The genetic distance within species ranged from 0.00 to 1.43%, the largest within L. fasciatus. The overall mean genetic distance was 10.11%. The genetic distance from the new species and congeners ranged from 3.93 to 14.77%, the closest congener being L. bleheri. The genetic distance between the latter species and congeners ranged from 3.93 to 12.42. These data showed that, apart from L. jatuncochi and L. desmotes, interspecific distances were much larger than intraspecific variation, and all species (except L. desmotes and L. jatuncochi) represent distinct species, according to the phylogenetic or evolutionary species concept (Groves et al., 2017). Both species delimitation analyses (ASAP and PTP) recovered Leporinus friderici, L. pearsoni, L. multimaculatus, L. altipinnis, L. octomaculatus, and L. tigrinus as distinct and unique MOTUs (Molecular Taxonomic Units) (Fig. 1). On the other hand, Leporinus desmotes and L. jatuncochi were recovered in the same MOTU in both analyses. Leporinus enyae and L. villasboasorum were recovered from each separate MOTU in PTP and ASAP, partition 2. In contrast, all four species (L. desmotes, L. enyae, L. jatuncochi, and L. villasboasorum) were recovered in a single MOTU in ASAP, partition 1. Leporinus affinis was recovered as a separate MOTU in PTP and ASAP, partition 2, whereas it was recovered in the same MOTU as all samples of L. fasciatus in ASAP, partition 1. PTP recovered the samples of L. fasciatus in four MOTUs, whereas the second partition of ASAP recovered them in two MOTUs. Leporinus bleheri and the new species were recovered each as separate MOTUs in PTP and the second ASAP partition. In contrast, the first partition of the latter recovered them in the same MOTU. The ASAP-score of the first and second partitions were respectively 1.5 and 2.5.

TABLE 3 |
Pairwise K2P+G genetic distances among species of Leporinus analyzed based on COI sequences, including Leporinus sp. n. described below. Numbers in bold and diagonal represent the genetic distance within samples; below the diagonal are the mean genetic distances between samples, and above the diagonal are standard error values. N = number of specimens.

	L. sp. n.	L. affinis	L. altipinnis	L. bleheri	L. desmotes	L. enyae	L. fasciatus	L. jatuncochi	L. pearsoni	L. tigrinus	L. villasboasorum	L. octomaculatus	L. multimaculatus	L. friderici
L. sp. n. (n = 2)	0.00	1.92	2.10	1.03	2.42	2.44	1.84	2.39	2.40	1.66	2.10	1.93	1.85	2.33
L. affinis (n = 9)	11.18	0.58	2.27	1.61	2.09	2.02	0.80	2.01	1.96	1.57	2.01	1.82	1.90	1.97
L. altipinnis (n = 1)	12.83	15.99	n/c	1.87	2.71	2.56	2.23	2.72	2.56	1.96	2.43	2.02	2.05	2.37
L. bleheri (n = 2)	3.93	9.28	12.42	0.00	1.96	1.85	1.42	1.83	1.96	1.40	1.62	1.63	1.73	1.87
L. desmotes (n = 1)	15.11	14.50	19.98	12.31	n/c	1.09	2.30	0.37	1.82	2.07	1.26	2.00	1.80	1.95
L. enyae (n = 2)	15.51	13.34	18.58	11.65	5.22	0.00	2.05	0.95	1.73	1.99	1.01	1.85	1.76	2.10
L. fasciatus (n = 16)	10.65	4.08	16.28	8.01	16.77	14.09	1.43	2.15	1.92	1.47	1.87	1.64	1.93	1.81
L. jatuncochi (n = 3)	14.76	13.69	19.98	11.33	0.69	4.41	15.62	0.00	1.70	1.95	1.15	1.89	1.69	1.96
L. pearsoni (n = 1)	14.67	13.10	18.89	12.19	11.68	11.04	13.01	10.69	n/c	1.90	1.69	1.79	1.86	1.64
L. tigrinus (n = 12)	9.37	9.33	13.42	7.68	13.70	12.91	9.24	12.72	11.97	0.43	1.99	1.33	1.63	2.05
L. villasboasorum (n = 2)	13.20	14.07	18.34	10.11	7.32	5.06	13.63	6.47	11.24	13.25	0.17	1.91	2.03	1.71
L. octomaculatus (n = 4)	11.35	11.61	13.53	9.42	13.09	11.73	10.91	12.12	11.14	7.62	12.70	0.65	1.48	1.98
L. multimaculatus (n = 5)	10.93	11.34	12.71	10.09	10.80	10.35	12.39	9.98	10.67	9.59	12.89	8.07	0.00	2.35
L. friderici (n = 18)	14.77	13.14	16.45	11.59	13.14	14.07	12.36	13.14	10.23	13.67	11.36	12.66	14.58	0.05

The new species was recovered as a sister to Leporinus bleheri in our multi-loci B.I. analysis with posterior probability of 1.0 (Fig. 2). The two species were estimated to have diverged approximately at 4.4 mya. That date is similar to the speciation event that originated L. affinis and L. fasciatus and is older than the branching of lineages within any of the studied species, including L. fasciatus, that represents a species complex still in need of further investigation (Burns et al., 2017). The analysis corroborated the species L. affinis, L. fasciatus, L. altipinnis, L. jatuncochi, and L. villasboasorum as monophyletic units. On the other hand, the monophyly of the L. fasciatus species group was not supported, corroborating previous studies (Ramirez et al., 2016; Sidlauskas et al., 2022). Nevertheless, a clade including L. affinis, L. bleheri, L. fasciatus, L. tigrinus, and the new species was supported.

