Two new miniature species of the fish genus Priocharax from the Rio Tapajós and Amazonas drainages, Pará, Brazil (Teleostei: Characiformes: Characidae)

George M.T. Mattox; Flávio C. T. Lima; Ralf Britz; Camila S. Souza; Claudio Oliveira

doi:10.3897/vz.74.e130038

Research Article

Two new miniature species of the fish genus Priocharax from the Rio Tapajós and Amazonas drainages, Pará, Brazil (Teleostei: Characiformes: Characidae)

George M.T. Mattox^‡, Flávio C. T. Lima^§, Ralf Britz^|, Camila S. Souza^¶, Claudio Oliveira^¶

‡ Universidade Federal de São Carlos, Sorocaba, Brazil

§ Universidade Estadual de Campinas, Campinas, Brazil

| Museum of Zoology, Dresden, Germany

¶ Universidade Estadual Paulista, Botucatu, Brazil

Corresponding author: George M.T. Mattox ( gmattox@ufscar.br )

Academic editor: Uwe Fritz

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Mattox GMT, Lima FCT, Britz R, Souza CS, Oliveira C (2024) Two new miniature species of the fish genus Priocharax from the Rio Tapajós and Amazonas drainages, Pará, Brazil (Teleostei: Characiformes: Characidae). Vertebrate Zoology 74: 533-550. https://doi.org/10.3897/vz.74.e130038

ZooBank: urn:lsid:zoobank.org:pub:5868E801-154F-4D94-A4F3-BECAD03033C8

Abstract

The miniature fish genus Priocharax currently comprises seven valid species: P. ariel, P. britzi, P. marupiara, P. nanus, P. pygmaeus, P. toledopizae and P. varii. Except for P. ariel and P. pygmaeus, all the species are endemic to Brazil. Priocharax is characterized by several paedomorphic features such as reductions in the laterosensory system, number of fin rays, and ossification of parts of the skull. The most striking reductive character of Priocharax is the larval rayless pectoral fin in which most of its ossified endoskeletal elements are absent. We describe herein two new species of Priocharax from the vicinity of Santarém municipality, Pará state, Brazil. Both new species are distinguished from each other and from congeners by a combination of morphological features (i.e., osteological, morphometric, and meristic data) and molecular information (i.e., DNA barcode). We also present an updated maximum likelihood tree which now includes all nine species of Priocharax.

Key words

Anatomy, DNA barcode, Neotropics, integrative taxonomy, miniature tetra, systematics

Introduction

Priocharax Weitzman & Vari, 1987 is a genus of miniature fishes (sensu Weitzman and Vari 1988) from the Amazon and Orinoco basins that until recently included only two species: P. ariel Weitzman & Vari, 1987 and P. pygmaeus Weitzman & Vari, 1987, both described from the upper Ríos Orinoco and Negro in Venezuela, and from the Río Amazonas in Letícia, Colombia, respectively (Weitzman and Vari 1987). The diversity of this group was clearly underestimated, as five other species were discovered in the last ten years: Priocharax nanus Toledo-Piza et al., 2014 from the Rio Negro, P. varii Mattox et al., 2020 from the Rio Madeira system, P. britzi Mattox et al., 2021 from the Rio Purus system, and P. marupiara Mattox et al., 2023 and P. toledopizae Mattox et al., 2023, both from the Juruá system in the state of Acre. These five species were all described from Brazil and are currently known from relatively restricted areas within their respective river systems.

All species of Priocharax are characterized by a series of morphological developmental truncations (e.g., Mattox et al. 2016) and the genus can be diagnosed by the following combination of characters: presence of a larval pectoral fin in adults (i.e., a rayless pectoral-fin bud with a cartilaginous pectoral radial plate and predominantly cartilaginous endoskeletal pectoral girdle skeleton), a fully toothed maxilla, a single series of conical teeth in the premaxilla, maxilla and dentary, a tiny size, translucid body in life, and 5–6 branched pelvic-fin rays (Weitzman and Vari 1987; Mattox et al. 2016, 2023). Examination of material recently collected from the surroundings of Santarém, Pará State, Brazil in the Rio Tapajós system and on islands of the Rio Amazonas at the confluence of the Rio Tapajós led to the discovery of yet two additional species, new to science, and described herein.

Methods

Morphological analysis

Counts and measurements follow Fink and Weitzman (1974), Weitzman and Vari (1987), and Menezes and Weitzman (1990) and were taken on the left side of each specimen whenever possible. All measurements other than standard length (SL) are expressed as percentages of SL, except for subunits of the head which are expressed as percentages of head length (HL). Caudal-peduncle depth is also expressed as a percentage of caudal-peduncle length (Weitzman and Vari 1987) and snout length as a percentage of orbital diameter (e.g., Mattox et al. 2021). Measurements were taken point to point with a precision of 0.1 mm from digital photographs of specimens taken under a Zeiss Discovery V20 stereomicroscope. In text and tables, SD is used for standard deviation. Counts of vertebrae, supraneurals, teeth, gill-rakers, procurrent caudal-fin rays, and information about osteological characters were obtained from specimens cleared and double stained for cartilage and bone following the protocol of Taylor and Van Dyke (1985). Total number of vertebrae includes the four vertebrae of the Weberian apparatus counted as separate elements and all post Weberian vertebrae including the compound ural centrum, which was counted as a single vertebra. The gill raker at the junction of ceratobranchials and epibranchials is considered as the posteriormost gill raker on the lower limb of the respective gill arch. Information on meristic and morphometric data of Priocharax ariel, P. pygmaeus, P. nanus, P. varii, P. britzi, P. marupiara and P. toledopizae were taken from Weitzman and Vari (1987), Toledo-Piza et al. (2014), and Mattox et al. (2020, 2021, 2023). Identification of infraorbitals and postcleithra for Priocharax ariel is based on Mattox et al. (2016, 2022).

To better understand morphometric data and putative differences between the two species described below, the 16 measures taken from 119 specimens were analyzed further with a Principal Component Analysis (PCA) to determine the morphometric variables with the highest amount of variability, followed by a Linear Discriminant Analysis (LDA) to access the level of jack-knifed success of correct reclassification of individual specimens according to their original groupings (i.e., hypothesized species) (Manly 2008). These two analyses were done in PAST 4.10 (Hammer et al. 2001).

Photographs were taken with a Zeiss Axiocam digital camera attached to a Discovery V20 stereomicroscope. Images of the live specimens were taken right after capture in a photography tank with a DSLR Nikon camera and Nikkor Macro lenses. Osteological terminology follows Weitzman (1962) with updates summarized in Mattox et al. (2014). In the description, the frequency of each count is provided in parentheses after the respective count, with the count of the holotype indicated by an asterisk. Specimens examined are deposited in Instituto Nacional de Pesquisas da Amazônia (INPA), Laboratório de Biologia e Genética de Peixes, Universidade Estadual Paulista, Botucatu (LBP), Museu de Ciências da Pontifícia Universidade Católica do Rio Grande do Sul (MCP), Museu Paraense “Emílio Goeldi” (MPEG), Museu de Zoologia da Universidade de São Paulo (MZUSP), Museu de Diversidade Biológica, Universidade Estadual de Campinas, Campinas (ZUEC), Universidade Federal do Oeste do Pará (UFOPA-I), and University of Florida, Gainesville (UF) in addition to the specimens examined as comparative material listed in Mattox et al. (2021). Sampling for the present study was authorized by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) through permit number SISBIO/MMA 80165-1 and in accordance with the National Council for the Control of Animal Experimentation (CONCEA) approved by UNESP Ethics Committee on Use of Animals (CEUA), protocol number 1058.

