Morphological and Molecular Evidence Reveals the Longnose Skate Zearaja brevicaudata (Marini, 1933) to be a Senior Synonym of Dipturus lamillai Concha, Caira, Ebert & Pompert 2019

© 2022 Academia Sinica, Taiwan Open Access Morphological and Molecular Evidence Reveals the Longnose Skate Zearaja brevicaudata (Marini, 1933) to be a Senior Synonym of Dipturus lamillai Concha, Caira, Ebert & Pompert 2019 Valeria Gabbanelli1,* , Gavin Naylor2, Simon Weigmann3,4 , Lei Yang2, Diego Martín Vazquez1,5 , Peter Last6, Juan Martin Díaz de Astarloa1, and Ezequiel Mabragaña1 1Laboratorio de Biotaxonomia Morfológica y Molecular de Peces, Instituto de Investigaciones Marinas y Costeras (IIMYC), Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata–CONICET, CC1260, Funes 3350, 7600 Mar del Plata, Argentina. *Correspondence: [email protected] (Gabbanelli). E-mail: [email protected] (Díaz de Astarloa); [email protected] (Mabragaña) 2Florida Museum of Natural History, 1659 Museum Rd. Gainesville, FL 32611, USA. E-mail: [email protected] (Naylor); [email protected] (Yang) 3Elasmo-Lab, Elasmobranch Research Laboratory, Sophie-Rahel-Jansen-Str. 83, 22609 Hamburg, Germany 4Leibniz Institute for the Analysis of Biodiversity Change (LIB), Centre for Taxonomy and Morphology, Zoological Museum, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany. E-mail: [email protected] (Weigmann) 5Instituto Nacional de Limnología, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional del Litoral, Santa Fe, Argentina. E-mail: [email protected] (Vazquez) 6CSIRO The Commonwealth Scientific and Industrial Research Organisation, Division of Marine and Atmospheric Research, Castray Esplanade, Hobart, TAS, 7001, Australia. E-mail: [email protected] (Last) Received 23 December 2021 / Accepted 8 September 2022 / Published 23 December 2022 Communicated by Felipe Ottoni Longnose skates have great economic importance in South American fisheries, and in order to preserve them, it is important to have a well-defined taxonomic status of their species. Dipturus lamillai was recently described for Malvinas Islands waters based on morphological and molecular comparisons with Zearaja chilensis. Although D. lamillai has been compared with several congeneric species, it was not properly compared with the morphologically similar Zearaja brevicaudata, the most abundant longnose skate in the Southwest Atlantic. Here, these species were compared by morphological and molecular analyses in order to evaluate their conspecificity. Linear morphometric variables of holotype and paratypes of D. lamillai and 69 specimens of Z. brevicaudata were compared and investigated using Principal Component Analysis. In addition, thorn patterns, denticle distributions, color, and clasper morphology were compared. No body proportions or other single character that could differentiate D. lamillai from Z. brevicaudata were found. Molecular analyses comprised of the comparison of the Cytochrome oxidase subunit I (COI) and the NADH dehydrogenase subunit 2. The results of the Maximum Likelihood (ML) carried out for each molecular marker showed that sequences from D. lamillai clustered together with those of Z. brevicaudata, and the molecular distance determined by Kimura twoparameter were lower than the expected for different species. Additionally, the Automatic Barcode Gap Discovery method and the Bayesian implementation of the Poisson tree processes were carried out with COI sequences to explore species limits, and their results were consistent with ML analyses. In summary, the results obtained showed that there are no morphological or molecular differences between these nominal species of the valid skate genus Zearaja, leading to the conclusion that they are conspecific. Therefore, we designated Z. brevicaudata as a senior synonym of D. lamillai. Key words: Integrative taxonomy, Longnose skates, Rajiformes, Synonyms, Southwest Atlantic. Citation: Gabbanelli V, Naylor G, Weigmann S, Yang L, Vazquez DM, Last P, de Astarloa JMD, Mabragaña E. 2022. Morphological and molecular evidence reveals the longnose skate Zearaja brevicaudata (Marini, 1933) to be a senior synonym of Dipturus lamillai Concha, Caira, Ebert & Pompert 2019. Zool Stud 61:76. doi:10.6620/ZS.2022.61-76. Zoological Studies 61:76 (2022) doi:10.6620/ZS.2022.61-76 1 © 2022 Academia Sinica, Taiwan BACKGROUND Most of the recognized members of the family Rajidae (sensu Last et al. 2016a) were initially assigned to the