Morphological analyses. There were significant differences in the meristic data when samples were compared (Tab. 3). Samples from Machado and Aripuanã rivers presented fewer lateral line scales (35–37) overall than L. bleheri (36–38, with the majority of examined specimens possessing 38 scales). The former specimens also have fewer scale rows between the dorsal-fin origin and the lateral line (4 vs. 5). The number of scale rows around the caudal peduncle was also different in the samples, with specimens from the Machado and Aripuanã rivers possessing only 12 scale rows, and specimens of L. bleheri having 16.

FIGURE 1 |
Phylogenetic tree of Leporinus sp. n. (described below) and related species generated by maximum-likelihood based on cytochrome oxidase subunit I (COI) sequences. The colors represent the molecular operational taxonomic units found. Vertical bars on the left show the results of delimitation species analyses. Morph = morphology, PTP = Poisson Tree-Processes, ASAP = Assemble Species by Automatic Partitioning.

FIGURE 2 |
Phylogenetic tree of Leporinus sp. n. (described below) and related species generated by Bayesian Inference based on COI, 16S, Cytb, RAG1, RAG2 and Myh6. The colors represent each species; whereas number at node indicate posterior probability, and bars at node indicate the 95% age height.

The PCA analysis showed two clouds of points with little overlap. Principal Components 2 and 3 explained 1.22% and 0.0004% of the variance, respectively (Fig. 3; Tab. 4). The heaviest variable loading on PC2 was eye diameter (0.86573), whereas on PC3 was caudal peduncle length (0.62574).

The regression analysis that investigates the snout size in comparisons to the standard length showed that the snout is relatively larger in the new species in specimens of all sizes (Fig. 4). The p-values bellow the significance level of 0.05 indicate that the correlation between the two variables is significant in both species (Tab. 5).

FIGURE 3 |
Scatter plot of individual scores of Leporinus bleheri and Leporinus sp. n. (described below) on the second and third axes of Principal Component Analysis (PCA).

TABLE 4 |
Variables loadings, eigenvalues, and variance in percentages in the three first axes of Leporinus sp. n. (described below) and L. bleheri PCA; discriminant characters in bold.

	PC 2	PC 3
Eigenvalue	0.00053092	0.000427906
% variance	12.216	0.98457
Standard Length	-0.11099	-0.23765
Predorsal distance	-0.0017331	-0.11031
Dorsal-fin origin to adipose-fin origin	-0.021592	0.0015399
Prepelvic distance	-0.043667	-0.33657
Body depth	-0.21555	0.27888
Caudal peduncle depth	0.060198	-0.15355
Caudal peduncle length	-0.24451	0.62574
Head length	0.0099332	-0.34582
Preopercle length	0.035005	-0.13311
Snout length	-0.12256	-0.17265
Head depth	-0.27167	0.28345
Eye diameter	0.86573	0.27762
Bony interorbital	0.1892	0.02495

FIGURE 4 |
Reduced major axis regression of snout length and standard length on Leporinus bleheri (blue) and Leporinus sp. n. (described below) (red), p-values indicating significant association between variables.

Leporinus lignator, new species

urn:lsid:zoobank.org:act:2BD29D53-528F-47CE-A69E-649E8658675A

(Fig. 5; Tab. 5)

Holotype. MZUEL 21727, 152.96 mm SL, Igarapé Vasquin, a tributary of Comemoração River, Machado River drainage, Madeira River basin, 12°12’18”S 60°37’23”W, Pimenta Bueno, Rondônia, Brazil, 25 Jan 2022, W. M. Ohara.

Paratypes. All from Brazil, Madeira River basin. MCP 41342, 1, 92.0 mm SL, São Domingos River, tributary of Guaporé River, 12°03’18”S 63°59’51”W, São Domingos do Guaporé, Rondônia, 16 Jul 2004, R. E. Reis, F. Langeni, F. C. T. Lima, and E. H. L. Pereira. MZUEL 21726, 5, 91.74–117.01 mm SL; 1 CS, 91.74 mm SL, collected with holotype. NUP 7736, 1, 132.6 mm SL, 10°06’35”S 59°26’12”W, tributary of Guaribal River, Aripuanã River basin, Aripuanã, Mato Grosso, 19 May 2008, I. M. Fernandes. MNRJ 36227, 1, 78.1 mm SL, Aripuanã River, below Cachoeira de Dardanelos, 10°09’43”S 59°26’31”W, Aripuanã, Mato Grosso, 27 Oct 2007, F. Pupo. ZUEC-PIS 10514, 2,65.1–78.1 mm SL mm SL, Aripuanã River, below Cachoeira de Dardenelos, 10°09’43”S 59°26’31”W, Aripuanã, Mato Grosso, 7 Oct 2004, F. A. Machado et al.

TABLE 5 |
Morphometric data of Leporinuslignator and L. bleheri, with mean and ± standard deviation in parentheses. N = number of specimens.