Molecular analysis

Fifteen sequences of Priocharax specimens were newly generated for this study, and 34 sequences were obtained from Mattox et al. (2020, 2021, 2023) plus one sequence of the outgroup taxon Jupiaba keithi (Géry, Planquette & Le Bail, 1996) deposited in the GenBank database by Papa et al. (2021). Molecular voucher specimens are deposited and preserved in 95% ethanol in the collection of the LBP (Table 1). Genomic DNA extraction followed Ivanova et al. (2006). Partial sequences of the cytochrome c oxidase subunit I (COI) gene were amplified by polymerase chain reaction (PCR) with primers FishF1/FishR1 and FishF2/FishR2 (Ward et al. 2005), L6252-Asn/H7271-COXI (Melo et al. 2011) or FishF6 and FishR7 (Jennings et al. 2019). PCR amplifications were performed in a total volume of 12.5 µl, with 1.25 µl of 10X buffer, 0.25 μl of MgCl 2 (50 mM), 0.2 μl dNTPs (2 mM), 0.5 μl of each primer (5 mM), 0.1 μl of PHT Taq DNA polymerase (Phoneutria), 1.0 μl of genomic DNA (200 ng) and 8.7 μl ddH 2 O. PCR conditions consisted of an initial denaturation (5 min at 94°C), followed by 30 cycles of chain denaturation (50s at 94°C), primer hybridization (45s at 50-54°C) and nucleotide extension (1 min at 68°C), and a ﬁnal extension (10 min at 68°C). All PCR products were checked on 1% agarose gels and then purified with ExoSap-IT (USB Corporation) following the manufacturer’s instructions. The purified PCR products were submitted to sequencing reactions with the Sanger method using BigDye Terminator v 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Sequencing reactions were performed with a final volume of 7 μL, containing 3.90 μL of ultrapure water, 1.05 μL of sequencing buffer 5 ×, 0.35 μL of primer (10 μM), 0.70 μL of BigDye and 1 μL of purified PCR products. Sequencing reactions consisted of an initial denaturation (2 min at 96°C), followed by 35 cycles of chain denaturation (30s at 96°C), annealing (15s at 50°C), and a final extension (4 min at 60°C). The samples were precipitated in ethanol/EDTA, eluted with 10 μL Formamida Hi-Di and loaded onto an ABI 3130 DNA Analyzer automatic sequencer (Applied Biosystems).

Table 1.

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Lots, vouchers, locality information, and GenBank accession numbers of the analyzed specimens of Priocharax.

Species	Lot	Voucher	Basin	Locality	Coordinates	GenBank
Priocharax conwayi sp. nov.	LBP 31740	108405	Rio Tapajós	Igarapé do Henrique, Rio Maró, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°41’9.30”S 55°40’32.97”W	PP902468
Priocharax conwayi sp. nov.	LBP 31740	108406	Rio Tapajós	Igarapé do Henrique, Rio Maró, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°41’9.30”S 55°40’32.97”W	PP902469
Priocharax conwayi sp. nov.	LBP 31740	108407	Rio Tapajós	Igarapé do Henrique, Rio Maró, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°41’9.30”S 55°40’32.97”W	PP902470
Priocharax conwayi sp. nov.	LBP 31741	108408	Rio Tapajós	Igarapé do Henrique, Rio Maró, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°41’9.30”S 55°40’32.97”W	PP902471
Priocharax conwayi sp. nov.	LBP 31741	108409	Rio Tapajós	Rio Mentaí, in the vincinity of settlement Boca do Mentaí, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°37’52.51”S 55°34’40.98”W	PP902472
Priocharax conwayi sp. nov.	LBP 31741	108410	Rio Tapajós	Rio Mentaí, in the vincinity of settlement Boca do Mentaí, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°37’52.51”S 55°34’40.98”W	PP902473
Priocharax conwayi sp. nov.	LBP 31741	108411	Rio Tapajós	Rio Mentaí, in the vincinity of settlement Boca do Mentaí, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°37’52.51”S 55°34’40.98”W	PP902474
Priocharax conwayi sp. nov.	LBP 31741	108412	Rio Tapajós	Rio Mentaí, in the vincinity of settlement Boca do Mentaí, affluent of Rio Arapiuns, Santarém, PA, Brazil	02°37’52.51”S 55°34’40.98”W	PP902475
Priocharax phasma sp. nov.	LBP 31742	108418	Rio Amazonas	Lago Santana, Ilha de Marimarituba, Rio Amazonas, Santarém, PA, Brazil	02°11’12.31”S 55°02’12.00”W	PP902476
Priocharax phasma sp. nov.	LBP 31742	108419	Rio Amazonas	Lago Santana, Ilha de Marimarituba, Rio Amazonas, Santarém, PA, Brazil	02°11’12.31”S 55°02’12.00”W	PP902477
Priocharax phasma sp. nov.	LBP 31742	108420	Rio Amazonas	Lago Santana, Ilha de Marimarituba, Rio Amazonas, Santarém, PA, Brazil	02°11’12.31”S 55°02’12.00”W	PP902478
Priocharax phasma sp. nov.	LBP 31742	108421	Rio Amazonas	Lago Santana, Ilha de Marimarituba, Rio Amazonas, Santarém, PA, Brazil	02°11’12.31”S 55°02’12.00”W	PP902479
Priocharax phasma sp. nov.	LBP 31743	108428	Rio Amazonas	Lago Pajaú, Ilha Nazareth, Rio Amazonas, Santarém, PA, Brazil	02°11’28.53”S 54°51’27.93”W	PP902480
Priocharax phasma sp. nov.	LBP 31743	108429	Rio Amazonas	Lago Pajaú, Ilha Nazareth, Rio Amazonas, Santarém, PA, Brazil	02°11’28.53”S 54°51’27.93”W	PP902481
Priocharax phasma sp. nov.	LBP 31743	108430	Rio Amazonas	Lago Pajaú, Ilha Nazareth, Rio Amazonas, Santarém, PA, Brazil	02°11’28.53”S 54°51’27.93”W	PP902482
Priocharax toledopizae	LBP 31744	108432	Rio Juruá	Igarapé Preto, tributary of Rio Moa, Cruzeiro do Sul, AC, Brazil	07°35’11”S 72°45’20”W	OP257279
Priocharax toledopizae	LBP 31744	108435	Rio Juruá	Igarapé Preto, tributary of Rio Moa, Cruzeiro do Sul, AC, Brazil	07°35’11”S 72°45’20”W	OP257280
Priocharax toledopizae	LBP 31745	108439	Rio Juruá	Balneário at Igarapé Preto near road BR-307, Cruzeiro do Sul, AC, Brazil	07°35’45”S 72°45’16’’W	OP257278
Priocharax toledopizae	LBP 31745	108440	Rio Juruá	Balneário at Igarapé Preto near road BR-307, Cruzeiro do Sul, AC, Brazil	07°35’45”S 72°45’16’’W	OP257282
Priocharax toledopizae	LBP 31745	108441	Rio Juruá	Balneário at Igarapé Preto near road BR-307, Cruzeiro do Sul, AC, Brazil	07°35’45”S 72°45’16’’W	OP257281
Priocharax toledopizae	LBP 31746	108443	Rio Juruá	Igarapé das Piabas, tributary of Rio Moa, Cruzeiro do Sul, AC, Brazil	07°31’15”S 72°53’48”W	OP257283
Priocharax marupiara	LBP 31747	108445	Rio Juruá	Igarapé Canela Fina, Cruzeiro do Sul, AC, Brazil	07°34’02”S 72°39’40”W	OP257285
Priocharax marupiara	LBP 31747	108446	Rio Juruá	Igarapé Canela Fina, Cruzeiro do Sul, AC, Brazil	07°34’02”S 72°39’40”W	OP257286
Priocharax marupiara	LBP 31747	108447	Rio Juruá	Igarapé Canela Fina, Cruzeiro do Sul, AC, Brazil	07°34’02”S 72°39’40”W	OP257284
Priocharax varii	LBP 28495	96981	Rio Madeira	Rio Preto, affluent of Rio Jamari, Candeias do Jamari, RO, Brazil	08°52’53.5”S 63°37’50.8”W	MT754786
Priocharax varii	LBP 28495	96982	Rio Madeira	Rio Preto, affluent of Rio Jamari, Candeias do Jamari, RO, Brazil	08°52’53.5”S 63°37’50.8”W	MT754785
Priocharax varii	LBP 28495	96984	Rio Madeira	Rio Preto, affluent of Rio Jamari, Candeias do Jamari, RO, Brazil	08°52’53.5”S 63°37’50.8”W	MT754783
Priocharax varii	LBP 28495	96985	Rio Madeira	Rio Preto, affluent of Rio Jamari, Candeias do Jamari, RO, Brazil	08°52’53.5”S 63°37’50.8”W	MT754784
Priocharax ariel	LBP 28442	98284	Rio Negro	Igarapé Tibarrá on left side of Rio Negro, Santa Isabel do Rio Negro, AM, Brazil	00°26’28.1”S 64°56’57.5”W	MT754780
Priocharax ariel	LBP 28442	98285	Rio Negro	Igarapé Tibarrá on left side of Rio Negro, Santa Isabel do Rio Negro, AM, Brazil	00°26’28.1”S 64°56’57.5”W	MT754781
Priocharax ariel	LBP 27704	98286	Rio Negro	Igarapé Tapage, Rio Urubaxi, approximatelly 1 hour from mouth of river, S. I. Rio Negro, AM, Brazil	00°30’05.3”S 64°49’11.7”W	MT754778
Priocharax ariel	LBP 27704	98287	Rio Negro	Igarapé Tapage, Rio Urubaxi, approximatelly 1 hour from mouth of river, S. I. Rio Negro, AM, Brazil	00°30’05.3”S 64°49’11.7”W	MT754782
Priocharax ariel	LBP 27704	98288	Rio Negro	Igarapé Tapage, Rio Urubaxi, approximatelly 1 hour from mouth of river, S. I. Rio Negro, AM, Brazil	00°30’05.3”S 64°49’11.7”W	MT754779
Priocharax ariel	LBP 25858	96383	Rio Negro	Igarapé Uacatuna, São Gabriel da Cachoeira, AM, Brazil	00°03’38.0’’S 67°05’45.0’’W	MT754777
Priocharax nanus	LBP 28490	98283	Rio Negro	Igarapé Tibarrá on left side of Rio Negro, Santa Isabel do Rio Negro, AM, Brazil	00°26’28.1”S 64°56’57.5”W	MT754766
Priocharax pygmaeus	LBP 22464	96986	Rio Amazonas	Quebrada La Ponderosa, Letícia, Colombia	04°08’24.4’’S 69°56’53.4’’W	MT754771
Priocharax pygmaeus	LBP 22464	96987	Rio Amazonas	Quebrada La Ponderosa, Letícia, Colombia	04°08’24.4’’S 69°56’53.4’’W	MT754774
Priocharax pygmaeus	LBP 22464	96988	Rio Amazonas	Quebrada La Ponderosa, Letícia, Colombia	04°08’24.4’’S 69°56’53.4’’W	MT754769
Priocharax pygmaeus	LBP 22464	96998	Rio Amazonas	Quebrada La Ponderosa, Letícia, Colombia	04°08’24.4’’S 69°56’53.4’’W	MT754768
Priocharax pygmaeus	LBP 22739	96989	Rio Amazonas	Quebrada Pichuna, Letícia, Colombia	04°07’33.8’’S 70°00’28.9’’W	MT754772
Priocharax pygmaeus	LBP 22739	96990	Rio Amazonas	Quebrada Pichuna, Letícia, Colombia	04°07’33.8’’S 70°00’28.9’’W	MT754773
Priocharax pygmaeus	LBP 22739	96991	Rio Amazonas	Quebrada Pichuna, Letícia, Colombia	04°07’33.8’’S 70°00’28.9’’W	MT754776
Priocharax pygmaeus	LBP 22739	96992	Rio Amazonas	Quebrada Pichuna, Letícia, Colombia	04°07’33.8’’S 70°00’28.9’’W	MT754770
Priocharax pygmaeus	LBP 22739	96993	Rio Amazonas	Quebrada Pichuna, Letícia, Colombia	04°07’33.8’’S 70°00’28.9’’W	MT754775
Priocharax britzi	LBP 28493	98295	Rio Purus	Marginal lake to Rio Ipixuna, Canutama, AM, Brazil	07°31’11.5’’S 63°20’59.6’’W	MW374298
Priocharax britzi	LBP 28493	98296	Rio Purus	Marginal lake to Rio Ipixuna, Canutama, AM, Brazil	07°31’11.5’’S 63°20’59.6’’W	MW374297
Priocharax britzi	LBP 28493	98297	Rio Purus	Marginal lake to Rio Ipixuna, Canutama, AM, Brazil	07°31’11.5’’S 63°20’59.6’’W	MW374296
Priocharax britzi	LBP 28493	98298	Rio Purus	Marginal lake to Rio Ipixuna, Canutama, AM, Brazil	07°31’11.5’’S 63°20’59.6’’W	MW374300
Priocharax britzi	LBP 28493	98299	Rio Purus	Marginal lake to Rio Ipixuna, Canutama, AM, Brazil	07°31’11.5’’S 63°20’59.6’’W	MW374299
Jupiaba keithi	MHNG 2718.031	SU08650	Tapanahony River	Wawapsi Creek, Sipaliwini, Suriname	3°10’42.0”N 55°25’09.1”W	MZ052052.1