genus Raja, which was later found to consist of multiple subgenera (McEachran and Dunn 1998). In their morphologically based phylogenetic study of the group, the family Rajidae (at subgeneric level) was found to consist of 15 genera; five of these were elevated from the subgenera of Raja (i.e., Amblyraja, Malm 1877; Dipturus Rafinesque, 1810; Leucoraja Malm, 1877; Okamejei Ishiyama, 1958; and Rajella Stehmann, 1970). These taxa remain in use at the generic level but there have been several amendments to the classification. Dipturus was described by Rafinesque (1810), who characterized the genus by the presence of two dorsal fins in the tail, and the absence of a caudal fin, and considered Dipturus batis (as Raja batis Linnaeus, 1758) the type species. Whitley (1939) described Zearaja Whitley, 1939, providing only the description of the type species Zearaja nasuta, which was originally described as Raja nasuta Müller and Henle, 1841. Considering the neurocranium morphology of this species, Stehmann and Bürkel (1990) considered Zearaja as a synonym of Dipturus. Last and Gledhill (2007) resurrected the genus Zearaja from synonymy with Dipturus, including their new species Zearaja maugeana Last and Gledhill, 2007, as well as Raia chilensis Guichenot, 1848 and Raja nasuta. Some taxonomic issues have been addressed with the elevation of Zearaja from Dipturus based mainly on morphological data (Last and Gledhill 2007) and supported by unpublished molecular data (Naylor, Chondrichthyan Tree of Life Project). The main morphological characteristic that differentiates Zearaja species from other rajid skates is the unique combination and anatomical configuration of components forming the clasper (Last and Gledhill 2007). Earlier, Naylor et al. (2012) had called into question the validity of the genus Zearaja based on molecular data (NADH2), stating that the genus Dipturus would be monophyletic only if the genus Zearaja was included within Dipturus. In contrast, Vargas-Caro (2017), by means of a complete mitochondrial genome analysis, found that both genera (Dipturus and Zearaja) were monophyletic. More recently, Concha et al. (2019), based on a molecular analysis of the mitochondrial gene NADH dehydrogenase subunit 2 (NADH2) and morphological features of the clasper, synonymized Zearaja with Dipturus, relocating all Zearaja species within Dipturus. Unfortunately, the Concha et al. (2019) molecular tree (Concha et al. 2019: fig. 13) is extremely limited in the number of Dipturus taxa represented (only 2 of 38 species of Dipturus treated in Last et al. 2016a, and not including the type species, Raja batis). Hence, this study does not factor in the rich diversity of Dipturus as defined by McEachran and Dunn (1998, 29 species) when the subgenus was first elevated to generic status. However, this nomenclatural decision is not widely accepted and was not adopted in the revision of skates and batoids of the world, which maintains the validity of the genus Zearaja, mostly based on clasper morphology and the particular importance of this structure in the taxonomy of skates (Last et al. 2016a). In support of this argument, comprehensive molecular analyses from the Chondrichthyan Tree of Life Project, including the type species of the genus, confirm the paraphyly of Dipturus as defined by Concha et al. (2019). This taxon includes Zearaja and several presently undefined genera (Naylor unpubl. data; Awruch et al. 2021). Hence, to avoid creating additional confusion in the literature, we have chosen to follow the nomenclature used in the most recent taxonomic review of the group (Last et al. 2016a). Moreover, the Catalog of Fishes currently presents Zearaja as a valid genus (Fricke et al. 2022). Skates of the genera Zearaja Whitley, 1939 and Dipturus Rafinesque, 1810 (Rajidae) are medium to large-sized skates, characterized by a rhombic disc and a medium to long snout with stiff rostral cartilage (Last et al. 2016a). They occur from cold temperate waters to tropical seas, and on continental shelves and slopes where they have their greatest diversity. Until 2018, eight longnose skate species had been reported in South American waters: D. argentinensis Díaz de Astarloa, Mabragaña, Hanner and Figueroa, 2008, D. bullisi (Bigelow and Schroeder, 1962), D. ecuadoriensis (Beebe and Tee-Van, 1941), D. leptocauda (Krefft and Stehmann, 1975), D. mennii Gomes and Paragó, 2001, D. teevani (Bigelow and Schroeder, 1951), D. trachyderma (Krefft and Stehmann, 1975) and Z. chilensis. Weigmann (2016), based on White (pers. comm. 2014), also assigned D. argentinensis to Zearaja, considering molecular data provided in the original description of the species. Nonetheless, the main morphological characteristic that differentiates Dipturus and Zearaja species is clasper morphology, which is still unknown for D. argentinensis. Therefore, we continue using the original designation until further studies of the clasper morphology are provided. Within the last few years, two more species had been added to the Southwest Atlantic (SWA): Z. brevicaudata (Marini, 1933) and D. lamillai Concha, Caira, Ebert and Pompert, 2019. The former is distributed in the SWA, from southern Brazil (34°S) to southern Argentina (Gabbanelli et al. 2018). Although Z. brevicaudata was described in 1933 (under the name Raia brevicaudata Marini, 1933), it was identified as Z. chilensis for almost a century, since it was placed in synonymy page 2 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan with this species by Norman (1937). Recently, Raia brevicaudata was resurrected from synonymy with Z. chilensis from the Southeast Pacific (SEP) and placed in Zearaja based on an integrative taxonomic analysis including DNA barcode (Cytochrome oxidase subunit I, COI), external morphology and skeletal features (Gabbanelli et al. 2018). Dipturus lamillai was described, based on morphological and molecular analyses of the mitochondrial gene NADH2, from specimens collected around the Malvinas/Falkland Islands in the SWA (Concha et al. 2019). According to the authors, individuals of this species had previously been identified as Z. flavirostris by Naylor et al. (2012). Concha et al. (2019) also redescribed Z. chilensis and designated a neotype for this species. The description of D. lamillai was largely based on the comparison of both morphological and molecular features of Z. chilensis from Chile. Concha et al. (2019) also compared, at the morphological level, D. lamillai with three congeners reported from waters off Malvinas/ Falkland Islands: D. argentinensis, D. leptocauda, and D. trachyderma. However, a comprehensive comparison with the recently resurrected Z. brevicautada, the most abundant and widespread longnose skate species from the SWA, was not provided. Indeed, Concha et al. (2019) only compared the coloration of D. lamillai with that of the holotype of Z. brevicaudata from the original description of Marini (1933) and with the coloration described by Gabbanelli et al. (2018). In addition, Concha et al. (2019) stated that morphometric comparisons between D. lamillai and Z. brevicaudata were difficult to make. The reasons the authors provided were that measurements presented by Gabbanelli et al. (2018) combined juvenile, subadult, and adult specimens of both sexes and this did not allow comparisons of the same size or sex of both species. In addition, Concha et al. (2019) pointed out that “although measurements are presented for the holotype specimen, that specimen is a juvenile female (32.4 cm in TL) and those measurements are not comparable to those for the holotype of D. lamillai sp. nov., which is an adult male.” Since Gabbanelli et al. (2018) and Concha et al. (2019) did not use the same genetic markers (COI and NADH2, respectively), molecular comparisons between both species have not been made. Interestingly, Concha et al. (2019) pointed out that the two complete mitochondrial genomes registered in GenBank under nos. KJ913073 and KF648508 (both identified as Z. chilensis) represented Z. chilensis and D. lamillai, respectively. One of the mitochondrial sequences (KJ913073) was obtained from a specimen collected in Chile (Vargas-Caro et al. 2014). The other sequence (KF648508), was obtained from a raw fillet sample of Z. chilensis collected from a restaurant in Korea (Jeong and Lee 2016). As expected, the mitochondrial genome contains, in addition to other genes, both the COI and the NADH2 genes, that could be also used to identify the specimen KF648508. Given the economic importance of longnose skates in SWA fisheries (Agnew et al. 2000; Colonello et al. 2002; Massa et al. 2004a b; Cedrola et al. 2005; Estalles et al. 2011; Arkhipkin et al. 2012) and their vulnerability to overexploitation (Stevens et al. 2000; Frisk et al. 2001; Dulvy et al. 2014), it is necessary to make a thorough taxonomic comparison between Z. brevicaudata and D. lamillai in order to