Integrative diagnosis and review of the conservation status of a Near Threatened species of the “Psalidodon scabripinnis complex”

Priscilla Caroline Silva¹ , Karine Orlandi Bonato¹, Vinícius A. Bertaco² and Luiz Roberto Malabarba¹

PDF: EN    XML: EN | Supplementary: S1 S2 S3 | Cite this article

Abstract​

---

Introduction​

---

Low ray count in the anal fin, massive head, elongated body, deeper, and heavier in the middle of the pectorals is a body shape common to species that live in fast currents (Bertaco, Malabarba, 2001). Based on the difficulty of searching for characters to diagnose characid species that present this generalized pattern, Bertaco, Lucena (2006) gather and list fifteen of them as part of the so-called “Psalidodon scabripinnis species complex”. This list includes three species of characids from southern Brazil: Psalidodon laticeps (Cope, 1894) from the coastal river drainages of Uruguay and Brazil to the south of the State of Paraná, and from the Uruguai River drainage (Bertaco, Lucena, 2010); Astyanax brachypterygium Bertaco & Malabarba, 2001, from the upper tributaries of the Jacuí River and Uruguai River drainages and Psalidodon cremnobates (Bertaco & Malabarba, 2001), from the upper tributaries of the Jacuí River and Maquiné River drainages.

As is usual in these Astyanax-like forms that live in swift currents, the three species are not diagnosable among themselves by discrete variations in fin ray or scale counts, or even by discrete variations in body proportions. Psalidodon laticeps was diagnosed mainly by the oval-shaped and horizontally elongated humeral spot, rather than the vertically elongated humeral spot as observed in the other two species (Bertaco, Lucena, 2010). Astyanax brachypterygium and Psalidodon cremnobates, however, were diagnosed from each other (Bertaco, Malabarba, 2001) by overlapping but statistically significant differences in anal fin ray counts (iii–v, 12–16, median = 14 in A. brachypterygium, versus iii–v, 14–18, median = 16 in P. cremnobates), proportions of orbital diameter and interorbital width in relation to head length and proportion of caudal peduncle depth in relation to standard length.

Here we investigated the identity of a population of characid fish that inhabit rapids in the Mampituba River drainage, which has a low anal fin ray count and a body shape typical of Astyanax-like forms that live in fast-flowing streams. The Mampituba is a coastal river that flows directly into the South Atlantic Ocean and is located on the border of the states of Rio Grande do Sul and Santa Catarina (SEMA, 2024). According to an analysis of the DPSIR socio-economic and environmental indicators matrix (Driving Force, Pressure, State, Impact and Response), Mampituba River drainage is classified in a regular situation (Porto el al., 2019). The main anthropic activities on the basis are agriculture, silviculture, and livestock farming, covering an area of 22.4%, 3.0% and 40.0% of the drainage, respectively (Porto el al., 2019). This drainage, together with the Tramandaí and Araranguá river drainages, make up one of the world’s freshwater ecoregions proposed by Abell et al. (2008) based on the distribution and endemism of fish species.

The use of COI sequences has been proved to be efficient to recognize species among more than 70 species previously referred to Astyanax Baird & Girard, 1854 (including species now referred to Psalidodon Eigenmann, 1911), with 12% of fail in the dataset considered (Rossini et al., 2016). The analysis of mini barcode sequences of COI combined with morphological data have also allowed to recover the identity of early described species based on historical DNA techniques of long preserved type-specimens as exemplified in Tetragonopterus taeniatus Jenyns, 1842 (currently Deuterodon taeniatus)by Silva et al. (2019). The resurrection of some species of the Astyanax bimaculatus group that are currently in the synonym of A. lacustris Lütken, 1875 has been recently suggested by Gavazonni et al. (2024) using integrative taxonomy considering barcode DNA and morphology.

In order to address the lack of discrete morphological data to adequately identify the species found in the Mampituba River drainage, we provide and test the efficiency of cytochrome c oxidase subunit 1 (COI) gene sequences to identify this population, as well as to diagnose the three species of the so called “Psalidodon scabripinnis complex” (A. brachypterygium, P. cremnobates and P. laticeps) that occurs in southern Brazil.

Material and methods

---

Morphological analyses. Specimens were counted and measured following Bertaco, Malabarba (2001). In order to standardize the data and avoid the influence of measurements taken by different people, the measurements of the the specimens under analysis from the Mampituba River drainage were taken by the same person (VAB) that measured the type-specimens of Psalidodon cremnobates and Astyanax brachypterygium. Institutional codes follow Sabaj (2023).

Meristic data that vary among species or populations compared herein do not have a normal distribution, which is expected for meristic data taken from fish populations. In this case we chose the Kruskal-Wallis analysis to test the null hypothesis that population samples are equal for two meristic data (branched anal-fin ray and lateral line scales). If this test indicated a significant difference among the populations, the pairwise multiple comparisons were done using the Dunn test. Violin graphs with boxplots were generated to display the distribution of the data. All tests and the violin plot were performed in the R environment v. 4.4.1 (R Development Core Team, 2024) and R Studio v. 2024.09.0+375 (R Studio Team, 2024).