Raw sequences were assembled to consensus using Geneious Prime Software (v. 2022.2.2) and aligned with MUSCLE (Edgar 2004) under default parameters. Substitution saturation was evaluated in DAMBE v7 (Xia 2018). Nucleotide variation and substitution patterns were estimated in Geneious Prime Software. A maximum likelihood (ML) analysis was performed under RAxML HPC-PTHREADS-SSE3 (Stamatakis 2014) using five random parsimony trees with the GTRGAMMA model on Brycon server at LBP/UNESP. Species delimitation analyses included two approaches: Assemble Species by Automatic Partitioning (ASAP) and Bayesian implementation of the Poisson Tree Process model (bPTP; Zhang et al. 2013). ASAP was performed through the web server (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html) using Kimura (K80; 2.0); and bPTP using the best ML tree as input, 100,000 generations, and other parameters at default in the bPTP web server (http://species.h-its.org/ptp). Overall and pairwise genetic distances were estimated based on the Kimura 2-parameter model (K2P) + Gamma using MEGA v11 and the order of groups was based on the species delimitation analyses results. ASAP, bPTP and genetic distances were performed excluding the outgroup taxon Jupiaba keithi.

Results

Priocharax conwayi sp. nov.

https://zoobank.org/45B7FA84-A69D-4976-8811-181B8FFD9FC1

Figures 1, 2, 3

Holotype

MZUSP 129745, 12.2 mm SL, Brazil, Pará State, Santarém municipality, Rio Mentaí, in the vicinity of settlement Boca do Mentaí, affluent of Rio Arapiuns, Rio Tapajós drainage, 02°37’52.51”S 55°34’40.98”W, 04 Nov 2021, G.M.T. Mattox, F.C.T. Lima, M. Lima, E. Cerdeira.

Paratypes

All from Brazil, Pará State, Santarém municipality, Rio Tapajós basin: LBP 31741 (5, not measured), INPA 60217 (20, 8.4–14.2 mm SL), MPEG 39636 (20, 10.8–12.9 mm SL), MZUSP 129746 (190, 10.8–19.0 mm SL; 18 c&s, 10.3–12.3 mm SL), UFOPA-I 1367 (20, 9.2–12.3 mm SL) and ZUEC 17853 (30, 9.6–12.8 mm SL), collected with holotype; MCP 55310 (3, 11.4–11.9 mm SL); UF 249762 (2, 11.0–11.6 mm SL); ZUEC 11648 (10, 11.2–17.5 mm SL), same locality as holotype, 21–24 Nov 2015, F.C.T Lima, E. Cerdeira, B.B. Calegari; LBP 31740 (3, not measured) and MZUSP 129747 (47, 11.1–14.5 mm SL), Igarapé do Henrique, Rio Maró, affluent of Rio Arapiuns, Rio Tapajós drainage, 02°41’9.30”S 55°40’32.97”W, 05 Nov 2021, G.M.T. Mattox, F.C.T. Lima, M. Lima, E. Cerdeira; ZUEC 8774 (1, 11.1 mm SL), same locality as previous, 19 Nov 2013, F.C.T. Lima, W.G.R. Crampton, J.S. Ready, E. Cerdeira.