	L. lignator (holotype)	N	L. lignator (paratypes)	N	L. bleheri
Standard length (mm)	152.96	5	91.74–117.01 (101.99±11.90)	10	86.02–123.97 (109.07±10.53)
Percentages in standard length
Predorsal distance	46.02	5	48.14–49.07 (48.61±0.35)	10	45.14–46.98 (46.18±0.57)
Dorsal-fin origin to adipose-fin origin	36.48	5	38.65–41.56 (39.58±1.17)	10	39.68–41.68 (40.44±0.70)
Prepelvic distance	48.62	5	48.58–50.82 (49.84±0.98)	10	47.40–49.92 (48.73±0.82)
Body depth	23.29	5	23.37–26.15 (25.20±1.10)	10	23.31–26.21 (24.53±0.88)
Caudal peduncle depth	8.43	5	9.24–9.69 (9.42±0.19)	10	8.87–9.67 (9.26±0.24)
Caudal peduncle length	12.45	5	12.32–13.18 (12.82±0.33)	10	10.70–12.96 (11.71±0.71)
Head lenght	29.04	5	28.33–29.47 (29.02 - 0.55)	10	25.37–26.77 (26.10±0.47)
Percentages in head length
Preopercle length	73.53	5	74.16–78.31 (75.35±1.68)	10	69.74–79.00 (75.36±2.56)
Snout length	41.29	5	39.62–42.71 (40.92±1.24)	10	39.82–43.36 (41.58±1.09)
Head depth	64.70	5	65.39–69.62 (67.0±1.22)	10	65.52–76.76 (69.83±3.63)
Eye diameter	16.46	5	16.59–19.70 (18.41±1.21)	10	19.89–23.46 (21.20±1.12)
Bony interorbital	30.23	5	29.96–34.78 (31.90±2.39)	10	36.94–40.66 (38.29±1.03)
Meristic data
Dorsal-fin unbranched rays	2	5	2 (5)	10	2 (10)
Dorsal-fin branched rays	10	5	10 (5)	10	10 (10)
Pectoral-fin branched rays	15	5	14 (2), 15 (3)	10	13 (1), 14 (2), 15 (5), 16 (2)
Pelvic-fin branched rays	9	5	9 (5)	10	9 (10)
Anal-fin unbranched rays	2	5	1 (2), 2 (3)	8	2 (8)
Anal-fin branched rays	9	5	9 (5)	8	9 (7), 10 (1)
Upper principal caudal-fin rays	10	5	10 (5)	8	10 (8)
Lower principal caudal-fin rays	9	5	9 (5)	8	9 (8)
Total lateral line scales	37	5	35 (1), 36 (3), 37 (1)	10	36 (1), 37 (1), 38 (8)
Upper transverse scales at dorsal-fin origin	4	5	4 (5)	10	5 (10)
Lower transverse scales at anal-fin origin	4	5	3.5 (1), 4 (4)	10	4 (4), 4.5 (6)
Circumpeduncular scales	12	5	12 (5)	10	16 (10)
Predorsal scales	9	5	9 (3), 10 (2)	10	9 (1), 10 (7), 11 (2)
Premaxilla teeth	3	5	3 (5)	10	3 (10)
Dentary teeth	4	4	4 (4)	7	4 (7)

Diagnosis.Leporinus lignatoris distinguished from all other anostomids except L. affinis, L. altipinnis, L. bleheri, L. desmotes, L. enyae, L. fasciatus, L. jatuncochi, L. pearsoni, L. tigrinus, L. villasboasorum, and L. y-ophorus, by having dark vertical bars encircling the body in adults and nine branched pelvic-fin rays (vs. horizontal bars, dark blotches or dark transversal bars not encircling the body, and eight branched pelvic-fin rays). Leporinus lignator is distinguished from the formers except for L. bleheri and L. tigrinus by having three unicuspid teeth on the premaxillary and four on the dentary (vs. 3/3 or 4/4). Leporinus lignator is distinguished from L. bleheri and L. tigrinus by having 12 scales around the caudal peduncle (vs. 16).

FIGURE 5 |
Leporinus lignator, MZUEL 21727, holotype, 152.96 mm SL (A), MZUEL 21726, paratype, 117.01 mm SL (B), and holotype in life (C), type-locality (D), Machado River, Madeira River basin, Brazil.

Description. Morphometric and meristic values in Tab. 5. Small-sized species for the genus, largest examined specimen 153.0 mm SL. Greatest body depth at dorsal-fin origin. Body rounded, slightly robust. Dorsal profile convex from snout tip to dorsal-fin origin, somewhat concave from dorsal-fin insertion to adipose-fin origin, then distinctly concave to caudal fin. Ventral profile convex from lower jaw to anal-fin origin, concave from anal fin to caudal fin. Three teeth on premaxillary and four on dentary, stair-like, unicuspid, and incisiform. Lateral line complete from supracleithrum to caudal-fin base. Dorsal-fin origin slightly anterior to middle of body. Adipose fin small, rounded, origin slightly behind anal-fin origin. Pectoral-fin origin at opercle border. Pelvic-fin origin slightly anterior to body middle. Anal-fin origin ahead of adipose-fin origin. Caudal fin forked, dorsal lobe a bit longer.

Osteology. Infraorbital series composed of six infraorbitals, nasal, antorbital, and supraorbital, all plate-like (Fig. 6A). Antorbital without canal, as long as first infraorbital, C-shaped and inclined relative to horizontal body axis, with small plate-like lamina. Nasal large, with a canal and two intermediate pores. Supraorbital located posteriorly to nasal and much smaller than latter, with no canals. First infraorbital somewhat triangular, immediately ventral to antorbital, with a small quadripartite canal located on anteriormost portion of plate-like bone. Second infraorbital triangular, with canal extended on middle of bone and bearing two intermediate pores. Third infraorbital largest, axe-shaped, with canal bearing an intermediate pore close to anteriormost opening. Fourth infraorbital somewhat rectangular, with canal bifurcate at dorsal portion and horizontal branch extended to posterior edge of bone. Fifth infraorbital with straight canal bearing a tiny branch at midlength. Sixth infraorbital smallest, bean-shaped, bearing Y-shaped canal.