Molecular analyses. We sequenced 670 base pairs of the COI gene from 31 individuals of P. laticeps, A. brachypterygium, P. cremnobates and four other species (Tab. S1 with Genbank accession numbers). Twenty-one sequences from six species were obtained from Genbank. The species were selected based on their relationship with the target species (Terán et al., 2020) or their occurrence in the Mampituba River (Tab. S1). DNA was extracted from gill filaments, muscles or liver tissue of the samples, with “Phire Animal Tissue Direct PCR Kit” developed by Thermo Scientific® under commercial recommendations. The COI was amplified with the primer cocktail FishF1t1 and FishR1t1 (Ivanova et al., 2007). The PCR reactions were carried out in a reaction volume of 20 µL [10.3 µL of H20, 2 µL of 10× reaction buffer (Platinum®Taq), 0.6 µL of MgCl2 (50 mM), 2 µL of dNTPs (2 mM), 2 µL of each primer (2 µM), 0.1 µL (5 U) of Platinum® Taq (Invitrogen), and 1 µL of template DNA]. The PCR conditions were: an initial DNA denaturation at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, at 52°C for 40 s, and at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were purified using enzymatic method Exosap (25% exonuclease, 25% Shrimp Alkaline Phosphatase and 50% of deionized water), and sequencing was performed by Macrogen Inc, Seoul, South Korea and by Ludwig Biotec at Porto Alegre, RS, Brazil.

Sequences were aligned using Muscle (Edgar, 2004) in MEGA 11 software (Tamura et al., 2021) and alignments were visually inspected for any obvious misalignments and then corrected. The p-distance was estimated twice, one time using the default conditions and the second time considering the best molecular evolutionary model; both analyzes were done in MEGA 11 software (Tamura et al., 2021). To illustrate the relationship between the sequences, a Neighbor Joining tree was used, considering the parameters suggested by Hebert (Hebert et al., 2003) using Mega11 (Tamura et al., 2021). A haplotype network was constructed with DnaSP 6.12 (Rozas et al., 2017) and Network 5.0 softwares (Fluxus technology Ltd.). All work was performed at the Molecular Laboratory of Departamento de Zoologia (UFRGS, Porto Alegre, RS, Brazil), with separately ordered primers (Ishida et al., 2011).

Data availability. The COI sequences used for this study are openly available in Genbank, GenBank accessions PQ341685-PQ341716. A BOLD project, i.e., a library consisting of voucher ID, collection numbers, photographs, and sequence data, is available for the data within this article. Morphological identifications and sampling localities are available in Tab. S1.

Results​

---

Astyanax brachypterygium was recovered under Neighbor Joining analyses (Fig. 1A) as closely related to Psalidodon cremnobates and P. laticeps, as well as deeply inserted in a clade that includes P. gymnodontus Eigenmann, 1911, type-species of the genus Psalidodon. Therefore, we now refer to this species here as Psalidodon brachypterygium (new combination). See further comments on discussion.

FIGURE 1| Genetic analyses of Astyanax, Psalidodon, Andromakhe Terán, Benitez & Mirande, 2020, and Oligosarcus Günther, 1864 genera based on COI gene. The yellow circles represent Astyanax cremnobates from Jacuí River drainage; the black circle represents A. cremnobates from Mampituba River drainage; the blue circles represent A. brachypterygium from Laguna dos Patosand the purple circles are A. laticeps from Tramandaí river drainage. A. Neighbor-joining tree of 54 sequences (Tab. S1) using genetic distance, with the branch lengths equivalent to the number of base substitutions per site. Bootstrap values (1000 replications) are indicated next to the branches. B. Haplotype network of the 11 taxa. Numbers in each branch refer to the number of mutational steps between haplotypes; branches with no number represent only one mutational step; size of the haplotypes increases according with the number of specimens that are part of it (i.e., higher sizes = more specimens). Astyanax jacuhiensis (following Gavazzoni et al., 2024) and A. mexicanus (De Filippi, 1853).

Molecular analysis. Under Neighbor Joining analyses (Fig. 1A), the COI sequence of one specimen from Mampituba River (Perdizes stream) from Mampituba forms a clade with Psalidodon cremnobates from Tramandaí and Jacuí river drainages. This clade is the sister group of a clade composed of Psalidodon brachypterygium and P. laticeps. The clade composed by P. brachypterygium, P. cremnobates, and P. laticeps is closer to species of the genus Psalidodon than with species of the genus Astyanax (Fig. 1A). The NJ result was the same in both analyses, considering default conditions for barcode analyses (Hebert et al., 2003) and the best molecular evolutionary model (Kimura 2 parameters with gamma).

The lowest p-distance (Tab. S2) of the sequence from Mampituba River specimen was 0.024 with P. cremnobates from Tramandaí River drainage, 0.026 with P. cremnobates from Jacuí River drainage, 0.029 with P. laticeps and 0.030 with P. brachypterygium. The highest was 0.17 with Astyanax jacuhiensis (Cope, 1894) (following Gavazzoni et al., 2024). The network analysis (Fig. 1B) shows the haplotype from Mampituba has fewer mutational steps with P. cremnobates haplotypes than with other Astyanax and Psalidodon species. Psalidodon cremnobates, P. brachypterygium and P. laticeps haplotypes showed fewer mutational steps with Psalidodon species than with Astyanax species.

Morphological analysis. A total of 124 specimens of Psalidodon cremnobates collected at Preá and Perdizes streams, tributaries of the Mampituba River, within the Parque Nacional de Aparados da Serra located in the municipality of Cambará do Sul, Rio Grande do Sul, Brazil (Fig. 2), were examined. These specimens easily differ from P. laticeps by showing a vertical humeral spot, instead of a horizontally elongated and oval humeral spot (Fig. 3).

FIGURE 2| Distribution of Psalidodon cremnobates. Red star corresponds to the type-locality from the Jacuí River drainage. Red and blue circles represent the localities of collection of the paratypes from the Jacuí and Maquiné river drainages, respectively. Yellow circles represent the new records from Mampituba River drainage. Orange represents the upland plateau located on Serra Geral and green represents the lowland areas of the river valleys and coastal plain.