Diagnosis

Priocharax conwayi sp. nov. is distinguished from all congeners except P. nanus and P. toledopizae by the presence of the claustrum (vs. absence). Priocharax conwayi sp. nov. is distinguished from all congeners except P. ariel by the presence of three infraorbitals, Ios 1+2+3 (vs. absence of infraorbitals in P. nanus, P. pygmaeus and P. varii; presence of a single infraorbital, Io 2, in P. britzi and P. marupiara; presence of two infraorbitals, Ios 1+2, in P. marupiara, P. phasma sp. nov. and P. toledopizae). Priocharax conwayi sp. nov. can be distinguished from P. nanus and P. varii by fewer premaxillary teeth (14–20 vs. 23–29), from P. ariel, P. nanus and P. varii by fewer maxillary teeth (24–29 vs. 32–45), and from P. ariel and P. varii by fewer dentary teeth (24–34 vs. 35–46). The presence of a single postcleithrum distinguishes P. conwayi sp. nov. from P. britzi, P. marupiara, P. nanus, P. toledopizae, and P. varii (vs. two), and from P. pygmaeus (vs. absence). Priocharax conwayi sp. nov. can be further distinguished from P. nanus and P. varii by five branched pelvic-fin rays (vs. six), and from P. varii by the absence of an adipose fin (vs. presence). Priocharax conwayi sp. nov. is further distinguished from P. phasma sp. nov. by the shorter anal-fin base (25–31 %SL vs. 32–38 %SL) and longer caudal peduncle (18–24 %SL vs. 13–19 %SL).

Description

For overall appearance, see Figure 1. Morphometric data are presented in Table 2. Body laterally compressed and elongated, greatest depth at vertical through pelvic-fin in small specimens, at vertical through dorsal-fin origin in larger specimens. Dorsal-fin origin approximately at midbody, at vertical through vent and slightly anterior to anal-fin origin. Pectoral-fin bud at vertical through anterior portion of pseudotympanum. Pelvic-fin origin slightly posterior to vertical at midway between posterior margin of opercle and anal-fin origin. Dorsal profile of head and body slightly convex from tip of snout to dorsal-fin origin. Dorsal profile of body along dorsal-fin base nearly straight, gently sloping posteroventrally; slope less conspicuous from latter point to caudal peduncle. Dorsal profile of caudal peduncle straight to base of dorsal procurrent rays. Ventral profile of head and body slightly convex from symphysis of lower jaw to vertical through pectoral-fin origin; straight to slightly convex from latter point to anal-fin origin. Ventral profile of body slightly concave and posterodorsally rising along anal-fin base, slightly concave from end of anal-fin base to origin of ventral procurrent rays. Caudal peduncle short and relatively robust. Pseudotympanum large, located anterior to rib of fifth vertebra.

Table 2.

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Morphometric data of Priocharax conwayi sp. nov. CPL = caudal-peduncle length; HL = head length; n = number of specimens; OD = orbital diameter; SD = Standard Deviation; SL = standard length. Range includes holotype.

	Holotype	n	Range	Mean	SD
Standard length (SL) (mm)	12.2	122	10.8–19.0	12.2	—
Percentages of Standard Length
Depth at dorsal-fin origin	23	122	19–25	22.9	1.0
Snout to dorsal-fin origin	54	122	51–57	54.2	1.0
Snout to pelvic-fin origin	40	122	37–42	39.7	1.0
Snout to anal-fin origin	53	122	50–57	53.3	0.9
Dorsal-fin length	24	121	21–28	24.5	1.0
Dorsal-fin base	12	122	9–15	11.8	1.2
Pelvic-fin length	12	122	9–15	11.0	1.0
Anal-fin length	21	121	20–24	21.7	0.9
Anal-fin base	29	120	25–31	28.5	1.0
Caudal-peduncle depth	7	121	7–9	7.9	0.5
Caudal-peduncle length	21	120	18–23	20.6	1.2
Percentages of head length
Head length (HL)	24	122	18–28	24.1	1.0
Orbital diameter	36	122	26–39	31.2	1.9
Interorbital distance	36	120	31–47	37.3	2.7
Snout length	21	122	17–28	23.0	2.0
Upper jaw length	60	121	44–72	60.6	4.1
Percentages of caudal-peduncle length (CPL)
Caudal-peduncle depth	36	120	31–47	38.4	2.9
Percentages of orbital diameter (OD)
Snout length	58	122	52–96	74.2	8.9

Figure 1.

Priocharax conwayi sp. nov. A Live paratype, MZUSP 129746, female, not measured, Brazil, Pará State, Santarém municipality: Rio Mentaí, in vicinity of settlement Boca do Mentaí, tributary of Rio Arapiuns, Rio Tapajós drainage. B Paratype, MZUSP 129746, male, 10.7 mm SL, collected with holotype. C Holotype, MZUSP 129745, female, 12.2 mm SL, Brazil, Pará State, Santarém municipality: Rio Mentaí, in vicinity of settlement Boca do Mentaí, tributary of Rio Arapiuns, Rio Tapajós drainage. D Paratype, same specimen as (B).

Snout round in lateral view. Eye diameter about one-third of head length. Antorbital and infraorbitals 1, 2 and 3 present (n = 8) (Fig. 2A). Infraorbitals 4 to 6 and supraorbital absent (n = 8). Mouth terminal. Tip of maxilla elongate, posterior border reaching vertical through midway between middle and posterior border of eye, but closer to middle of eye in largest males. Premaxillary teeth in single series, premaxilla with 15 (1), 18(4), or 19(3) teeth. Maxilla with 24(1), 26(4), 27(2), or 28(1) teeth. Dentary with 27(2), 28(1), 29(2), or 30(3) teeth. Dentary teeth in single series, with few anterior teeth slightly displaced from series anteriorly. A conspicuous, elongate foramen at the anterior portion of the dentary. All jaw teeth small, conical and curved lingually to a moderate extent (Fig. 2B).

Figure 2.

Priocharax conwayi sp. nov., paratype, MZUSP 129746, male, 12.1 mm SL, c&s. A Dorsolateral view of head showing infraorbital bones. B Jaws in lateral view. C Right pectoral girdle in lateral view, image flipped. D Weberian apparatus in lateral view. E Detail of claustrum. Abbreviations: Ana, anguloarticular; Ant, antorbital; Cl, cleithrum; Cla, claustrum; Cm, coronomeckelian; De, dentary; Ect, ectopterygoid; Exoc, exoccipital; Fr, frontal; Int, intercalarium; Io1–3, infraorbitals 1–3; LEt, lateral ethmoid; Mx, maxilla; NA3–4, neural arches 3–4; PecRdC, pectoral-fin radial cartilage; Pcl, postcleithrum; Pmx, premaxilla; Pt, posttemporal; OsS, os suspensorium; Qua, quadrate; Ra, retroarticular; Sc, scaphium; ScCoC, scapulocoracoid cartilage; Sn3, supraneural 3; Suc, supracleithrum; Tr, tripus. Scale bar: (A) 2 mm, (B) 0.5 mm, (C) 1 mm, (D) 0.5 mm, (E) 0.25 mm.

Dorsal-fin rays ii,8(7) or ii,9*(118). Endoskeletal part of pectoral fin and some thin exoskeletal bones of the pectoral girdle showing larval structure (Fig. 2C). Cartilaginous pectoral-radial plate with incomplete longitudinal middle fissure leaving upper and lower halves connected at base and tip; base articulating with vertically elongated scapulocoracoid cartilage and round distal margin with larval-like pectoral-fin fold supported solely by actinotrichia. Pectoral-fin rays absent. All bones of endoskeletal pectoral girdle absent, exoskeletal part with posttemporal, supracleithrum, cleithrum and one postcleithrum. Cleithrum with posteriorly directed, curved process immediately below ventral tip of supracleithrum. Pelvic-fin rays i,5*(125). Posterior tip of pelvic fin on vertical through vent in females and extending further posterior slightly beyond anal-fin origin in males. Anal-fin rays ii,16(1), ii,18(1), ii,19(19), ii,20(36), ii,21*(47), ii,22(17), or ii,23(1). Anal-fin margin concave with anterior lobe formed by elongated fin rays and posterior section of short rays. Principal caudal-fin rays 9,9(1), 10,8(1), 10,9*(115) or 10,10(1), dorsal procurrent rays 6(1), 7(2), or 8(5), ventral procurrent rays 5(1), 6(3), or 7(4). Caudal fin forked. Adipose fin absent.