FIGURE 6 |
A. Infraorbital bones of Leporinus lignator, MZUEL 21726, paratype, 91.74 mm SL and B.Leporinus bleheri, MZUEL 21725, 86.02 mm SL. C. and D. Premaxilla, maxilla, dentary, retroarticular, and anguloarticular of L. lignator and E. and F. of L. bleheri. G. Suspensorium of L. lignator and H. of L. bleheri.

Premaxillary bone trapezoidal with rounded borders, bearing three unicuspid incisiform teeth, decreasing gradually in size laterally (Figs. 6C–D). Maxillary bone located on posterolateral edge of premaxillary, positioned vertically relative to body axis, with acute dorsal end and enlarged ventral portion. Lower jaw trapezoidal, larger, and more rounded than premaxillary. Dentary with four unicuspid incisiform teeth, decreasing gradually in size laterally. Fourth dentary tooth considerably smaller than anteriormost ones. Retroarticular tiny; anguloarticular with dorsal portion slightly expanded and curved anteriorly, but not dorsally surpassing dorsal limit of dentary.

Suspensorium L-shaped, laterally compressed and with horizontal portion longer than vertical one (Fig. 6G). Autopalatine bearing a large and rounded anterodorsal process, ectopterygoid vertically aligned and constricted near its dorsal edge. Entopterygoid small, associated with quadrate, metapterygoid, ectopterygoid and autopalatine. Metapterygoid large, dorsal to quadrate, forming an irregular fenestra with quadrate. Metapterygoid-quadrate fenestra shaped as a fallen eight, with a median constriction. Quadrate large and triangular. Symplectic elongate and thin. Hyomandibular large, bearing a pointy process on ventral-anterior margin anteriorly-directed towards quadrate. Preopercle large and L-shaped, with a horizontal ventrolateral shelf. Two small canal-like bones between preopercle and lower jaw. Interopercle and subopercle small. Opercle large, posterior margin rounded, resembling a semicircle.

Mesethmoid triangular, anteriorly forming attachment site for premaxillae (Figs. 7A–C). Anterior and posterior cranial fontanel large and wide, encased by mesethmoid, frontals, parietals, and supraoccipital. Frontal large, rectangular-shaped. Lateral ethmoid long with a distinct ventrally-directed triangular process. Parietal wide, much shorter than frontal. Sphenotic and pterotic sightly triangular and forming ventrolateral acute projections of the neurocranium. Supraoccipital C-shaped anteriorly and with small triangular process on posterior border. Epiotic bears three distinct bony bridges at posterior border of neurocranium, exoccipital posterior to it. Vomer triangular in ventral view, rounded borders. Parasphenoid long and thin, extended from vomer to basioccipital and bearing two posteriorly elongated horizontal processes ventral to basioccipital. Orbitosphenoid, pterosphenoid, and prootic dorsal to parasphenoid, ventral to frontal and parietal, forming the ventral wall of the braincase. Basioccipital thin and rounded, posterior to prootic and ventral to exoccipital.

Hyoid arch composed of dorsal and ventral hypohyals, anterior and posterior ceratohyals, urohyal, interhyal, and four branchiostegals (Fig. 8A). Branchiostegals long and thin, spatula-shaped, three articulated to posterior ceratohyal, and last articulated with anterior ceratohyal. Branchial apparatus composed of basihyal, three basibranchials, three hypobranchials, five ceratobranchials, four epibranchials, and four pharyngobranchials (Figs. 8C–D). Basihyal long and thin, with a toothplate on anterior margin and posteriorly connected to the first basibranchial. Basibranchials rod-like, connected by cartilage to hypobranchials, which are square-shaped with rounded borders, also connected to ceratobranchials by cartilage. Four ceratobranchials long, rod-like, with small rakers on anterior and posterior margins, and the fifth ceratobranchial with rakers only on anterior margin, bearing a tooth plate on lenticular posterior expansion with two irregular rows of approximately ten acicular teeth. Four epibranchials, with the fourth bearing a tooth plate with two irregular rows of around eight acicular teeth, connected by cartilage to ceratobranchials and pharyngobranchials.

FIGURE 7 |
Cranium bones of Leporinus lignator, MZUEL 21726, paratype, 91.74 mm SL in A. dorsal, B. lateral, and C. ventral view, and of L. bleheri, MZUEL 21725, 86.02 mm SL in D. dorsal, E. lateral and F. ventral view.

Dorsal fin with two unbranched and ten branched rays (Fig. 9A), first pterygiophore inserted between neural spines of 9 and 10 vertebral centra. Pelvic bone supporting a pelvic splint, one unbranched ray, and nine branched rays; ischiatic process elongated (Fig. 9C).

Anal fin with two unbranched and nine branched rays (Fig. 10A), first pterygiophore inserted between haemal spines of 25 and 26 vertebral centra. Pectoral fin connected to neurocranium through extrascapular and posttemporal, with one unbranched plus thirteen to fifteen branched rays (Fig. 10C). Cleithrum large and somewhat triangular, with a small bump on anterior margin. Coracoid rectangular with triangular posterior process. Scapula large and somewhat triangular. Three postcleithra present, first rounded, posterior to cleithrum, second triangular, posterior to cleithrum and scapula, third largest with ventrally-directed thin process.

Caudal skeleton comprises a compound center, pleurostyle, three epurals, two uroneurals, opisthural cartilage, parhypural, and six hypurals (Fig. 11A). Parhypural, first and second hypurals on lower lobe. Opisthural cartilage, uroneurals, epurals, pleurostyle, and four hypurals on upper lobe.

