Bisecting the type series of Leporinus paranensis Garavello & Britski, 1987 (Characiformes, Anostomidae)

. The type series of Leporinus paranensis includes two distinct species, one of which is herein described as new. Leporinus paranensis is redescribed based on its holotype, two paratypes, and additional specimens recently sampled in tributaries of the Grande River, in the north portion of the upper Paraná basin in Brazil. A new species of Leporinus is described based on specimens collected in all major tributaries of the upper Paraná basin in Brazil, and tributaries of the Paraná River in Paraguay. Both species share the presence of three unicuspid teeth on the premaxillary and four on the dentary, a terminal mouth and three dark midlateral blotches on the body. The new species is distinguished from L. paranensis based on the number of scale series around the caudal peduncle (12 vs. 16). Despite the similar body shape, species delimitation analyses using DNA barcodes, applied to compare samples of the new species to congeners, corroborated the uniqueness of the new species. In addition, molecular data revealed that L. bahiensis, L. octofasciatus, and L. taeniatus are possibly closely related to the L. paranensis species group. Conservation statuses of L. paranensis and the new species are recommended based on the IUCN criteria.


INTRODUCTION
Leporinus Agassiz, 1829 is one of the most diverse genera of Characiformes encompassing approximately 76 species that range throughout South America from Colombia to Argentina (Toledo-Piza et al., 2024).Nevertheless, only a handful of species of Leporinus occur in the upper Paraná basin.Kner (1859) described Leporinus striatus from Orissanga, a locality in the Mogi-Guaçu River drainage within the upper Paraná.Amaral Campos (1945a) registered the occurrence of five species of Leporinus in the same river (Rio Mogi-Guaçu), and in the same year, Amaral Campos (1945b) diagnosed eleven species of Leporinus based on specimens from the La Plata basin deposited at the MZUSP, two of which were described as new: Leporinus aguapeiensis (a junior synonym of Megaleporinus obtusidens, Britski et al., 2012) from the Aguapeí River (Tietê drainage) and L. lacustris from the Mogi-Guaçu River (Grande drainage).For comments on the identifications provided by Amaral Campos see Britski et al. (2012).In 1978, based on samples from the upper Paraná, Britski & Garavello redescribed Leporinus octofasciatus, which was originally described based on specimens from a coastal river in Santa Catarina state.A decade later, Garavello & Britski (1987) described L. paranensis based on samples from the upper Paraná basin, including the Tietê and Grande rivers.The main diagnostic features of their new species were the reduced number of premaxillary teeth (three) and the color pattern composed of three dark midlateral blotches.In the same year, and possibly unaware of the recently described L. paranensis, Géry et al. (1987) revised the species of Leporinus from the Paraguay basin, identifying some specimens as "Leporinus aff.bahiensis."Leporinus bahiensis was described by Steindachner (1875) and is apparently endemic to isolated coastal rivers in eastern Brazil (Camelier & Zanata, 2015;Toledo-Piza et al., 2024).The color pattern composed of three dark midlateral blotches and the three unicuspid teeth on each side of the premaxillae of the Paraguayan specimens described by Géry et al. (1987) are two main diagnostic features shared by L. bahiensis and L. paranensis.In addition, Géry's Paraguayan specimens share the presence of 12 scale rows around the caudal peduncle with paratypes of L. paranensis, which led us to wonder if the specimens examined by Géry et al. (1987) from the Paraguay were conspecific with the ones described from the Paraná in the same year by two of us (HAB, JCG) as L. paranensis.
The present contribution describes the new species, diagnoses it from congeners, and redescribes L. paranensis, re-examining its diagnostic features and geographical distribution.In addition, we recommend a conservation status for both species based on the IUCN criteria.Britski & Garavello (1978) and Garavello (1979).Counts of unbranched fin rays were expressed in Roman symbols, and branched rays by Arabic symbols.The lateral-line scale count included the pored scales extending onto the base of the median caudal-fin rays; counts of the longitudinal scale rows above the lateral line exclude the lateral-line scale row and the middorsal scale row; counts of the longitudinal scale rows below the lateral line exclude the lateral-line scale row and include a half scale row when the pelvic-fin origin is immediately behind the middle of a scale.The pattern of radii was defined on scales immediately dorsal to the lateral line row at the vertical through the dorsal-fin origin.All examined specimens are alcohol preserved.Codes for scientific fish collections followed Sabaj (2020Sabaj ( , 2022)).