Squamation present in almost all specimens, but scales highly deciduous and easily lost during handling. Scales cycloid, very thin, with no obvious circuli or radii. Scales in midlateral row 19(5), 20(8), 21(19), 22(29), 23*(21), 24(20), 25(4), 26(5), 27(2), or 28(5); no canal-bearing lateral line scales. Scale rows between dorsal-fin origin and pelvic-fin origin 8(2) or 9*(8). Scale rows around caudal peduncle 10*(10). Predorsal scales typically absent but occasionally one or two scales present immediately anterior to dorsal fin. Caudal-fin squamation restricted to base of caudal-fin rays, no scales on caudal-fin lobes.

Total vertebrae 32(8) with 13(1), or 14(7) abdominal vertebrae and 18(7), or 19(1) caudal vertebrae. Total number of gill-rakers on first branchial arch 11(1), 12(4), 13(2) or 14(1), with 3(3) or 4(5) gill-rakers on upper limb, and 8(3), 9(4), or 10(1) gill-rakers on lower limb. Weberian apparatus well-developed, all components ossified including claustrum (Fig. 2D). Large gap between neural arches 3 and 4, with gap partially covered by dorsally projecting pointed process from vertebra 3. Inner arm of os suspensorium large, projecting forward to vertical through middle of second centrum. Supraneurals 5(5) or 6(3).

Color in alcohol

Overall body color pale yellow (Fig. 1B–D). Melanophores scattered on dorsal surface of head in region of brain. Horizontal line of two melanophores from tip of snout to anterior margin of orbital cavity in some specimens. A conspicuous melanophore approximately at articulation between premaxilla and maxilla, and another conspicuous melanophore ventral to that, on dentary. A line of 3-4 melanophores along posteroventral margin of dentary, with patch of melanophores concentrated on posterior margin of lower jaw. A few specimens with inconspicuous line of three deep melanophores radiating horizontally from posterior margin of orbital cavity. Deep melanophores along ventral margin of neurocranium. Thin line of melanophores along posteroventral margin of orbital cavity. Dorsal portion of eye black, with melanophores fading ventrally towards middle of eye. Overall coloration of eye silver with guanine. Deep vertical lines of 2-3 melanophores on anterior portion of heart chamber and halfway between heart chamber and pelvic-fin base. Line of superficial melanophores along pelvic-fin base, a single melanophore on ventral surface of body at vertical through middle of pelvic fin, and a deep inconspicuous vertical line of melanophores from anus along posterior portion of abdominal cavity. These vertical lines give a striped aspect to ventral portion of body along abdominal cavity. Thick patch of melanophores along dorsal surface of swim bladder, more visible through pseudotympanum. Irregular thin line of melanophores along dorsal-fin base. A few specimens with irregular line of melanophores along dorsal profile of body. Thin line of scattered melanophores along entire anal-fin base, and another thicker line of melanophores along ventral margin of hypaxial myomeres starting at vertical through 8-9th anal-fin ray, approaching thin line of anal-fin base posteriorly. Both lines continuing posteriorly to caudal-fin ventral procurrent rays. Two or three isolated melanophores along midlateral surface of caudal peduncle. Scattered melanophores on lateral surface of hypural plate. Fins mostly hyaline, with small dark patch on first rays of dorsal- and anal-fin bases. Dorsal, anal and caudal fins with scattered melanophores along rays.

Color in life

Body mostly translucent, with patterns of melanophores as described in alcohol specimens (Fig. 1A). Xanthophores scattered on dorsal surface of head and snout. Anterior tip of dentary with few xanthophores. Scattered xanthophores on dorsal midline of body anterior to dorsal fin and dorsal surface of swim bladder. Line of conspicuous superficial xanthophores extending from anterior portion of swim bladder to tip of caudal peduncle. Xanthophores along dorsal surface of vertebral column. Line of xanthophores along dorsal-fin base and small patch of xanthophores on base of anterior two or three dorsal-fin rays. Few xanthophores along anterior portion of anal-fin base approximately at level of first five anal-fin rays. Xanthophores along some of longest principal caudal-fin rays.

Sexual dimorphism

Hooks present on anal- and pelvic-fin rays of mature males as small as 11.1 mm SL and 12.4 mm SL, respectively (Fig. 3A, B). Hooks better developed on anal fin. Hooks on anal fin present in longest unbranched and subsequent branched rays up to seventh ray, one pair of hooks on posterior edge of six to nine distal segments of rays. Approximately six to ten small hooks on medial margin of four lateralmost branched pelvic-fin rays in most mature males (n = 3). Two specimens (11.1–13.5 mm SL) with a horizontal slit along the dorsal portion of the pelvic-fin musculature (Fig. 3A). Overall size of pelvic girdle in mature males larger than that of females, pelvic bone restricted posteriorly to level of rib of sixth vertebra in females, but reaching further anteriorly (e.g., halfway between ribs of fifth and sixth vertebra, or rib of fifth vertebra) in mature males. Basipterygium of males better ossified, more robust, and closer to contralateral part than in females. General color pattern more pronounced in males than in females.

Figure 3.

A Priocharax conwayi sp. nov., paratype, male, ZUEC 11648, 13.5 mm SL, lateral view of anal fin showing the bony hooks. B Priocharax conwayi sp. nov., paratype, female, MZUSP 129746, 11.1 mm SL, c&s, lateral view of the ventral portion of the body showing rib of fifth vertebra (red arrow), anterior tip of basipterygium (black arrow) and anal-fin rays without hooks. Anal fin slightly damaged and specimens with alizarin red faded. C Priocharax phasma sp. nov., paratype, male, MZUSP 129750, 12.8 mm SL, lateral view of anal fin showing the bony hooks. D Priocharax phasma sp. nov., paratype, female, MZUSP 129749, c&s, lateral view of the ventral portion of the body showing rib of fifth vertebra (red arrow), anterior tip of basipterygium (black arrow) and anal-fin rays without hooks. E Priocharax phasma sp. nov., same specimen as (C) showing longitudinal slit along the pelvic girdle musculature (black arrows).

Distribution

Priocharax conwayi sp. nov. is known from two localities in the upper Rio Arapiuns, Rio Tapajós system, Santarém municipality, Pará State, Brazil: rios Mentaí and Maró (Fig. 4).

Figure 4.

Map of South America showing details of lower Rio Tapajós and tributary Rio Arapiuns, and Rio Amazonas close to mouth of Rio Tapajós, in vicinity of Santarém municipality, Pará State, Brazil, with records of Priocharax conwayi sp. nov. (losangle and triangle) and Priocharax phasma sp. nov. (circle and star). Triangle and star represent type localities.

Ecological notes

Priocharax conwayi sp. nov. occurs in black water systems and was collected in two slightly different habitats, a shallow riverine area at the margin of the flooded forest presenting dense aquatic vegetation and a small, slow-flowing forest stream also presenting dense aquatic vegetation (Fig. 5A, B). Individuals were collected along the margins where riparian vegetation was still present. At the type locality, Priocharax conwayi sp. nov. was collected with the small scale pike characin Acestrorhynchus minimus Menezes, 1969, the characin tetras Charax condei (Géry & Knöppel, 1976), Microschemobrycon melanotus (Eigenmann, 1912), and Tyttobrycon xeruini Géry, 1973, the tetras Hemigrammus analis Durbin, 1909, Hemigrammus levis Durbin, 1908, Hemigrammus stictus (Durbin, 1909), Hyphessobrycon diancistrus Weitzman, 1977, the toothless characin Curimatopsis cryptica Vari, 1982, a juvenile of the wolf-fish Hoplias sp., the pencil fishes Nannostomus digrammus (Fowler, 1913), Nannostomus unifasciatus Steindachner, 1876, unidentified larvae of Characiformes, larvae and juveniles of the pacu Metynnis sp., the checkerboard cichlid Dicrossus maculatus Steindachner, 1875, the flag cichlid Mesonauta insignis (Heckel, 1840), the eleotrid Microphilypnus ternetzi Myers, 1927, the guppy Poecilia scalpridens (Garman, 1895), and the lampeye Fluviphylax simplex Costa, 1996.

Figure 5.

Type localities of Priocharax conwayi sp. nov. (A, B) and P. phasma sp. nov. (C). (A) General view of type locality: Rio Mentaí, in vicinity of settlement Boca do Mentaí, tributary of Rio Arapiuns (B) Detail of submerged macrophytes associated with Priocharax specimens in (A). (C) General view of type locality: Lago Santana, Ilha de Marimarituba, Rio Amazonas near the mouth of Rio Tapajós. Both localities in Santarém municipality, Pará State, Brazil.