FIGURE 3| Characid fish species of the “Psalidodon scabripinnis complex” from Rio Grande do Sul State, Southern Brazil. A. Psalidodon cremnobates, UFRGS 8229, 45.4 mm SL, arroio Pinto, Jacuí River drainage, São Francisco de Paula, Rio Grande do Sul, Brazil, 29^o23’04”S 50^o32’04”W. B. P. brachypterygium, UFRGS 21849, 33.2 mm SL, creek tributary to rio Pelotas, Uruguai River drainage, São José dos Ausentes, Rio Grande do Sul, Brazil, 28°40’26”S 49°58’00”W. C. P. laticeps, UFRGS 8828, 58.5 mm SL, Amola Faca River, Maquiné River drainage, Maquiné, Rio Grande do Sul, Brazil, 29°32’19”S 50°14’46”W. D. P. cremnobates, UFRGS 17203, 48.2 mm SL, Perdizes stream, Cambará do Sul, Mampituba River drainage, Rio Grande do Sul, Brazil, 29^o08’28”S 50^o04’55”W.

Meristic data of Mampituba River population are summarized in Tab. 1 and compared to data from the type-series of P. cremnobates and P. brachypterygium. Likewise Bertaco, Malabarba (2001), we found a unique count that differs between the two species. The Mampituba River population showed a number of branched anal-fin rays that ranges from 13 to 17, that is intermediate between the range described for P. cremnobates (14 to 18) and that described for P. brachypterygium (12 to 16). The median and the mode of the count of anal-fin rays in the Mampituba River population (median = 16, mode = 16), however, equals that of P. cremnobates (median = 16, mode = 16) and differ from that of P. brachypterygium (median = 14, mode = 14). The count of anal-fin rays of the three populations of P. cremnobates and the single population of P. brachypterygium was submitted to a the Kruskal-Wallis analysis and the null hypothesis was rejected, showing that there is a significant difference (X²₍₃₎= 70.14; p < 0.001). The Dunn test comparing each pair of populations showed that the P. brachypterygium population differed from all three populations of P. cremnobates when we compared the branched anal-fin ray, and that these three populations do not differ among themselves (Tab. 2; Fig. 4). The counts of lateral line scales differed P. brachypterygium population only from P. cremnobates population of the Jacuí drainage, and the Jacuí drainage’s population differs from Tramandaí and Mampituba populations (Tab. 3; Fig. 4).

TABLE 1 | Meristic data of Psalidodon cremnobates from Mampituba River drainage. UFRGS 16286 (10 of 22), 16290 (2 of 4), 16294 (4 of 8), 17203 (4 of 7), 17205 (3), 17212 (3 of 14). N = 26 specimens.

	Minimum	Maximum	Mode
Unbranched anal-fin rays	3	4	3
Branched anal-fin rays	13	17	16
Unbranched dorsal-fin rays	2	2	2
Branched dorsal-fin rays	9	9	9
Unbranched pelvic-fin rays	1	1	1
Branched pelvic-fin rays	7	7	7
Unbranched pectoral-fin rays	1	1	1
Branched pectoral-fin rays	11	13	12
Total caudal-fin rays	19	19	19
Lateral line scales	35	37	36
Scale rows between dorsal-fin origin and lateral line	6	6	6
Scale rows between lateral line and pelvic-fin origin	5	5	5
Predorsal scales	11	12	11
Scale rows around caudal peduncle	14	16	14
Scale sheath along anal-fin base	3	6	4
Maxilla teeth	1	2	1

TABLE 2 | Results of Dunn parwise multiple comparisons test for branched anal-fin ray and lateral line scales of the Psalidodon brachypterygium and P. cremnobates drainage populations.

Species	Branched anal-fin ray		Lateral line scales
	Statistic	p	Statistic	p
P. brachypterygium (Uruguai) x P. cremnobates (Jacuí)	8.13	< 0.05	3.01	< 0.05
P. brachypterygium (Uruguai) x P. cremnobates (Mampituba)	5.04	< 0.05	-0.42	> 0.05
P. brachypterygium (Uruguai) x P. cremnobates (Tramandaí)	5.00	< 0.05	2.58	> 0.05
P. cremnobates (Jacuí) x P. cremnobates (Mampituba)	-1.81	> 0.05	-3.02	< 0.05
P. cremnobates (Jacuí) x P. cremnobates (Tramandaí)	-1.68	> 0.05	0.13	> 0.05
P. cremnobates (Mampituba) x P. cremnobates (Tramandaí)	0.08	> 0.05	2.69	< 0.05

TABLE 3 | Median and inter-quartile range (iqr) for branched anal-fin ray and lateral line scales of the Psalidodon brachypterygium and P. cremnobates drainage populations. N = number of specimens.

Species/Meristic data	N	Median	iqr
Branched anal-fin ray
P. brachypterygium (Uruguai)	43	14	0
P. cremnobates (Jacuí)	50	16	0
P. cremnobates (Mampituba)	26	16	1
P. cremnobates (Tramandaí)	24	15	1
Lateral line scales
P. brachypterygium (Uruguai)	43	36	1
P. cremnobates (Jacuí)	50	36	1
P. cremnobates (Mampituba)	26	36	1
P. cremnobates (Tramandaí)	24	36.5	1

FIGURE 4| The violin plots and boxplots of the two meristic data (A= lateral line scales and B = branched anal-fin ray) for the Psalidodon brachypterygium of the rio Uruguai (U) and P. cremnobates of the Jacuí (J), Mampituba (M), and Tramandaí (T) drainages. The violin plot shows the density distribution of meristic data for each population; the central line in the box represents the median, the whiskers show the distribution outside the central quartiles (the lowest/highest value within 1.5 times inter-quartile range from the box), and the circle are outliners.