Measurements and counts followed
Morphometric and molecular comparisons were made between the two studied species.The body shape of the species was compared by examining the distribution individuals in a morphospace resulting from Principal Component Analysis (PCA).The analysis included the eight variables used in Garavello (1979) and Garavello & Britski (1988).Raw data were transferred to PAST v.4 (Hammer et al., 2001), log transformed and analyzed as a variance-covariance matrix.Leporinus lacustris was used for comparisons in the analysis, as it is a closely related sympatric species that exhibits a distinct overall body shape.
The genetic data used herein included an approximately 570 bp fragment of the cytochrome c oxidase subunit I (COI) barcode region.All DNA barcoding sequences (COI) were obtained from GenBank (https:// www.ncbi.nlm.nih.gov) or BOLD (https://www.boldsystems.org).The COI sequences matrix included 11 samples of L. inexpectatus, 1 of L. paranensis, 19 of L. octofasciatus, 2 of L. bahiensis, 20 of L. taeniatus, 11 of L. lacustris and 5 of L. friderici.The unique genetic sample of L. paranensis came from a larva collected in the Mogi Guaçu river.Due to the early developmental stage of this specimen, it was impossible to confirm its identification prior to the molecular analysis that consumed the entire voucher (Diogo Freitas Souza, personal communication).However, the identification of the latter sample is unequivocal based on its collection origin and the results obtained in the DNA analysis (see Results).The Mogi Guaçu river, where the larva came from, is also where most of the examined specimens of L. paranensis were collected (see Results).In fact, the fish fauna of the Mogi Guaçu is one of the most studied in South America (Godoy, 1954(Godoy, , 1975;;Meschiatti & Arcifa, 2009).There are only five species of Leporinus occurring in that basin: L. friderici, L. inexpectatus, L. lacustris, L. octofasciatus, L. paranensis, and L. striatus.A complete list of specimens, GenBank and BOLD accession numbers used in the genetic analyses is available in Table 1.All sequences were aligned using Muscle v3.8.4 (Edgar, 2004) in MEGA XI (Kumar et al., 2018;Stecher et al., 2020) with default parameters.The best model for nucleotide evolution (in our case K2+G) was estimated in MEGA XI.The molecular matrix was used to estimate the genetic distance over sequences within samples/species, and between species pairs, with standard error calculated via 1,000 bootstrap pseudoreplicates.The matrix was also used in a species delimitation analysis through the method ASAP, available online at https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html choosing Kimura K80 option as the substitution model to compute distances.A phylogenetic tree was generated using Maximum Likelihood in MEGA XI under the K2+G model.The tree was edited in FigTree v.1.4.4 (Rambaut, 2010) and used in a species delimitation analysis through the method PTP (Zhang et al., 2013), available online at https://species.h-its.org/ptp.Garavello & Britski, 1987 (Fig. 1)

Leporinus paranensis
Leporinus paranensis Garavello & Britski, 1987: 156  Description: Morphometric data in Table 2. Medium-sized species, largest known specimen 170 mm SL.Head and trunk elongate and moderately compressed.Dorsal profile strongly convex from tip of upper lip to anterior naris, straight from this point to tip of supraoccipital spine, somewhat straight from there to origin of dorsal fin; dorsal-fin base straight; slightly convex from terminus of dorsal-fin base to origin of adipose fin and distinctly concave on dorsal margin of caudal peduncle.Ventral profile convex from tip of lower lip to the vertical through anterior naris, straight from this point to vertical through pectoral-fin origin; straight or somewhat convex from latter point to origin of anal-fin; anal base straight and concave on ventral margin of caudal peduncle.Body depth greatest at origin of dorsal fin.Mouth terminal, opening longitudinally aligned with ventral margin of pupil in large specimens (100 mm SL or more).Lips smooth or slightly fringed.Premaxillary with three unicuspid teeth gradually decreasing in size away from symphyseal tooth.Dentary with four unicuspid teeth decreasing in size postero-laterally.
Dorsal fin ii, 10*(6); its origin slightly anterior to midpoint of standard length and anterior to vertical through pelvic-fin origin.Distal margin of dorsal-fin convex.Adipose fin small, its origin posterior to origin of anal fin by diameter of one or two scales.Pectoral fin i,14(3) or i,15*(3); its margin slightly convex; its tip when adpressed extending to fourth or fifth scale anterior to pelvic-fin origin.Pelvic fin i,8*(6); its margin concave.Anal fin ii,8*(6), distal margin concave in most specimens; rays not reaching the caudal fin base when adpressed.Caudal fin i,8,9,i*(6); fin distinctly forked with approximately symmetrical lobes.
Coloration: Body ground coloration brown to yellow, distinctly countershaded.Three large rounded to oval dark-brown blotches on midlateral portion of trunk, each larger than orbit (blotches nearly rectangular in a couple of specimens).Anteriormost blotch ventral to dorsal-fin base; median one between dorsal-fin terminus and adipose fin; and posterior one on posterior half of caudal peduncle.Trunk with eight to 12 dark transversal bars on dorsum and extending ventrally below lateral line; bars much less conspicuous than midlateral blotches, being fragmented in larger specimens forming inconspicuous blotches.Inconspicuous dark lines between scale series present on median and posterior half of lateral portion of trunk in some specimens, including the holotype, but absent in recently collected specimens.Dark inconspicuous spot on base of trunk lateral scales in some of the recently collected specimens (absent on holotype).Head distinctly countershaded; upper lip dark.Fins hyaline or uniformly tan, except for adipose fin with dark distal margin.Distribution: Leporinus paranensis is known from the type specimens collected in the Grande river, in an area currently flooded by the hydropower dam UHE Marimbondo; and from specimens collected in tributaries of the Mogi-Guaçu river, itself a tributary of the Grande river (Fig. 2).