determine whether both correspond to the same or different species. Therefore, the objective of this study is to compare these two nominal species through an integrative morphometric and molecular analysis. MATERIALS AND METHODS Examined material The examined material is listed in table S1. Given the impossibility of analyzing the type material as a consequence of global COVID-19 pandemic travel restrictions, data of D. lamillai were taken from Concha et al. (2019). These data constituted linear morphometric variables, expressed as a percentage of total length (TL), taken from the holotype (FA-46: mature male, 787 mm TL) and paratypes (FA-39: mature male, 915 mm TL, FA-47: mature female, 942 mm TL) of D. lamillai and were compared to the data from 69 specimens of Z. brevicaudata (216–1017 mm TL), including the holotype of Raia brevicaudata (MACN-ict569). Fortyseven of these specimens were previously analyzed in Gabbanelli et al. (2018), and the rest were revised for this study, (Table 1, Table S1). The maturation stage of individuals was assigned and they were classified as neonates, juveniles, subadults and adults. Females were dissected after examination to observe oviductal glands and oocytes size, and clasper calcification was registered in males following Paesch and Oddone (2008) and Colonello and Cortés (2014). Some of these specimens are stored at collections in different institutions, including: Museo Argentino Bernardino Rivadavia, Buenos Aires, Argentina (MACN), Universidad Nacional de Mar del Plata, Mar del Plata, Argentina (UNMDP), Instituto Nacional de Desarrollo Pesquero, Mar del Plata, Argentina (INIDEP), Instituto de Ciencias del Mar, Barcelona, España (IIPB). Specimens not stored in collections were analyzed under the field code RM (Table S1). For comparative purposes, data from 15 specimens of Z. chilensis were taken into account, and some of the specimen are stored page 3 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan at the ichthyological collection of the Museo Nacional de Historia Natural, Santiago de Chile (MNHN) (Table S1). Morphological analyses Morphological comparisons between D. lamillai and Z. brevicaudata were carried out. The variables were taken following the same measurement methodology of Last et al. (2008). Relationships between variables that were included in the diagnosis of D. lamillai were calculated for Z. brevicaudata (from the original measurements in mm), and compared with the results obtained by Concha et al. (2019). A normalization technique to scale the data that exhibited allometric growth was employed following Lleonart et al. (2000). This method was derived from theoretical equations of allometric growth and completely removes all information related to size, not only scaling all individuals to the same size, but also adjusting their shape to a standard form according to allometry. Total length (TL) was used as the independent variable, while the remaining measurements were considered dependent variables. TLo represents a reference value of size (570 mm for this study, regarding the TL of the specimens included in the analysis) to which all individuals were either reduced or amplified (Lleonart et al. 2000; Konan et al. 2010; Orlando et al. 2015; González-Castro et al. 2016; Mabragaña et al. 2020). After transformation, a new matrix was constructed, which contained the corrected matrices for each species, and a Principal Component Analysis (PCA) was performed with the transformed variables using the FactoMineR package (Lê et al. 2008) in the software R 4.0.2 (R Core Team 2020). Data from neonates were excluded and only data from juveniles and adults were considered for this analysis. Measurements of 14 juveniles and one adult of Z. chilensis were also included in the analyses for comparative purpose. In addition, thorn patterns and denticle distribution following Gravendeel et al. (2002), color, and clasper morphology following Hulley (1972) of the two nominal species were compared. Molecular analyses COI analyses Sequences of Cytochrome oxidase subunit I (COI) of D. argentinensis (n = 7), Z. brevicaudata (n = 25), Z. chilensis (n = 10), Z. nasuta (Banks, 1841) (n = 15), D. lamillai (n = 14) were included in the molecular analyses. In addition, the placement of the COI sequence from the complete mitochondrial genome of Z. chilensis sequenced by Jeong and Lee (2016) (KF648508) was analyzed. Sequences of D. argentinenesis, Z. brevicaudata and