Morphometric data further corroborates that specimens from Mampituba River belong to P. cremnobates (Tab. S3). Among measurements used to distinguish the two species by Bertaco, Malabarba (2001), specimens from Mampituba River have a shallower caudal peduncle depth (10.1–12.1% in SL, mean = 11.2) with similar range and mean to that observed in P. cremnobates (10.9–12.6%, mean = 11.7), and different from that of P. brachypterygium (13.7–15.3%, mean = 14.3). The larger orbital diameter (30.2–35.4% of head length, mean = 33.1) also fits with P. cremnobates (32.2–35.0%, mean = 33.7) and not with P. brachypterygium (26.3–31.2%, mean = 29.9). We were not able, however, to classify the specimens from Mampituba River based on the interorbital width, once they showed intermediate range and mean (26.1–31.9% of head length, mean = 29.3), comparatively to those observed in P. cremnobates (25.7–27.7%, mean = 26.6) and in P. brachypterygium (29.6–33.6%, mean = 31.4).

Material examined. All from Brazil: Rio Grande do Sul State. Psalidodon brachypterygium: Uruguai River drainage. MCP 8627, 1 paratype (c&s), 36.1 mm SL, rio Tourinhos, Bom Jesus, ca. 28°42’S 50°08’W, 30 Sep 1980, C. A. Lucena. MCP 11661, 5 paratypes, 31.8–55.5 mm SL, headwaters of arroio Lageado Bonito, Bom Jesus, ca. 28°38’S 50°33’W, 4 May 1985, C. A. Lucena, L. R. Malabarba & R. E. Reis; MCP 14367, 24 paratypes, 39.2–50.9 mm SL, rio Manoel Leão, Bom Jesus, ca. 28°44’S 50°02’W, 14 Jan 1989, C. A. Lucena, E. H. L. Pereira & P. V. Azevedo; MCP 14391, 26 (5 c&s), 39.4–58.3 mm SL; MCP 22296, 10 paratypes, 24.5–43.7 mm SL, tributary of rio Manoel Leão, São José dos Ausentes, ca. 28°43’S 50°00’W, 17 Dec 1998, R. E. Reis, A. R. Cardoso, P. A. Buckup & F. Melo; MCP 26094, holotype, 40.8 mm SL, arroio Água Branca, Bom Jesus, ca. 28°36’S 50°24’W, 15 Jan 1989, C. A. Lucena, E. H. L. Pereira & P. V. Azevedo. MZUSP 62713, 20, 37.2–59.5 mm SL. NMW 94544, 20, 37.7–55.1 mm SL. UFRGS 4950, 20, 39.6–58.5 mm SL; UFRGS 21849, 1, 33.2 mm SL, creek tributary to rio Pelotas, São José dos Ausentes, 28°40’26”S 49°58’00”W, 29 Apr 2016, L. R. Malabarba, J. Ferrer, T. P. Carvalho, R. Agrizani & E. Wendt. USNM 364303, 20, 36.3–62.5 mm SL; paratypes and same data of holotype. Psalidodon cremnobates: Jacuí River drainage. MCP 11142, 25 (5 c&s), 40.7–71.5 mm SL; MCP 11650, 34 paratypes (2 c&s), 13.5–81.9 mm SL, arroio Camisa, Cambará do Sul, ca. 29°11’S 50°12’W, 1 May 1985, C. A. Lucena, L. R. Malabarba & R. E. Reis; MCP 21101, 12 paratypes, 31.6–59.9 mm SL, rio Contendas, Tainhas, ca. 29°17’S 50°14’W, 23 Sep 1997, W. Bruschi & G. Vinciprova. MCP 26093, holotype, 43.8 mm SL, tributary of rio Santa Cruz, São Francisco de Paula, ca. 29°23’S 50°32’W, 16 May 1987, R. E. Reis, L. A. Bergman & P. V. Azevedo. MCP 22295, 78 paratypes, 22.1–57.6 mm SL, tributary of rio Tainhas, Tainhas, ca. 29°16’S 50°20’W, 16 Dec 1998, R. E. Reis, A. R. Cardoso, P. A. Buckup & F. Melo. MCP 22297, 9 paratypes, 25.2–45.9 mm SL, headwaters of rio Lageado Grande, ca. 29°13’S 50°28’W, 16 Dec 1998, R. E. Reis, A. R. Cardoso, P. A. Buckup & F. Melo. MCP 25660, 15 paratypes, 30.7–41.4 mm SL, rio Contendas, São Francisco de Paula, ca. 29°17’S 50°16’W, 20 Mar 2000, W. Bruschi. MZUSP 62712, 20, 37.5–64.9 mm SL; NMW 94543, 20, 35.7–49.4 mm SL; UFRGS 4949, 20, 37.2–54.1 mm SL; USNM 364302, 20, 36.8–57.6 mm SL; paratypes and same data as holotype. UFRGS 8229, 1, 45.4 mm SL, arroio Pinto, São Francisco de Paula, 29^o23’04”S 50^o32’04”W, 11 Sep 2009, J. A. Anza, J. Ferrer, L. R. Malabarba & G. Neves. Mampituba River drainage: UFRGS 16282, 30, 24.5–45.4 mm SL, tributary of arroio Preá, 29^o10’22”S 50^o 06’11”W, 10 May 2012, J. Ferrer, T. Guimarães, M. Severo & L. Santos. UFRGS 16283, 2, 25.1–49.8 mm SL, Quebrada Funda, tributary of arroio Perdizes, 29^o10’22”S 50^o02’43”W, 12 May 2012, J. Ferrer, T. Guimarães, M. Severo, L. Santos, P. Lehmann & A. Souza. UFRGS 16286, 22, 28.6–63.6 mm SL, arroio Preá, 29^o09’49”S 50^o05’21”W, 12 May 2012, T. Guimarães, L. Santos, P. Lehmann & A. Souza. UFRGS 16290, 4, 33.7–59.3 mm SL, UFRGS 16294, 8, 35.5–48.4 mm SL, arroio Perdizes, 29^o09’25”S 50^o03’03”W, 12 May 2012, J. Ferrer, T. Guimarães, M. Severo, L. Santos, P. Lehmann & A. Souza. UFRGS 16291, 5, 21.5–47.5 mm SL, tributary of arroio Preá, 29^o10’21”S 50^o06’43”W, 10 May 2012, J. Ferrer, T. Guimarães, M. Severo, L. Santos & A. Souza. UFRGS 16292, 23, 27.5–54.5 mm SL, floodplain that drains to arroio Preá, 29^o10’27”S 50^o07’00”W, 10 May 2012, J. Ferrer, T. Guimarães, M. Severo & L. Santos. UFRGS 16293, 2, 29.1–33.5 mm SL, tributary of arroio Preá, 29^o10’22”S 50^o07’00”W, 10 May 2012, J. Ferrer, T. Guimarães, M. Severo, L. Santos. UFRGS 16296, 1, 48.5 mm SL, tributary of arroio Preá, 29^o09’49”S 50^o05’51”W, 10 May 2012, J. Ferrer, T. Guimarães, M. Severo & L. Santos. UFRGS 17203, 8, 28.1–49.2 mm SL, stream that drains to arroio Perdizes, 29^o08’28”S 50^o04’55”W, 11 Oct 2012, A. Fregonezi, F. G. Becker & T. Guimarães. UFRGS 17205, 3, 45.0–63.2 mm SL, arroio Perdizes, 29^o10’21”S 50^o02’41”W, 11 Oct 2012, A. Fregonezi, F. G. Becker & T. Guimarães. UFRGS 17211, 2, 20.5–40.5 mm SL, arroio Perdizes, 29^o09’28”S 50^o04’47”W, 10 Oct 2012, A. Fregonezi, F. G. Becker & T. Guimarães. UFRGS 17212, 14, 28.7–51.2 mm SL, arroio Preá, 29^o09’47”S 50^o05’50”W, 11 Oct 2012, A. Fregonezi, F. G. Becker & T. Guimarães. Psalidodon laticeps: Maquiné River drainage: UFRGS 8828, 1, 58.5 mm SL, Amola Faca River, Maquiné, 29°32’19”S 50°14’46”W, 6 Jan 2007, L. R. Malabarba, J. Ferrer & C. B. Fialho.