Conservation status:
The apparently rarity of Leporinus paranensis is puzzling.The distribution of the species is at best speculative and its abundance and biology in the wild are completely unknown.The fact that the type series was collected almost 50 years ago in an area that has been largely altered by the construction of a hydroelectric dam is a concern.However, the putative distribution of the species (in at least the Grande River basin) is relatively broad, encompassing parts of the states of São Paulo, and Minas Gerais.The species has been recently recorded (2003)(2004)(2005)(2006)(2007) from tributaries of the Mogi-Guaçu river in areas where no potential impacts are expected in the near future.Furthermore, the species is not commercially relevant and does not suffer under any direct anthropological impact (such as overfishing, for example).Therefore, given the IUCN criteria, the species is herein recommended to be considered as Least Concern (LC).

Remarks:
The paratypes of Leporinus paranensis MZUSP 14456 (16 alc), and MZUSP 37410 (1 alc), that likely belonged to Leporinus inexpectatus, were not found at MZUSP (Oyakawa, 1996: 477).Géry et al. (1987: 387) mention a juvenile (c.55 mm SL) from Río Negro, 6 km north of Chaco-I, Paraguay basin, Paraguay, as belonging to Leporinus aff.bahiensis (= L. inexpectatus).This specimen was examined by one of us (HAB) and likely belongs to Leporinus lacustris because it has four teeth on each premaxillary and dentary, in addition to the three dark midlateral blotches, 34 lateral-line scales and 16 scale series around the caudal peduncle.Interestingly, the first specimen of Leporinus inexpectatus deposited in fish collections was sampled in 1908 (FMNH 71240), more than a hundred years ago, by J.D. Haseman in the Salto do Avanhadava, a waterfall that was flooded by a hydroelectric plant during the 1970's.Head and trunk elongate and moderately compressed.Dorsal profile slightly arched from anterior margin of snout to dorsal-fin insertion; declining in an almost straight line from dorsal-fin origin to adipose fin; and concave at caudal peduncle.Ventral profile in a slight convex line from lower jaw to anal-fin insertion; straight on anal-fin base, and concave on caudal peduncle.Body depth greatest at origin of dorsal fin.Mouth terminal, opening horizontally aligned with ventral margin of eye in larger specimens (100 mm SL or more).Lips smooth or slightly fringed.Premaxillary with three unicuspid teeth gradually decreasing in size from symphyseal tooth.Dentary with four unicuspid teeth decreasing in size postero-laterally.
Dorsal fin ii,10*(21); its origin slightly anterior to half of standard length and anterior to pelvic-fin origin.Distal margin of dorsal-fin convex.Adipose fin small, its origin posterior to vertical through origin of anal fin by diameter of one or two scales.Pectoral fin i,14*(15), or i,15(6); its margin slightly convex; its tip when adpressed extending to fourth or fifth scale anterior to pelvic fin.Pelvic fin i,8*(21); its margin concave.Anal fin ii,8*( 21), distal margin concave in most specimens; rays not reaching the caudal fin base when adpressed.Caudal fin i,8,9,i*(21); fin distinctly forked with approximately symmetrical lobes.