two sequences of Z. chilensis belong to Laboratorio de Biotaxonomía Morfológica y Molecular de Peces, Instituto de Investigaciones Marinas y Costeras (CONICETUNMdP), all sequences from D. lamillai belong to Chondrichthyan Tree of Life Project, all sequences of Z. nasuta and eight sequences of Z. chilensis were downloaded from Barcode of Life Data System (BOLD), and a sequence of KF648508 was downloaded from GenBank. Sequences of COI and NADH2 used in this work are listed in the tables S2 and S3, respectively. Sequences were aligned by Muscle (Edgar 2004) in the Table 1. Data of specimens of Zearaja brevicaudata and Z. chilensis analyzed Stage Sex N TL range Zearaja brevicaudata Neonates M 2 216–228 F 1 254 Juveniles M 24 310–617 F 25 324–705 Subadults M 4 645–776 F 2 815–942 Adults M 3 787–884 F 8 760–1017 Zearaja chilensis Juveniles M 8 294–534 F 6 305–507 Adults M 1 896 F: female, M: male, N: number of specimens, TL: total length, in mm. page 4 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan software MEGA X (Kumar et al. 2018). A best-fit model of nucleotide evolution was estimated using Akaike information criterion in MEGA X and was determined to be Kimura twoparameter (K2P). Distance divergences were calculated using K2P. A Maximum Likelihood (ML) was carried out for cluster analyses using K2P, and 1000 bootstrap pseudoreplicates were conducted to estimate node support values. A sequence of the rajid species Amblyraja doellojuradoi (Pozzi, 1935), from the Argentine Sea, was used as the outgroup. Distance divergences and ML analyses were performed in MEGA X. The COI sequences of D. lamillai and KF648508 were compared with BOLD, and BIN assignment was analyzed. The BIN analysis clusters barcode sequences algorithmically to create Operational Taxonomic Units (OTUs) that show high concordance with species. Species limits were also explored using the Automatic Barcode Gap Discovery method (ABGD) (Puillandre et al. 2012) and the Bayesian implementation of the Poisson tree processes (bPTP) (Zhang et al. 2013). The ABGD automatically finds the distance at which a barcode gap occurs and sorts the sequences into putative species based on this distance. Therefore, as with BIN analysis, ABGD is applicable as an independent tool without an a priori species hypothesis, and it provides insight into whether the taxonomic identification based on morphological features has any genetic support. The ABGD was run with the default settings (P min = 0.001, P max = 0.1, steps = 10, X relative gap width = 1.5, Nb bins = 20) and K2P distance on the web server https://bioinfo.mnhn.fr/abi/public/abgd/. The bPTP uses nucleotide substitutions to estimate intraand interspecific processes. It identifies the shift among intraand interspecific processes by using one parameter for coalescence and another for speciation. It was conducted at the web server https://species. h-its.org/ptp/; the parameters for the run were 100,000 MCMC generations, a thinning interval of 100 and 10% of burn-in. NADH2 analyses Sequence data from the protein-coding gene NADH dehydrogenase subunit 2 (NADH2) was generated for Z. brevicaudata in order to compare them with those of D. lamillai. Tissue samples of Z. brevicaudata collected in the SWA, Argentine Sea (n = 4) were stored in 95% ethanol prior to DNA extraction using the E.Z.N.A. Tissue DNA Kit (Omega BioTek, Inc., Georgia USA). Universal primers were used to amplify the mitochondrial NADH2 fragment by Polymerase Chain Reaction (PCR) for all samples prior to purification and Sanger sequencing, following Naylor et al. (2012). Sequences of NADH2 provided by Concha et al. (2019) were downloaded from Genbank (MK613969, MK613970, MK613971, MK613972, MK613973, MK613967, MK613966, MK613968) as well as the NADH2 sequence from the complete mitochondrial genome sequenced by Jeong and Lee (2016) (KF648508). Also, other sequences identified as D. lamillai belonging to the Chondrichthyan Tree of Life Project were taken into account. For comparative purposes, sequences of D. argentinensis, Z. chilensis, Z. nasuta, and D. olseni (Bigelow and Schroeder, 1951) (as outgroup) were included in the following analyses (sequence data in Table S3). DNA sequences were aligned and distance divergences were calculated using K2P. Maximum Likelihood (ML) analysis of clusters was carried out using the GTR+G model, as it was determined to be the best model under Akaike criterion and 1000 rapid bootstrap replicates. These