FIGURE 8 |
A. Hyoid arc of Leporinus lignator, MZUEL 21726, paratype, 91.74 mm SL and B.L. bleheri, MZUEL 21725, 86.02 mm SL. C. and D. Branchial apparatus of L. lignator and E. and F. of L. bleheri.

Coloration in alcohol. Body with a light tan background and dark brown vertical bars. Two bars on head, nine bars on trunk. First bar at snout, second along eyes, third at opercle, fourth and fifth before dorsal-fin origin, connected only in their middle portion, forming an “X”. Sixth bar at dorsal fin and seventh bar posterior to dorsal fin, also connected only at their middle portion forming an “X”. Eighth bar between dorsal fin and adipose fin, bifurcated dorsally, “V” shaped. Ninth bar at adipose fin and tenth bar between adipose and caudal fins, connected only at their middle portion, forming an “X”. Eleventh bar on caudal peduncle separated from ninth bar. Dorsal and adipose fins dark brown at base, adipose fin with a dark strip at tip, yellow in middle. Pelvic and anal fins with a dark brown stripe in middle, yellow at base. Caudal fin slightly yellow at base (Figs. 5A–B).

FIGURE 9 |
A. Dorsal fin of Leporinus lignator, MZUEL 21726, paratype, 91.74 mm SL and B.L. bleheri, MZUEL 21725, 86.02 mm SL. C. Pelvic fin of L. lignator and D. of L. bleheri.

Coloration in life. Live specimens yellowish, slightly darker dorsally than ventrally. Dark black vertical bars conspicuous. Dorsal fin dark at base. Adipose fin black at base and tip, yellow in-between. Pelvic and anal fin with a black strip in the middle, yellow at the base. Caudal fin slightly yellow at the base (Fig. 5C).

Geographical distribution.Leporinus lignator is only known from the tributaries of the Madeira drainage, including the Guaporé, the Aripuanã, and the Machado basins in the Amazon basin (Fig. 12).

Etymology. The specific epithet, lignator, is allusive to its type-locality, the Machado River, part of the Madeira River basin. In Portuguese, Machado means axe, and Madeira means wood. Lignator is Latin (m.) for a lumberjack who cuts trees into logs, often using axes. A noun in apposition.

FIGURE 10 |
A. Anal fin of Leporinus lignator, MZUEL 21726, paratype, 91.74 mm SL and B.L. bleheri, MZUEL 21725, 86.02 mm SL. C. Pectoral fin of L. lignator and D. of L. bleheri.

FIGURE 11 |
A. Caudal fin of Leporinus lignator, MZUEL 21726, paratype, 91.74 mm SL and B.L. bleheri, MZUEL 21725, 86.02 mm SL.

Ecological notes. The type-locality of Leporinus lignator is a clear water stream, 3–5 m wide, 0.3–1.8 m deep, with rocks, pebbles, sand bottoms, and riparian vegetation, located at 300 m altitude (Fig. 5). Sintopic species included Ancistrus verecundus Fisch-Muller, Cardoso, da Silva & Bertaco, 2005, Cichlasoma boliviense Kullander, 1983, Moenkhausia mikia Marinho & Langeani, 2010, Moenkhausia cf. pankilopteryx Bertaco & Lucinda, 2006.

Conservation status.Leporinus lignatoris known only from a few specimens collected in a few sites, all of which are located in areas relatively well-preserved and close to several preservation areas, including the Parque do Aripuanã, the Parque Estudual de Corumbiara, and many indigenous regions. Therefore, although the species distribution is poorly known, we suggest that the conservation status of Leporinus lignator is likely to be Least Concern (LC) at this moment, according to IUCN criteria (IUCN, 2022).

FIGURE 12 |
Partial map of South America (as indicated by the red square in the upper-right corner) showing the distribution where Leporinus lignator (red, star = holotype) has been recorded, and L. bleheri (yellow, star = holotype) based on material examined in the present study.

Leporinus bleheriGéry, 1999

urn:lsid:zoobank.org:act:2C5CC7DD-78E5-49A1-9417-7D919BCC5A7D

(Fig. 13; Tab. 5)

Leporinus bleheri Géry, 1999:108–12 (holotype MHNG 2599.60, 142 mm SL; type-locality: Verde River, Iténez-Guaporé basin, Bolivia at the border with Brazil, 14°8’S 60°30’W, 11 Set 1996, H. Bleher). —Jégu et al., 2012:119 (checklist, Verde River). —Sarmiento et al., 2014:38, 183 (checklist, Verde River). —Ramirez, 2015:14, 25, 27, 31, 57, 75-76, 105–106 (comparative molecular material, Guaporé River). —Britski, Birindelli 2016:26–27, 37 (comparative material, Verde and Tiquié rivers). —Ramirez et al., 2016:3, 5 (fig. 1–d), 8 (comparative molecular material, Guaporé River). —Ramirez et al., 2017:3 (comparative molecular material, Guaporé River). —Burns et al., 2017:1, 11, 15, 19 (comparative material, Verde River). —Dagosta, Pinna, 2019:69 (new reports, Guaporé, Negro and Amazonas Estuary). —Oliveira et al., 2020:3, 5 (checklist, Jamari and Madeira rivers). —Ramirez et al., 2020:7 (comparative molecular material, Guaporé River).

Leporinus sp.1. —Lima et al., 2005:145 (checklist and brief description, Tiquié River).