Coloration:
In alcohol-preserved specimens, body ground coloration brown to yellow, distinctly counter- Britski, H.A. et al.: Bisecting Leporinus paranensis Pap. Avulsos Zool., 2024;v.64: e202464023 8/13 shaded.Three large rounded to oval dark-brown blotches midlateral on trunk, larger than orbit.Anteriormost blotch below dorsal-fin base; median one dorsal to anal-fin origin; and posterior one on posterior half of caudal peduncle (posterior one always less conspicuous than anterior two, and sometimes completely confluent with last dark transversal bar).Trunk with eight to 14 dark transversal bars on dorsum extending ventrally below lateral line; bars much less conspicuous than midlateral blotches; bars fragmented in some specimens (especially on specimens larger than 80 mm SL) forming inconspicuous blotches.Small specimens (up to 60 mm SL) with eight dark transversal bars on trunk, first one not extending past lateral line scale series.Head distinctly countershaded; upper lip dark.Fins hyaline or uniformly tan except for adipose fin with dark distal margin.
Distribution: Leporinus inexpectatus is known from the entire upper Paraná river basin (Fig. 2), occurring in the main channel of large rivers such as the Paranapanema and the Tietê rivers, and also in smaller tributaries, in the São Paulo, Paraná, and Minas Gerais states of Brazil.The species was also sampled in the Acaray river, a Paraná-river tributary, a few kilometers below the Itaipu dam, and from tributaries of the Itaipu reservoir in Paraguay (Géry et al., 1987).
Conservation status: Given the large distribution of the species encompassing almost the entire upper Paraná basin, its relatively common occurrence in fish collections and inventories, and its presence on smaller tributaries of large rivers, the species is herein recommended to be considered as Least Concern, following the IUCN Criteria.

Etymology:
The specific name inexpectatus is a Latinized adjective in allusion to the surprising previous inclusion of this species in the type series of Leporinus paranensis, and the fact that it was undescribed, even though it occurs in one of the most sampled area in South America, the upper Paraná basin.

Comparative analyses
The body shape analysis of samples of L. paranensis, L. inexpectatus and L. lacustris showed at least two different morphometric patterns (Fig. 4), one of L. lacustris and another of L. inexpectatus and L. paranensis.Data of latter two species was largely overlapping, showing that their general body shape is very similar, and quite distinct when compared to that of L. lacustris.92.623% of data variation was included in Principal Component 1 (PC1), whereas 4.0701% was in PC2 and 1.4637% in PC3 (each remaining component includes less than 1% of the variance).The most important variables on PC2 were bony interorbital (0.51884), followed by standard length (−0.46083), predorsal distance (−0.43925), and eye diameter (0.41572) (Table 3).On PC3, the most important variables were eye diameter (0.83778) followed by bony interorbital (−0.46973), and body depth (−0.19824) (Table 3).
Molecular data included 570 base pairs of cytochrome c oxidase subunit I (COI) from 69 samples.The molecular analyses clearly distinguished the new species from closely related congeners, including L. bahiensis, L. paranensis L. octofasciatus and L. taeniatus, as well as all these nominal species (Fig. 5).The genetic distance, based on Kimura-2-parameter model, ranges from 0.023 ± 0.006 between L. paranensis and L. inexpectatus to 0.094 ± 0.014 between L. taeniatus and L. lacustris (Table 4).The COI gene tree recovered L. inexpectatus and L. paranensis as closely related species forming a clade sister to the clade including L. bahiensis and L. taeniatus, and this larger clade sister to L. octofasciatus.On the other hand, L. friderici and L. lacustris were recovered at the base of the tree.Both species delimitation analyses (ASAP, PTP) confirmed the identifications based on external morphology, and supported the validity of L. inexpectatus and L. paranensis.These results also confirm the identification of the larva (accession number PP335714) as L. paranensis.Indeed, the identification of the latter sample is unequivocal based on its collection origin and the results obtained in the DNA analysis,

Figure 2 .
Figure 2. Geographical distribution of Leporinus paranensis (circles) and L. inexpectatus (squares) in southeast South America.Open symbols represent the type localities.

Figure 4 .
Figure 4. Principal Components Analysis showing the body shape variation in L. paranensis, L. inexpectatus, and L. lacustris.

Figure 5 .
Figure 5. Phylogenetic analysis based on COI sequences and Maximum Likelihood of L. inexpectatus and closely related species, showing results of species delimitation analyses based on morphological (MORPH), and molecular (ASAP, bPTP) methods (bars on right).

Table 1 .
Sampling information for DNA Barcode molecular analyses, including voucher number, locality, accession number in GenBank or BoldSystem, and first publication using each COI sequence.

:
Leporinus inexpectatus is distinguished from congeners, except L. amae, L. amblyrhynchus, L. bahiensis, inexpectatus is distinguished from L. paranensis by having 12 scale rows around caudal peduncle (vs.16); and from L. bahiensis by having a terminal mouth (mouth cleft longitudinally aligned with ventral border of eye) in specimens larger than 100 mm SL (vs.subterminal, mouth cleft longitudinally aligned with ventral border of infraorbital series), body pale brown to silver and slightly yellow to hyaline fins in life (vs.body dark brown and bright orange fins in life).Morphometric data in Table 2. Medium-sized species, largest known specimen 155.6 mm SL.