Discussion​

---

The samples collected at Mampituba proved to belong to Psalidodon cremnobates both in the morphological and molecular analyses, enlarging the distribution of this species to that drainage. Psalidodon cremnobates has been considered an endemic species from the “Campos de Altitude” (highland grasslands of southern Brazil), and has was found only in a small area at altitudes from 800 to 1.000 m a.s.l. in the headwaters of the Maquiné River and Laguna dos Patos drainages (Becker et al., 2023). Such a restricted distribution and the occurrence of Oncorhynchus mykiss (Walbaum, 1792), an exotic invasive species (Bertaco et al., 2023) lead the species to be considered Near Threatened (NT) in a regional list (RS, 2014) under criteria B1ab(iii). Our new records come from altitudes from 892 to 1,029 m a.s.l., but from different river drainage. The register of the species in Mampituba extends the geographic distribution of the species to the north from the known distribution. The additional locality is further important because it is inside of the protected area the Parque Nacional de Aparados da Serra, and near to another protected area the Parque Nacional da Serra Geral. The species has been recently reclassified as of Least Concern (LC) in the National list, based on new distribution records in the Maquiné and Jacuí river drainages and review of the extent of occurrence (EOO) criteria (ICMBio, 2024), but has not been reevaluated yet on the regional list. Our new records to the Mampituba River drainage corroborates ICMBio (2024) decision of changing the conservation status to Least Concern (LC), and allows us to recommend the same review of its status on the Rio Grande do Sul State regional list.

Integrative use of multiple operational criteria considering genes, morphology and color pattern showed it to be important in the recognition of three morphologically similar and closely related species, as well as of a new population of P. cremnobates. Based on these results, the use of a recommended 2% limit of genetic divergence (Hebert et al., 2003) to recognize different species deserves further discussion. We found genetic divergence between A. brachypterygium and P. laticeps ranges from 1.6 to 1.9%, while Mampituba River sample of P. cremnobates differ by 2.4% from Tramandaí River and 2.6% from Jacuí River samples of the same species. The three populations of P. cremnobates, however, constitute a single lineage, a unifying criteria for species recognition, and despite the p-distance values larger than 2% among the three populations, they lack additional criteria to split each population as a separate species. Species evolve in different rates (Melkikh, 2023) so the speciation process is occurring in different ways, printing (or not printing) differences on dataset in many different forms (Malabarba et al., 2021), varying among species (e.g., morphological differences and low genetic divergence or high genetic divergence and stable morphology). The use of a standard genetic distance to recognize species has not been supported in several studies (e.g., Pedraza-Marrón et al., 2019; Queiroz et al., 2020; Lehmann et al., 2024). Therefore, our dataset accurately points to the population of Mampituba as being P. cremnobates and validates the three species: P. cremnobates, P. brachypterygium, and P. laticeps.