Etymology

Priocharax conwayi sp. nov. honours Dr. Kevin W. Conway, esteemed friend and notable ichthyologist. Dr. Conway has greatly contributed to our knowledge of miniature fish taxonomy and morphology. A noun in the genitive case.

Priocharax phasma sp. nov.

https://zoobank.org/4BFFF288-F386-4FA5-B5A0-B3483D9DC213

Figures 3, 6, 7

Holotype

MZUSP 129748, 10.2 mm SL, Brazil, Pará State, Santarém municipality, Lago Santana, Ilha de Marimarituba, Rio Amazonas near Rio Tapajós mouth, 02°11’12.31”S 55°02’12.00”W, 07 Nov 2021, G.M.T. Mattox, F.C.T. Lima, M. Lima, E. Cerdeira.

Paratypes

All from Brazil, Pará State, Santarém municipality, Rio Amazonas basin: LBP 31742 (10, 9.2–11.0 mm SL), INPA 60218 (20, 9.5–11.0 mm SL), MPEG 39637 (20, 9.2–10.7 mm SL), MZUSP 129749 (348, 9.5–13.8 mm SL; 28 c&s, 10.1–11.2 mm SL), UFOPA-I 1368 (20, 9.3–12.9 mm SL) and ZUEC 17854 (30, 8.7–12.0 mm SL), collected with holotype; LBP 31743 (3, 9.7–12.1 mm SL) and MZUSP 129750 (5, 9.9–12.8 mm SL), Lago Pajaú, Ilha Nazareth, Rio Amazonas near mouth of Rio Tapajós, 02°11’28.53”S 54°51’27.93”W, 06 Nov 2021, G.M.T. Mattox, F.C.T. Lima, M. Lima, E. Cerdeira; ZUEC 8998 (2, 9.3–10.3 mm SL), Lago Pajaú (or Tamoatá), Ilha Nazareth, Rio Amazonas near mouth of Rio Tapajós, 2°11’29”S 54°51’28”W, 21 Sep 2013, F.C.T. Lima, J.S. Ready, W.G.R. Crampton, E. Cerdeira; MCP 55309 (2, 10.4–12.2 mm SL); UF 249763 (2, 10.1–10.3 mm SL); ZUEC 9017 (5, 10.1–12.3 mm SL), same locality as holotype, 21 Sep 2013, F.C.T. Lima, J.S. Ready, W.G.R. Crampton, E. Cerdeira.

Diagnosis

Priocharax phasma sp. nov. is distinguished from all congeners by the complete lack of pigmentation on the body (vs. presence of at least some chromatophores), and by fewer maxillary teeth in slightly irregular series (13–21 vs. 21–45). Priocharax phasma sp. nov. is distinguished from all congeners except P. conwayi sp. nov. by fewer dentary teeth (18–26 vs. 26–55). It is distinguished from all congeners except Priocharax ariel and P. conwayi sp. nov. by the presence of a single postcleithrum (vs. two in most congeners and absence in P. pygmaeus). The presence of two infraorbitals, Ios 1+2, distinguishes Priocharax phasma sp. nov. from most congeners except P. marupiara and P. toledopizae (vs. three infraorbitals, Ios 1+2+3 in P. ariel and P. conwayi sp. nov.; one infraorbital, Io 2, in P. britzi; and absence of infraorbitals in P. nanus, P. pygmaeus and P. varii). Priocharax phasma sp. nov. has more branched anal-fin rays than P. ariel (21–26 vs. 16–21), and fewer branched pelvic-fin rays than P. nanus and P. varii (five vs. six). P. phasma sp. nov. is further distinguished from P. conwayi sp. nov., P. nanus and P. toledopizae by the absence of the claustrum (vs. presence), and from P. varii by the absence of the adipose fin (vs. presence). It also has a longer anal-fin base (32–38 vs. 25–31) and a shorter caudal peduncle (13–19 vs. 18–23) than Priocharax conwayi sp. nov.

Description

For overall appearance, see Figure 6. Morphometric data are presented in Table 3. Body laterally compressed and elongated, greatest depth at vertical through dorsal-fin origin. Dorsal-fin origin approximately at midbody, at vertical through anal-fin origin. Pectoral-fin bud at vertical through anterior portion of pseudotympanum. Pelvic-fin origin approximately midway between posterior margin of opercle and anal-fin origin. Dorsal profile of head and body slightly convex from tip of snout to dorsal-fin origin. Dorsal profile of body along dorsal-fin base nearly straight, gently sloping posteroventrally; slope less conspicuous from latter point to caudal peduncle. Dorsal profile of caudal peduncle straight to base of dorsal procurrent rays. Ventral profile of head and body slightly convex from symphysis of lower jaw to vertical through pectoral-fin origin; straight to slightly convex from latter point to anal-fin origin. Ventral profile of body slightly concave and posterodorsally rising along anal-fin base, and straight from latter point to base of ventral procurrent rays. Caudal peduncle narrow and elongate. Pseudotympanum located anterior to the rib of the fifth vertebra.

Table 3.

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Morphometric data of Priocharax phasma sp. nov. CPL = caudal-peduncle length; HL = head length; n = number of specimens; OD = orbital diameter; SD = Standard Deviation; SL = standard length. Range includes holotype.

	Holotype	n	Range	Mean	SD
Standard length (SL) (mm)	10.2	79	9.3–13.8	10.8	—
Percentages of Standard Length
Depth at dorsal-fin origin	23	79	20–28	23.9	1.5
Snout to dorsal-fin origin	54	79	51–56	53.3	1.1
Snout to pelvic-fin origin	41	79	35–44	40.2	1.4
Snout to anal-fin origin	51	79	48–55	52.0	1.3
Dorsal-fin length	26	78	21–31	25.6	1.5
Dorsal-fin base	11	78	9–15	12.0	1.2
Pelvic-fin length	10	79	7–15	9.6	1.7
Anal-fin length	22	77	20–26	23.0	1.3
Anal-fin base	34	73	32–38	34.3	1.4
Caudal-peduncle depth	8	79	7–10	8.3	0.6
Caudal-peduncle length	17	73	13–19	16.3	1.3
Percentages of head length
Head length (HL)	26	79	23–27	25.0	0.9
Orbital diameter	30	78	28–39	31.0	2.0
Interorbital distance	41	79	33–45	39.1	2.9
Snout length	26	79	16–26	22.2	2.0
Upper jaw length	58	79	48–63	57.7	2.6
Percentages of caudal-peduncle length (CPL)
Caudal-peduncle depth	48	73	37–64	51.5	5.7
Percentages of orbital diameter (OD)
Snout length	85	78	48–88	71.8	9.0

Figure 6.

Priocharax phasma sp. nov. A Live paratype, MZUSP 129749, female, not measured, collected with holotype. B Paratype, MZUSP 129749, male, 10.7 mm SL, collected with holotype. C Holotype, MZUSP 129748, female, 10.2 mm SL, Brazil, Pará State, Santarém municipality: Lago Santana, Ilha de Marimarituba, Rio Amazonas near the mouth of Rio Tapajós. D Paratype, same specimen as (B).

Snout rounded in lateral view. Eye diameter about one-third of head length. Antorbital and infraorbitals 1 and 2 present, infraorbitals 3–6 and supraorbital absent (n = 18) (Fig. 7A). Mouth terminal. Tip of maxilla elongate, posterior border almost reaching vertical through posterior border of eye. Premaxillary teeth in single series, with 15(3), 16(2), 17(5), 18(3), 20(1), 22(1), or 23(1) teeth. Maxilla with 12(1), 13(4), 15(1), 16(1), 17(2), 18(2), 19(1), or 20(3) teeth. Dentary with 17(1), 18(2), 19(3), 20(4), 21(1), 22(3), 23(1), 24(2) or 25(1) teeth. Dentary teeth in single series, with few anterior teeth slightly displaced from series anteriorly. Conspicuous elongate foramen at anterior portion of dentary. All jaw teeth small, conical and curved lingually to moderate extent (Fig. 7B).