Diagnosis.Leporinus bleheri is distinguished from all other anostomids except L. affinis, L. altipinnis, L. desmotes, L. enyae, L. fasciatus, L. jatuncochi, L. personi, L. tigrinus, L. villasboasorum, L. y-ophorus, and L. lignator by having dark vertical bars encircling the body in adults and nine branched pelvic-fin rays (vs. horizontal bars, dark spots or dark transversal bars not encircling the body, and eight branched pelvic-fin rays). Leporinus bleheri is distinguished from the formers except for L. tigrinus and L. lignator by having three teeth on the premaxillary and four on the dentary (vs. 3/3 or 4/4). Leporinus bleheri is distinguished from L. tigrinus by having nine dark transversal bars on the trunk (vs. eight) and from L. lignator by having 16 circumpeduncular scales (vs. 12).

Description. Morphometric and meristic values in Tab. 5. Small-sized species for the genus, largest examined specimen 123.97 mm SL. Greatest body depth at dorsal-fin origin. Body rounded, slightly robust. Dorsal profile convex from snout tip to dorsal-fin origin, somewhat concave from end of dorsal fin to adipose-fin origin, then distinctly concave to caudal fin. Ventral profile convex from lower jaw to anal-fin origin, concave from anal fin to caudal fin. Three teeth on the premaxillary and dentary, stair-like, unicuspid, and incisiform. Lateral line complete from supracleithrum to base of caudal fin. Dorsal-fin origin a bit anterior to body middle. Adipose fin small, rounded, origin slightly behind anal-fin origin. Pectoral-fin origin at the border of opercle. Pelvic-fin origin a bit behind dorsal-fin origin. Anal-fin origin slightly ahead of adipose-fin origin. Caudal fin forked, dorsal lobe slightly longer than ventral one.

FIGURE 13 |
Leporinus bleheri, MZUEL 21725, 123.97 mm SL, Capivari River, Madeira river basin, Brazil; specimen photographed preserved in alcohol (above), and living (below).

Osteology. It is the same as Leporinus lignator (Figs. 6-7; 9-12) with the following exceptions. Canal on infraorbital one robust and curved. Third infraorbital less wide, more rectangular (Fig. 6B). On suspensorium, smaller anterodorsal process on autopalatine. Metapterygoid-quadrate fenestra slightly bigger (Fig. 6H). On branchial apparatus, tooth plate of fifth ceratobranchial with two irregular rows of approximately twelve acicular teeth (Fig. 8E). Tooth plate of fourth epibranchial with two irregular rows of around six acicular teeth (Fig. 8F).

Coloration in alcohol. Coloration as in L. lignator.

Coloration in life. Live specimens yellowish, darker dorsally, and lighter ventrally. Vertical bars conspicuously black. Fins colors similar to those of preserved specimens (Fig. 13).

Geographical distribution.Leporinus bleheri is distributed in different tributaries of the Amazonas rivers, including the Madeira, Negro, and Purus rivers, in Brazil and Bolivia (Fig. 12).

Conservation status. Most of the distribution area of L. bleheri is relatively well-preserved and includes several areas of protection, such as indigenous land and national or state parks. The conservation status of Leporinus bleheri is likely a Least Concern (LC) according to IUCN criteria (IUCN, 2022).

Material examined. Bolivia: MNHN 2001-0495, 1 paratype, ca. 14°08’S 60°29’W, 11 Sep 1996, Bleher. Brazil: CPUFMT 2820, 1, 122.7 mm SL, Córrego Corgão, tributary of Galera River (Guaporé basin), 14°27’25”S 59°30’30”W, Nova Lacerda, Mato Grosso, 4 Nov 2011, G. Figueiredo.MPEG 10790, 2, 104.5–135.3 mm SL, Urucu River, tributary of Solimões River, 04°52’4.7”S 65°07’25”W, Coari, Amazonas, 16 Aug 2006, W. B. Wosiacki. MPEG 10791, 4, 128.1–154.3 mm SL, Urucu River, tributary of Solimões River, 04°52’04.7”S 65°07’25”W, Coari, Amazonas, 21 Aug 2006, W. B. Wosiacki. MPEG 16764, 2, 169.0–180.6 mm SL, Urucu River, tributary of Solimões River, 04°52’04.7”S 65°07’25”W, Coari, Amazonas, 5 Aug 2009, B. S. Prudente. MZUEL 21725, 10, 123.97–86.02 mm SL; 1 CS, 86.02 mm SL, Capivari River, Madeira River basin, 14°42’45.41”S 60°14’59.77”W, Vila Bela da Santíssima, Mato Grosso, 17 Feb 2020, W. M. Ohara. MZUSP 66676, 1, 130.0 mm SL, Tiquié River, Negro River basin, 00°16’27”N 69°54’56”W, Santa Izabel do Rio Negro, Amazonas, Mai-Jun 2000, Tukano indians. MZUSP 85374, 1, 168.3 mm SL, Tiquié River, Negro River basin, 00°15’22”N 69°50’24”W, Santa Izabel do Rio Negro, Amazonas, 2004, Tarcísio. MZUSP 93445, 1, 168.0 mm SL, Tiquié River, Negro River basin, ca. 00°10’N 69°07’W, Santa Izabel do Rio Negro, Amazonas, Nov 2006, F. C. T. Lima. NUP 19446, 3, 131.7–46.4 mm SL, Jamari River, 10°05’01.0”S 63°08’52.0”W, Monte Negro, Rondônia, 1 Jun 2016, J. R. Gonçalves. NUP 23852, 1, 93.7 mm SL, Jamari River, 10°20’10”S 63°15’12”W, Monte Negro, Rondônia, 15 Jul 2022, J. R. Gonçalves.