Astyanax has long been considered non-monophyletic (Eigenmann, 1917; Rosen, 1972; Janovillo et al., 2010; Schmitter-Soto, 2016; Mirande, 2019). Recently, Terán et al. (2020) proposed a new hypothesis of the phylogenetic relationships of Astyanax species and proposed changes in the generic allocation of some species. They resurrected and expanded the genus Psalidodon, and identified the clade composed of Astyanax cremnobates, A. laticeps, and A. serratus Garavello & Sampaio, 2010 as the sister group to Psalidodon. More recently, Melo et al. (2024) transferred these three species to Psalidodon. None of the previous authors have examined A. brachypterygium. We found A. brachypterygium is closely related to P. cremnobates and P. laticeps, as well as included in the same clade with P. gymnodontus, type-species of the genus Psalidodon. In order to reflect these relationships and to maintain Psalidodon monophyletic, we now refer to this species here as P. brachypterygium.

Psalidodon gymnodontus was recovered as paraphyletic in our analysis. The samples are from two different locations in the Iguaçu River drainage, whose sequences were obtained on the NCBI platform. Differences found in the barcodes may suggest a possible species complex or an incorrect identification of one of the samples. Regardless of these facts, both clades of P. gymnodontus are closely related to other Psalidodon species (Fig. 1), which does not affect the results presented here. A taxonomic revision of P. gymnodontus shows a high polymorphism in morphological characters (Pavanelli, Oliveira, 2009), nevertheless molecular data was not included to compare the different morphotypes. An integrative taxonomic work is recommended to evaluate the genetic diversity, and also to help in the determination of the species boundaries.

The haplotype network showed that the sample from the Preá stream (Mampituba River) is more closely related to the P. cremnobates specimens from Maquiné (Tramandaí River drainage) than to the specimens from the Jacuí River drainage (Fig. 1). The Mampituba and Maquiné drainages are included in the Tramandai-Mampituba Freshwater Ecoregion (TMF) (Abell et al., 2008 – FEOW 335). The TMF ecoregion hosts many endemic species (Malabarba, Isaia, 1992; Reis, Schaefer, 1998; Albert et al., 2011; Ferrer et al., 2015; Delapieve et al., 2020) mainly due to shared geological historical changes. The region was highly impacted by changes in sea level in the Pleistocene that culminated in a division of areas and connections mainly to capture streams, modeling a pattern in the distribution of species that inhabit TMF (Hirschmann et al., 2015, 2017; Thomaz et al., 2015, 2017; Thomaz, Knowles, 2018).

Acknowledgments​

---

We thank all colleagues that sampled the studied specimens. Claudia Malabarba for all support during molecular framework. Marcos S. Silveira for helping with the figures layout. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) supported PCS (grant number: 381062/2024–4) and LRM (grant number: 308026/2021–7) are partially funded by the CNPq.

References​

---

Abell R, Thieme ML, Revenga C, Bryer M, Kottelat M, Bogutskaya N et al. Freshwater ecoregions of the world: a new map of biogeographic units for freshwater biodiversity conservation. BioScience. 2008; 58:403–14. https://doi.org/10.1641/B580507

Albert JS, Carvalho TP. Neogene assembly of modern faunas. In: Albert JS, Reis RE, editors. Historical biogeography of Neotropical freshwater fishes. Berkeley: University of California Press; 2011. p.119–36.

Becker FG, Ferrer J, Loureiro M, Reis RB, Malabarba LR. Fish diversity and conservation in a Neotropical Grassland Region. In: Overbeck GE, Pillar VP, Müller SC, Bencke GA, editors. South Brazilian grassland: ecology and conservation of the Campos Sulinos. Springer; 2023. p.319–48. https://doi.org/10.1007/978-3-031-42580-6_12

Bertaco VA, Azevedo MA, Malabarba LR. Peixes de água doce não-nativos e seus impactos sobre a ictiofauna do estado do Rio Grande do Sul, Brasil. Bio Diverso. 2023; 3:1–80. https://seer.ufrgs.br/index.php/biodiverso/article/view/127020

Bertaco VA, Lucena CAS. Two new species of Astyanax (Ostariophysi: Characiformes: Characidae) from eastern Brazil with a synopsis of the Astyanax scabripinnis species complex. Neotrop Ichthyol. 2006; 4(1):53–60. https://doi.org/10.1590/S1679-62252006000100004

Bertaco VA, Lucena CAS. Redescription of the Astyanax obscurus (Hensel, 1870) and A. laticeps (Cope, 1894) (Teleostei: Characidae): two valid freshwater species originally described from rivers of Southern Brazil. Neotrop Ichthyol. 2010; 8(1):7–20. https://doi.org/10.1590/S1679-62252010000100002

Bertaco VA, Malabarba LR. Description of two species of Astyanax (Teleostei: Characidae) from headwater streams of Southern Brazil, with comments on the “A. scabripinnis species complex”. Ichthyol Explor Freshw. 2001; 12:221–34.

Delapieve ML, Carvalho TP, Reis RE. Species delimitation in a range-restricted group of cascudinhos (Loricariidae: Epactionotus) supports morphological and genetic differentiation across coastal rivers of southern Brazil. J Fish Biol. 2020; 97(6):1748–69. https://doi.org/10.1111/jfb.14538

Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32:1792–97. https://doi.org/10.1093/nar/gkh340

Eigenmann CH. American Characidae – Part 1. Mem Mus Comp Zool. 1917; 43:1–102.