Figure 7.

Priocharax phasma sp. nov., paratype, MZUSP 129749, male, 11.0 mm SL, c&s. A Dissected infraorbital bones, right lateral view, image flipped. B Jaws in right lateral view, image flipped. C Left pectoral girdle in lateral view. D Weberian apparatus in lateral view. Abbreviations: Ana, anguloarticular; Ant, antorbital; Cl, cleithrum; Cm, coronomeckelian; De, dentary; Exoc, exoccipital; Int, intercalarium; Io1–2, infraorbitals 1–2; Mx, maxilla; NA3–4, neural arches 3–4; PecRdC, pectoral-fin radial cartilage; Pcl, postcleithrum; Pmx, premaxilla; Pt, posttemporal; OsS, os suspensorium; Ra, retroarticular; Sc, scaphium; ScCoC, scapulocoracoid cartilage; SN3, supraneural 3; Soc, supraoccipital; Suc, supracleithrum; Tr, tripus. Scale bar: 0.2 mm, except for (D): 0.5 mm.

Dorsal-fin rays ii,8(2), ii,9*(72), or ii,10(1). Endoskeletal part of pectoral fin and some thin exoskeletal bones of the pectoral girdle showing larval structure (Fig. 7C). Cartilaginous pectoral-radial plate with incomplete longitudinal middle fissure leaving upper and lower halves connected at base and tip; base articulating with vertically elongated scapulocoracoid cartilage and round distal margin with larval-like pectoral-fin fold supported solely by actinotrichia. Pectoral-fin rays absent. All bones of endoskeletal pectoral girdle absent, exoskeletal part with posttemporal, supracleithrum, cleithrum and one postcleithrum. Cleithrum with posteriorly directed, curved process immediately below ventral tip of supracleithrum. Pelvic-fin rays i,4(1) or i,5*(77). Posterior tip of pelvic fin on vertical through vent in females and extending further posterior to anal-fin origin in males. Anal-fin rays ii,21(5), ii,22(8), ii,23*(16), ii,24(20), ii,25(16), ii,26(6), or ii,27(2). Anal-fin margin concave with anterior lobe formed by elongated fin rays and posterior section of short rays. Principal caudal-fin rays 10,9*(69), dorsal procurrent rays 8(4), 9(10), or 10(2), ventral procurrent rays 6(7), 7(10), or 8(1). Caudal fin forked. Adipose fin absent.

Squamation present in almost all specimens, but scales highly deciduous and easily lost during handling. Scales cycloid, very thin, with no obvious circuli or radii. Scales in midlateral row 20(1), 21(8), 22(6), 23 (14), 24*(14), 25(14), or 26(5); no canal-bearing lateral line scales. Scale rows between dorsal-fin origin and pelvic-fin origin 7(1) or 8*(9). Scale rows around caudal peduncle 8 (2) or 10*(8). Predorsal scales typically absent, occasionally with one or two scales immediately anterior to the dorsal fin. Caudal-fin squamation restricted to base of caudal-fin rays, no scales on caudal-fin lobes.

Total vertebrae 32(2), 33(15), or 34(2) with 14(12), or 15(6) abdominal vertebrae and 17(1), 18(5), 19(11), or 20(1) caudal vertebrae. Total number of gill-rakers on first branchial arch 8(2), 9(3), 10(4), 11(5), 12(3), or 13(1), 2(13), or 3(5) gill-rakers on upper limb, and 6(2), 7(5), 8(2), 9(7), or 10(2) gill-rakers on lower limb. Weberian apparatus well-developed, all components ossified except for claustrum, which is absent (Fig. 7D). Large gap between neural arches 3 and 4, partially covered by dorsally projecting pointed process from vertebra 3. Inner arm of os suspensorium large, projecting forward to vertical through middle of second centrum. Supraneurals 5(7), 6(5), 7(5), or 8(1).

Color in alcohol

Overall body color pale yellow (Fig. 6B–D). Melanophores absent from most parts of body, except for thin inconspicuous superficial line along ventral portion of hypaxial myomeres starting at vertical through 10-11th anal-fin ray and extending to last anal-fin ray. Scattered melanophores forming inconspicuous line along base of caudal-fin rays. Eye silvery with guanine, with thin line of melanophores along dorsal margin.

Color in life

Body mostly translucent, with patterns of melanophores as described in alcohol specimens (Fig. 6A). Thin line of melanophores along anal-fin base and ventral profile of caudal peduncle up to level of ventral procurrent caudal rays. Patch of xanthophores on dorsal surface of head and along vertebral column. All fins hyaline.

Sexual dimorphism

Hooks present either on anal-fin (10.7–12.3 mm SL, n = 7) or on both anal- and pelvic-fin rays (10.7–13.4 mm SL, n = 11) of mature males (Fig. 3C, D). Hooks better developed on anal fin. Anal-fin hooks present on longest unbranched ray and anterior three branched rays, one pair of hooks at posterior edge of distal four or five segments of branched rays, and two pairs in middle segments on longest unbranched ray. One or two poorly developed hooks on medial margin of first and second branched pelvic-fin rays in most mature males (11 out of 18 males). One specimen (12.8 mm SL) with a horizontal slit along the dorsal portion of the pelvic-fin musculature (Fig. 3E). This specimen has the ventral profile of body along the anal fin sinuous. Overall size of pelvic girdle in mature males larger than that of females, pelvic bone restricted posteriorly to level of rib of sixth vertebra in females, but reaching further anteriorly (e.g., halfway between ribs of fifth and sixth vertebra, or at level of rib of fifth vertebra) in mature males.

Distribution

Priocharax phasma sp. nov. is known from floodplain lakes on two islands, Ilha Marimarituba and Ilha Nazareth, in the Rio Amazonas slightly upstream from the Rio Tapajós mouth, Santarém municipality, Pará State, Brazil (Figs 4, 5).

Ecological notes

Priocharax phasma sp. nov. was sampled among marginal vegetation (presumably Echinochloa sp. or Paspalum sp. grasses) in both lakes, at depths approximately between 0.5–1m (Fig. 5). Other fish species sampled with Priocharax phasma sp. nov. at the type locality were a larva of the pike characin Acestrorhynchus sp., the characin tetras Phenacogaster sp., Roeboides descalvadensis Fowler, 1932, the tetras Aphyocharax nattereri (Steindachner, 1882), Hemigrammus hyanuary Durbin, 1918, Hemigrammus sp., Hyphessobrycon sp., Prionobrama filigera (Cope, 1870), Serrapinnus micropterus (Eigenmann, 1907), the hatchet characin Triportheus albus Cope, 1872, the armored catfish Farlowella amazona (Günther, 1864), the flag cichlid Mesonauta insignis (Heckel, 1840), juveniles of the demon eartheater Satanoperca sp, and the eleotrid Microphilypnus ternetzi Myers, 1927.

Etymology

The name phasma is derived from the Greek word φάσμα which means ghost. It alludes to the almost completely transparent appearance of the new species, resembling a ghost. A noun in apposition.

Molecular data analysis

The molecular dataset included 50 sequences with 526 bp and 230 variable sites (43.7%). The nucleotide frequencies were 23.9% adenine, 16.8% guanine, 33% thymine, and 26.2% cytosine. DAMBE indicated no saturation for either transitions or transversions in both asymmetrical (Iss.cAsym) and symmetrical (Iss.cSym) topologies. The maximum likelihood (ML) tree showed high bootstrap values supporting each of the analyzed species (Fig. 8). The best partition proposed by ASAP recognized 9 species (asap-score: 2.00) supporting Priocharax conwayi sp. nov. and Priocharax phasma sp. nov. as new species. ML solution of the bPTP analysis recovered identical results (Figs 8, S1). The overall mean of genetic distances (K2P) among Priocharax species was 21.3%. Intraspecific genetic distances ranged from 0% within Priocharax pygmaeus and P. marupiara to 0.2% within P. ariel, P. conwayi sp. nov., P. phasma sp. nov. and P. varii. The values of interspecific distances ranged from 8.8% between Priocharax pygmaeus and P. toledopizae to 31.8% between P. pygmaeus and P. ariel. The genetic distance between Priocharax conwayi sp. nov. and P. phasma sp. nov. was 26.4% (Table 4).

Table 4.