DISCUSSION

Both morphological and molecular data support the hypothesis that Leporinus lignator is a distinct species close to L. bleheri. The genetic distance between both species is 3.9%, and between any of the two and their closest congener (L. tigrinus) is 7.68%. On the other hand, the genetic distance within the studied species ranges from 0.0 to 1.43%. These results are similar to those observed for other species of Anostomidae, including other genera such as Hypomasticus (Birindelli et al., 2020b); Megaleporinus (Ramirez et al., 2017a; Birindelli et al., 2020a), Laemolyta (Ramirez, Galetti Jr., 2015) and Schizodon (Ramirez et al., 2020; Garavello et al., 2021). The delimitation analyses also support the distinctiveness of both species, at least in most analyses. The PTP and the second partition of ASAP analyses recovered both species as separate MOTUs. However, the first partition of ASAP (the one with the lowest ASAP-score) considered that both species likely belong to the same MOTU. According to Puillandre et al. (2020), ASAP users should consider the partition with the best score and the few subsequent ones. In our case, the second partition was similar to the species delimited by the morphological-based identification of specimens and identical to the results obtained via PTP analysis.

The phylogenetic analysis recovered a monophyletic group composed of Leporinus fasciatus, L. affinis, L. bleheri, L. lignator, and L. tigrinus. This clade was previously recovered in part by Ramirez et al. (2015, for L. bleheri, L. fasciatus, L. tigrinus; Ramirez et al., 2017a, for L. affinis, L. bleheri, L. fasciatus, L. tigrinus), and Sidlauskas et al. (2022, L. fasciatus, L. tigrinus). On the other hand, in these studies, other similar-looking congeners, including L. altipinnis, L. desmotes, L. enyae, L. jatuncochii, L. pearsoni, and L. villasboasorum, were not recovered as closely related to the L. fasciatus species group. That result is surprising given that all these species have dark vertical bars encircling the body and nine branched rays in the pelvic fin, two exclusive features among Anostomidae (Sidlauskas, Vari, 2008). Nevertheless, the non-monophyly of the banded Leporinus was already recovered by Ramirez et al. (2015, 2017a) and Sidlauskas et al. (2022). Leporinus fasciatus and closely related species (i.e., L. affinis, L. bleheri, L. lignator, and L. tigrinus) seem to be more closely related to L. octomaculatus (and other closely related species; see Ito et al., 2023), than to other vertically banded congeners such as, for example, L. desmotes.

The distribution of Leporinus bleheri and L. lignator is also interesting. Both species occur in tributaries of the Madeira River, with L. bleheri being more widespread and occurring in other tributaries of the Amazon basin. However, neither species is apparently sympatric. This interesting fact could be related to an ecological preference of one or both species, which could be tested once more data is available. The speciation event that led to L. bleheri and L. lignator was estimated to have happened approximately four million years ago (Fig. 2), a time in which both species may have expanded their original distribution, forming what today seems a common distribution area.

The number of teeth is an essential characteristic of the genus (Britski, Birindelli, 2008). Previously, all species of the L. fasciatus group presented either three teeth on the premaxilla and dentary (L. desmotes, L. enyae, L. jatuncochi, and L. villasboasorum) or four in both (L. affinis, L. altipinnis, L. fasciatus, L. pearsoni and L. y-ophorus). The only exceptions were L. bleheri and L. tigrinus, which have three teeth on the premaxilla and four on the dentary, just like L. lignator. In Anostomidae, the number of scale rows around the caudal peduncle is also a significant feature, with many species presenting 16 scale rows, including the ones of Leporinus. Only four species of Anostomidae show 14 scale rows: Abramites eques (Steindachner, 1878), A. hypselonotus (Günther, 1868), L. jatuncochi, and some specimens of L. desmotes and L. taeniatus Lütken, 1875, where it varies from 14 to 16 rows, a rare case of polymorphism (Birindelli et al., 2016). The presence of 12 scale rows, as seen in L. lignator, is the only case in the L. fasciatus group, although present in many other species of Leporinus, Megaleporinus Ramirez, Birindelli & Galetti, 2017 and Hypomasticus Borodin, 1929 (Birindelli et al., 2016). The number of scale rows is not indicative of phylogenetic proximity, considering the lack of pattern in the family; however, it is helpful for species differentiation.

Leporinus bleheri and L. lignator share the coloration pattern composed of nine bars on the trunk, with second and third, and fourth and fifth bars connected forming an “X”, and the sixth bifurcated dorsally and “V” shaped. Trunk dark bars differ slightly in L. desmotes, L. enyae, L. jatuncochi, and L. villasboasorum, in which only the second and third bars are connected and “X” shaped. Furthermore, these species have dark dorsal and pelvic fins base. Leporinus tigrinus and L. pearsoni are also similar, having most bars shaped as “Y”. Leporinus fasciatus, L. affinis, and L. altipinnis have eight to fourteen dark bars, all shaped as an “I”, with dorsal and ventral ends slightly bifurcated in some specimens, pending on specimen development. Leporinus y-ophorus also has a unique color pattern, with seven trunk bars, and the third is “Y” or “V” shaped. Géry (1999) described L. bleheri and other species of the group using many letters such as “J”, “D” and “H”. Here, we are maintaining only the letters “I”, “Y”, “X” and “V” to describe the group. Following that nomenclature, the new species and L. bleheri can be described as “I-X-X-V-X-I”.