Ferrer J, Donin LM, Malabarba LR. A new species of Ituglanis Costa & Bockmann, 1993 (Siluriformes: Trichomycteridae) endemic to the Tramandaí-Mampituba ecoregion, southern Brazil. Zootaxa. 2015; 4020(2):375–89. https://doi.org/10.11646/zootaxa.4020.2.8

Gavazzoni M, Brezinski FC, Pedroso TH, Pavanelli CS, Graça WJ, Blanco DR et al. Integrative taxonomy suggests resurrection of species of the Astyanax bimaculatus group (Characiformes, Characidae). Zebrafish. 2024; 21(4):300–09. https://doi.org/10.1089/zeb.2024.0132

Hebert PD, Cywinska A, Ball SL, deWaard JR. Biological identifications through DNA barcodes. Proc Royal Soc B. 2003; 270:313–21. https://doi.org/10.1098/rspb.2002.2218

Hirschmann A, Fagundes NJ, Malabarba LR. Ontogenetic changes in mouth morphology triggers conflicting hypotheses of relationships in characid fishes (Ostariophysi: Characiformes). Neotrop Ichthyol. 2017; 15(1):e160073. https://doi.org/10.1590/1982-0224-20160073

Hirschmann A, Malabarba LR, Thomaz AT, Fagundes NJR. Riverine habitat specificity constrains dispersion in a Neotropical fish (Characidae) along Southern Brazilian drainages. Zool Scr. 2015; 44(4):374–82. https://doi.org/10.1111/zsc.12106

Instituto Chico Mendes de Biodiversidade (ICMBio). Sistema de avaliação do risco de extinção da biodiversidade – SALVE. 2024. Available from: https://salve.icmbio.gov.br/

Ishida Y, Demeke Y, van Coeverden de Groot PJ, Georgiadis NJ, Leggett KE, Fox VE et al. Distinguishing forest and savanna African elephants using short nuclear DNA sequences. J Heredity. 2011; 102(5):610–16. https://doi.org/10.1093/jhered/esr073

Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes. 2007; 7:544–48. https://doi.org/10.1111/j.1471-8286.2007.01748.x

Javonillo R, Malabarba LR, Weitzman SH, Burns JR. Relationships among major lineages of characid fishes (Teleostei: Ostariophysi: Characiformes), based on molecular sequence data. Mol Phylogenet Evol. 2010; 54(2):498–511. https://doi.org/10.1016/j.ympev.2009.08.026

Lehmann A. P, Bartzen CS, Malabarba LR. The barcode trap – description of a new species of Microglanis, with a review of the status of Microglanis cibelae (Siluriformes: Pseudopimelodidae). J Fish Biol. 2024; 105(1):110–23. https://doi.org/10.1111/jfb.15764

Malabarba LR, Chuctaya J, Hirschmann A, Oliveira EB, Thomaz AT. Hidden or unnoticed? Multiple lines of evidence support the recognition of a new species of Pseudocorynopoma (Characidae: Corynopomini). J Fish Biol. 2021; 98(1):219–36. https://doi.org/10.1111/jfb.14572

Malabarba LR, Isaia EA. The fresh water fish fauna of the rio Tramandaí drainage, Rio Grande do Sul, Brazil, with a discussion of its historical origin. Com Mus Ciênc Tecnol PUCRS, Sér Zool. 1992; 5:197–223.

Melkikh AV. Aging and group selection: new arguments in favor of partially directed evolution. BioSystems. 2023; 234:105061. https://doi.org/10.1016/j.biosystems.2023.105061

Melo BF, Ota RP, Benine RC, Carvalho FR, Lima FC, Mattox GM et al. Phylogenomics of Characidae, a hyper-diverse Neotropical freshwater fish lineage, with a phylogenetic classification including four families (Teleostei: Characiformes). Zool J Linn Soc. 2024; 202(1):zlae101. https://doi.org/10.1093/zoolinnean/zlae101

Mirande JM. Morphology, molecules and the phylogeny of Characidae (Teleostei, Characiformes). Cladistics. 2019; 35(3):282–300. https://doi.org/10.1111/cla.12345

Pavanelli CS, Oliveira CAM. A redescription of Astyanax gymnodontus (Eigenmann, 1911), new combination, a polymorphic characid fish from the rio Iguaçu basin, Brazil. Neotrop Ichthyol. 2009; 7(4):569–78. https://doi.org/10.1590/S1679-62252009000400003

Pedraza-Marrón CDR, Silva R, Deeds J, Van Belleghem SM, Mastretta-Yanes A, Domínguez-Domínguez O et al. Genomics overrules mitochondrial DNA, siding with morphology on a controversial case of species delimitation. Proc Royal Soc B. 2019; 286(1900):20182924. https://doi.org/10.1098/rspb.2018.2924

Porto DT, Basso LA, Strohaecker TM. Diagnóstico ambiental da bacia hidrográfica do Rio Mampituba, região Sul do Brasil, utilizando a matriz FPEIR. Geosul. 2019; 34(72):28–50. https://doi.org/10.5007/1982-5153.2019v34n72p28

Queiroz LJ, Cardoso Y, Jacot-des-Combes C, Bahechar IA, Lucena CA, Py-Daniel LR et al. Evolutionary units delimitation and continental multilocus phylogeny of the hyperdiverse catfish genus Hypostomus. Mol Phylogenet Evol. 2020; 145:106711. https://doi.org/10.1016/j.ympev.2019.106711

R Development Core Team. R: a language and environment for statistical computing, version 4.4.1. Vienna, Austria: R Foundation for Statistical Computing; 2024. Available from: https://www.r-project.org/

Reis RE, Schaefer AS. New cascudinhos from southern Brazil: systematics, endemism, and relationships (Siluriformes, Loricariidae, Hypoptopomatinae). Am Mus Novit. 1998; 3254:1–25.