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Pairwise K2P genetic distances among species of Priocharax. Intraspecific genetic variations highlighted in bold. Numbers below diagonal are values of interspecific distances. Values shown in percentages, followed by standard deviation.

	1	2	3	4	5	6	7	8	9
1 P. nanus	-
2 P. varii	21.1 ± 2.8	0.2 ± 0.1
3 P. britzi	22.2 ± 3.1	26.2 ± 3.3	0.1 ± 0.1
4 P. ariel	21.8 ± 3.0	23.0 ± 3.1	26.1 ± 3.5	0.2 ± 0.1
5 P. marupiara	18.7 ± 2.7	21.4 ± 2.9	19.5 ± 2.6	18.5 ± 2.7	0
6 P. pygmaeus	22.0 ± 3.2	23.3 ± 3.1	24.6 ± 3.2	31.8 ± 4.2	22.2 ± 3.0	0
7 P. toledopizae	20.8 ± 3.0	23.1 ± 3.1	23.0 ± 3.1	29.6 ± 3.9	22.1 ± 3.1	8.8 ± 1.5	0.1 ± 0.1
8 P. phasma sp. nov.	22.1 ± 3.2	21.1 ± 2.7	19.0 ± 2.7	28.6 ± 3.7	26.2 ± 3.5	25.6 ± 3.5	23.5 ± 3.3	0.2 ± 0.2
9 P. conwayi sp. nov.	22.4 ± 3.1	18.3 ± 2.5	27.0 ± 3.6	24.1 ± 3.3	20.5 ± 2.8	30.7 ± 4.1	25.6 ± 3.5	26.4 ± 3.3	0.2 ± 0.1

Figure 8.

Maximum likelihood tree of species in Priocharax based on partial sequences of cytochrome oxidase c subunit 1 gene (526 bp). Vertical bars represent number of species delimited by ASAP and bPTP. Green and blue bars represent new species. Black bars represent other species of Priocharax analyzed. Numbers near nodes represent bootstrap support. Codes after species names represent voucher numbers.

Discussion

We describe here two new miniature species of the genus Priocharax, raising the number of valid species to nine. One of them, Priocharax phasma sp. nov., is so far restricted to islands in Rio Amazonas near the mouth of Rio Tapajós in Santarém municipality, Pará state. The other species, Priocharax conwayi sp. nov., is also known from a restricted area in the Rio Arapiuns, an affluent of the Rio Tapajós, Santarém municipality, Pará State. So far, these two new species follow a similar pattern as all the other known Brazilian species of Priocharax: they are geographically restricted to a single or few locations (Mattox et al. 2020, 2021, 2023), making them especially vulnerable to threats caused by anthropogenic impacts.

A common and interesting theme regarding the species level diversification in Priocharax is the difference in body shape whenever two species occur in close geographical vicinity. Mattox et al. (2023) reported two new species from the Juruá system, Acre state, one of them relatively elongate and the other rather short and deep-bodied (e.g., Priocharax marupiara and P. toledopizae, respectively). A similar morphological pattern is seen with the pair Priocharax conwayi sp. nov. and P. phasma sp. nov., with the former more streamlined and the latter shorter and taller (Figs 9, S2, Tables S1, S2). Also, the LDA succesfully recovered the species assigned originally (Fig. 9, Tables S3, S4)

Figure 9.

A Principal component analysis plot of 16 morphometric data showing grouping of specimens in two subsets (i.e., Priocharax phasma sp. nov. and P. conwayi sp. nov.). B Linear discriminant analysis plot of the same morphometric characters showing almost perfect separation of specimens of two species recognized herein. Abbreviations: AB, anal-fin base; AL, anal-fin length; BD, body depth; CPD, caudal-peduncle depth; CPL, caudal-peduncle length; DB, dorsal-fin base; DL, dorsal-fin length; HL, head length; IOD, interorbital width; OD, orbital diameter; PA, pre-anal distance; PD, pre-dorsal distance; PV, pre-pelvic distance; SNL, snout length; UJL, upper-jaw length; VL, pelvic-fin length.

Another interesting feature discovered herein concerns the sexual dimorphism of both species. One mature male of Priocharax phasma sp. nov. and two of P. conwayi sp. nov. showed a similar unusual sexual dimorphism as mature males of P. toledopizae (Mattox et al. 2023), consisting of a horizontal slit along the pelvic-fin musculature separating it from the main hypaxial muscle mass of the body (Fig. 3E). The specimen of Priocharax phasma sp. nov. even has the sinuous ventral profile of body along the anal fin described earlier for P. toledopizae (Mattox et al. 2023). Cases of conspicuous sexual dimorphism are common in progenetic miniaturized lineages (e.g., Conway and Britz 2007; Britz and Conway 2016). The functional role of this character is unknown and its phylogenetic significance will be explored in the future.

Both species described herein present a wide range in the number of scales in the midlateral row (19–28 for Priocharax conwayi sp. nov. and 20–26 for P. phasma sp. nov.), with some variation. This may be due to the difficulty in counting the scales in species of Priocharax, so the lower counts must be interpreted as tentative (e.g., Weitzman and Vari 1987).

It is generally thought that miniature fishes are more commonly associated with low-nutrient, highly acidic black waters (e.g., Weitzman and Vari 1988; Carvalho et al. 2006; Perkin et al. 2022). Although this may be true to most Priocharax species discovered until today, there are exceptions such as P. britzi and P. phasma sp. nov. which occur in white water floodplains (Mattox et al. 2020; present work), and also for some species associated with clear water streams within white-water systems, as P. marupiara and P. pygmaeus (Mattox et al. 2023; G.M.T. Mattox, pers. obs.). As Perkin et al. (2022) noticed, there is still much to be learned about the ecology of miniature fishes.

Acknowledgements

Most of this study was conducted at the Departamento de Biologia, Universidade Federal de São Carlos – UFSCar, campus Sorocaba, and Museum of Zoology, Senckenberg Dresden, which provided space and access to facilities. Molecular studies were conducted at Departamento de Biologia Estrutural e Funcional, UNESP, campus Botucatu. The authors are grateful to Marcos Lima (UFOPA) for valuable help during the recent fieldwork. André Canto and Frank Ribeiro (UFOPA) provided important infrastructure aid to the fieldwork, and curatorial assistance. Maurício Cetra (DCA-UFSCar) aided in the statistical analyses. Michel Gianeti and Osvaldo Oyakawa (MZUSP), Lúcia Py-Daniel (INPA), Wolmar Wosiacki (MPEG), Carlos Lucena (MCP), and Rob Robins (UF) provided curatorial assistance. Giovana Hackmann (LISO-UFSCar) helped to identify fishes sampled with both species. GMTM was funded by Fundação de Apoio à Pesquisa do Estado de São Paulo – FAPESP (grant 2017/01970-4). Material of both species was obtained for the first time during expeditions of the “Aquatic Faunal Survey of the Lower Amazon” (NSF grant DEB-1146734 to William G.R. Crampton), from which one of the authors (FCTL) took part. FCTL is grateful to William G.R. Crampton, Jonathan S. Ready, Bárbara B. Calegari, and Elias Cerdeira for their help in the field. CS was funded by FAPESP (grant 17/06551-0). CO received financial support of Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (grant 2020/13433-6) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (proc. 306054/2006-0). Amanda Pinion, Kole Kubicek and a third anonymous reviewer gave valuable suggestions to an earlier version of the manuscript.

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Supplementary materials

Supplementary material 1

Figure S1, S2

Mattox GMT, Lima FCT, Britz R, Souza CS, Oliveira C (2024)

Data type: .pdf

Explanation notes: Figure S1. Maximum Likelihood solution of the Poisson Tree Process model (bPTP) analysis. — Figure S2. Principal Component Analysis (PCA) loading plots.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

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Supplementary material 2

Tables S1–S4

Mattox GMT, Lima FCT, Britz R, Souza CS, Oliveira C (2024)

Data type: .pdf

Explanation notes: Table S1. Summary of the detailed results of the Principal Component Analysis (PCA). — Table S2. Principal Component Analysis (PCA) loadings. — Table S3. Summary of the detailed results of the Linear Discriminant Analysis (LDA). — Table S4. Results of the reclassification of the Linear Discriminant Analysis (LDA).

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