The osteology of both species herein studied are very similar. The infraorbital series has minor differences regarding the size of the antorbital (thinner in L. lignator), first infraorbital (larger in L. bleheri), and third infraorbital withdistinct axe shape in L. lignator (verified on both sides of the single cleared and stained specimen examined). As in most anostomids, the fourth and fifth infraorbitals are not fused (see the condition in L. fasciatus in Sidlauskas, Vari, 2008: fig. 9). The two species share with L. tigrinus the combination of three teeth on the premaxilla and four on the dentary. In the original description, Géry (1999) described L. bleheri as having only three teeth on the dentary but left open the possibility of a small fourth tooth that he couldn’t see without damaging the specimen, which has been confirmed. Leporinus lignator also presents four teeth on the dentary, with the last one being considerably complicated to see but still present in all specimens analyzed. On the suspensorium, the metapterygoid-quadrate window is slightly smaller on L. lignator.

This is another contribution that shows that the diversity of Neotropical fishes is yet underestimated, especially in the Amazon, where there are still large unexplored areas. The discovered of the new species, and also, the new hypotheses on its phylogenetic relationship and evolution was strengthen by applying distinct methods and using molecular and morphological data.

Comparative material.Leporinus affinis. MZUEL 20672, 9 alc, not measured, 13º16’35”S 50º36’28”W, Araguaia River in Luiz Alves, São Miguel do Araguaia, Goiás, Brazil, 15 Oct 2019, J. Birindelli, N. Narezzi, E. Santana & A. Souza. Leporinus altipinnis. MZUEL 20164, 2 alc, not measured, unknow locality, 9 Feb 2021, J. Birindelli. Leporinus desmotes. MZUSP 40734, 2 alc, 119.0–127.6 mm SL, 13º27’S 47º04’59”W, Paranã River, tributary of Tocantins River, Monte Alegre de Goiás, Goiás, Brazil. 1988, J. C. Oliveira & W. J. M. Costa. Leporinus enyae. MZUEL17001, 1 alc, not measured, 04º13’07”N 66º25’26”W, Ventuari River, Amazonas basin, 205 km southeast of Puerto Ayacucho, Venezuela, 15 Apr 2010, J. Birindelli, N. Lujan & V. Meza. Leporinus fasciatus. MZUEL 14698, 9 alc, not measured, 02º43’02”S 60º44’42”W, Negro River in Lago do Prato, Anavilhanas National Park, Novo Airao, Amazonas, Brazil, 3 May 2016, J. Birindelli et al. Leporinus jatuncochi. MZUEL 10207, 13 alc, not measured, 00º53’16”S 59º34’28”W, Pitinga River, Uatumã River basin, Presidente Figueiredo, Amazonas, Brazil, 19 Sep 2014, J. Birindelli et al. Leporinus pearsoni.FMNH 102145, 2 alc, 150.0–184.0 mm SL, 01°05’24”S 77°34’18”W. Arajuno River, Napo, Ecuador. Leporinus tigrinus. MZUEL 20673, 17 alc, not measured, 13º16’35”S 50º36’28”W, Araguaia River in Luiz Alves, São Miguel do Araguaia, Goiás, Brazil, 15 Oct 2019, J. Birindelli, N. Narezzi, E. Santana & A. Souza. Leporinus villasboasorum. MZUEL 17000, 5 alc, not measured, 13º51’01”S 53º15’33”W, Culuene River, Xingu River basin, Campinapolis, Mato Grosso, Brazil, 21 Aug 2006, J. Birindelli, L. Sousa & A. Akama. Leporinus y-ophorus. MZUEL 20449, 1 alc, not measured, ca. 03º56’N 73º03’W, Metica River, 1.5 km east of Rajote (Plancha 267 Departamento del Meta, Colômbia, 14 Mar 1973, J. E. Bohlke, W. G. Saul & W. F. Smith-Vaniz.

ACKNOWLEDGEMENTS

We thank Oscar Akio Shibatta, Jorge Ramirez, Bruno Melo, and Brian Sidlauskas for their corrections and suggestions on the manuscript. Thanks are due to Karsten Hartel and Andrew Williston (MCZ), Alexandre Ribeiro and Katiane Ferreira (CPUFMT), Wolmar Wosiacki and Alberto Akama (MPEG), Carla Pavanelli, and Wefferson Graça (NUP), Patrice Provost (MNHN), Roberto Reis and Carlos Lucena (MCP), Marcelo Britto, Paulo Buckup and Cristiano Moreira (MNRJ), Flávio Lima (ZUEC-PIS), and Mario de Pinna, Aléssio Datovo, Osvaldo Oyakawa, and Michel Gianetti (MZUSP) for allowing the study of specimens under their care. JLOB received a research grant from CNPq (process 308846/2023–0) and Taxonline (Fundação Arauacária process 2020061000012); MPB received grants from CAPES (MSc, through Programa de Pós-Graduação em Ciências Biológicas da UEL).

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ADDITIONAL NOTES

• Ethical Statement
  Not applicable.
• HOW TO CITE THIS ARTICLE
  Boaretto MP, Ohara WM, Souza-Shibatta L, Birindelli JLO. New banded Leporinus (Characiformes: Anostomidae) from the Madeira River basin, Brazil, and redescription of L. bleheri, based on integrative taxonomy. Neotrop Ichthyol. 2024; 22(4):e240028. https://doi.org/10.1590/1982-0224-2024-0028

• Edited-by
  Bruno Melo

Publication Dates

• Publication in this collection
  10 Jan 2025
• Date of issue
  2024

History

• Received
  21 Oct 2024
• Accepted
  04 Nov 2024