Rio Grande do Sul (RS). Decreto n° 51.797, de 8 de setembro de 2014. Declara as espécies da fauna silvestre ameaçadas de extinção no Estado do Rio Grande do Sul. Diário Oficial do Estado do Rio Grande do Sul; 2014. Available from: https://www.al.rs.gov.br/filerepository/replegis/arquivos/dec%2051.797.pdf

Rosen DE. Origin of the characid fish genus Bramocharax and a description of a second, more primitive, species in Guatemala. Am Mus Novit. 1972; 2500:1–21.

Rossini BC, Oliveira CAM, Melo FAG, Bertaco VA, Astarloa JMD, Rosso JJ et al.Highlighting Astyanax species diversity through DNA barcoding. PLoS ONE. 2016; 11:e0167203. https://doi.org/10.1371/journal.pone.0167203

R Studio Team. R Studio: Integrated Development for R [Internet]. Boston: R Studio, PBC; 2024. Available from: http://www.rstudio.com/

Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 2017; 34:3299–302. https://doi.org/10.1093/molbev/msx248

Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021; 38:3022–27. https://doi.org/10.1093/molbev/msab120

Sabaj MH. Codes for natural history collections in ichthyology and herpetology (online supplement). Version 9.5; 2023. Available from: https://www.asih.org/resources/standard-symbolic-codes

Schmitter-Soto JJ. A phylogeny of Astyanax (Characiformes: Characidae) in Central and North America. Zootaxa. 2016; 4109(2):101–30. https://doi.org/10.11646/zootaxa.4109.2.1

Secretaria Estadual de Meio Ambiente e Infraestrutura do Rio Grande do Sul (SEMA). L050 – Bacia hidrográfica do rio Mampituba. 2024. Available from: https://sema.rs.gov.br/l050-bh-mampituba

Silva PC, Malabarba MC, Malabarba LR. Integrative taxonomy: morphology and ancient DNA barcoding reveals the true identity of Astyanax taeniatus, a tetra collected by Charles Darwin during the Beagle’s voyage. Zool Anz. 2019; 278:110–20. https://doi.org/10.1016/j.jcz.2018.12.007

Terán GE, Benitez MF, Mirande JM. Opening the Trojan horse: phylogeny of Astyanax, two new genera and resurrection of Psalidodon (Teleostei: Characidae). Zool J Linn Soc. 2020; 190(4):1217–34. https://doi.org/10.1093/zoolinnean/zlaa019

Thomaz AT, Knowles LL. Flowing into the unknown: inferred paleodrainages for studying the ichthyofauna of Brazilian coastal rivers. Neotrop Ichthyol. 2018; 16(3):e180019. https://doi.org/10.1590/1982-0224-20180019

Thomaz AT, Malabarba LR, Bonatto SL, Knowles LL. Testing the effect of palaeodrainages versus habitat stability on genetic divergence in riverine systems: study of a Neotropical fish of the Brazilian coastal Atlantic Forest. J Biogeogr. 2015; 42(12):2389–401. https://doi.org/10.1111/jbi.12597

Thomaz AT, Malabarba LR, Knowles LL. Genomic signatures of paleodrainages in a freshwater fish along the southeastern coast of Brazil: genetic structure reflects past riverine properties. Heredity. 2017; 119:287–94. https://doi.org/10.1038/hdy.2017.46

Authors

---

Priscilla Caroline Silva¹ , Karine Orlandi Bonato¹, Vinícius A. Bertaco² and Luiz Roberto Malabarba¹

^[1]    Programa de Pós-Graduação em Biologia Animal, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9090, 91540-000 Porto Alegre, RS, Brazil. (PCS) pricarola@gmail.com (corresponding author), (KOB) kakabio2005@yahoo.com.br, (LRM) malabarb@ufrgs.br.

^[2]    Museu de Ciências Naturais, Secretaria do Meio Ambiente e Infraestrutura, Rua Dr. Salvador França, 1427, 90690-000 Porto Alegre, RS, Brazil. (VAB) vbertaco@gmail.com.

Authors’ Contribution

---

Priscilla Caroline Silva: Conceptualization, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review and editing.

Karine Orlandi Bonato: Conceptualization, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review and editing.

Vinícius A. Bertaco: Formal analysis, Investigation, Methodology, Writing-review and editing.

Luiz Roberto Malabarba: Formal analysis, Investigation, Methodology, Project administration, Resources, Writing-review and editing.

Ethical Statement​

---

Not applicable.

Competing Interests

---

The author declares no competing interests.

How to cite this article

---

Silva PC, Bonato KO, Bertaco VA, Malabarba LR. Integrative diagnosis and review of the conservation status of a Near Threatened species of the “Psalidodon scabripinnis complex”. Neotrop Ichthyol. 2024; 22(4):e240069. https://doi.org/10.1590/1982-0224-2024-0069

Copyright​

---

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Distributed under

Creative Commons CC-BY 4.0

© 2024 The Authors.

Diversity and Distributions Published by SBI

Accepted November 11, 2024 by Priscila Camelier

Submitted July 18, 2024

Epub January 10, 2025