analyses were performed in MEGA X (Kumar et al. 2018). RESULTS Morphological analyses Morphometrics The specimens of Z. brevicaudata analyzed ranged from 216 to 1017 mm TL, and included neonates, juveniles, and adults. Those of D. lamillai ranged from 485 to 1140 mm, but measurements were given only for the adult holotype and paratypes, which ranged from 787 to 940 mm TL (Table 2). Table 2 summarizes measurements expressed as percentages of TL of D. lamillai and Z. brevicaudata. No variables distinguish D. lamillai from Z. brevicaudata, since all measurements of D. lamillai are included within the range of Z. brevicaudata. On the other hand, table 3 summarizes the relationships between variables included in the diagnosis and description of D. lamillai for both species. In the same way, all respective values of D. lamillai are within the range of Z. brevicaudata. Moreover, the values are the most similar when only adult ranges of the two species are compared (Table 2 and 3). Ratios of tail length in relation to TL for males and females of Z. brevicaudata range from 38.4 to 40.3% TL and from 36.3 to 38.4% TL, respectively, similar to the data for D. lamillai by Concha et al. (2019) (37.9% and 38.4% TL for two males, 35.1% TL for one female). The PCA generated for standardized body measurements, including Z. brevicaudata, Z. chilensis, and D. lamillai, produced three eigenvalues greater than 1 (data not shown). The first three principal components explained 44.3% of the variance of morphometric data (24.8%, 12.6%, and page 5 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan 6.8%, respectively). The PCA allowed a clear separation of individuals from the SEP and the individuals from the SWA when comparing PC1 vs. PC2, and with a small degree of overlap when comparing PC1 vs. PC3 (Fig. 1). Specimens from the SEP corresponded to Z. chilensis and specimens from the SWA corresponded to Z. brevicaudata and D. lamillai. Specimens of Z. brevicaudata and D. lamillai appeared together in one group, and there was no differentiation between them. Moreover, the three individuals of D. lamillai appeared scattered between specimens of Z. brevicaudata (Fig. 1a). Correspondingly, both nominal species differed from Z. chilensis by the same characteristics: larger preorbital length, greater snout-spiracle distance, larger head length (dorsal and ventral), greater spiracle length and the distance between them, greater snout to cloaca distance, larger preoral and prenasal length, and shorter cloaca to tail tip distance (Table 4). The variables with higher loadings that differentiate SWA specimens from those of the SEP are related to snout length. This strongly suggests that both D. lamillai and Z. brevicaudata have a longer snout and a shorter tail than specimens of Z. chilensis. Thorn and denticle patterns Thorn data of D. lamillai (Concha et al. 2019) were compared with those of Z. brevicaudata (Table 5). The thorn pattern of both species is similar, with thorns in orbital, nuchal, dorsal and caudal regions in all individuals, and in malar and alar regions in adult males. Moreover, the number of thorns present in each body Fig. 1. Principal component analysis based on morphological data expressed as percentage of total length. a) PC1 vs PC2 b) PC2 vs PC3. PC1: first principal component, PC2: second principal component, PC3: third principal component. ▲, ellipse with whole line: Zearaja brevicaudata; ■, holotype of Raia brevicaudata; ●, ellipse with dotted line: Z. chilensis; ◊, ellipse with dashed line: Dipturus lamillai. page 6 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan Table 2. Measurements for Dipturus lamillai (3 types) and Zearaja brevicaudata (holotype and 68 non-type specimens) expressed as percentages of total length. Clasper measurements are given for adult males of Z. brevicaudata (n = 3) Dipturus lamillai Zearaja brevicaudata Holotype FA-46 Paratype FA-39 Paratype FA-47 Holotype MACN-ict569 Total range Adults mm % Range Mean SD Range Mean SD Sex M M F F Total length (TL, mm) 787 915 940 324.0 – 216.0–1017.0 – – 760.0–1017.0 – – Measurements in % TL Disc width 75.2 73.2 78.9 250.0 77.2 61–83.1 77.3 3.3 74.3–82.3 77.3 2.8 Direct disc length 63.8 59.3 65.5 200.0 61.7 55.3–74.8 62.3 2.5 60–74.8 64.6 4.3 Snout to maximum width 41.7 39.1 45.2 122.0 37.7 32.3–42 37.3 2.3 35–41.4 39.1 1.8 Direct preorbital length 20.2 19.7 23.4 62.0 19.1 16.5–24 20.5 1.3 18.7–24 21.2 1.4 Snout to spiracle 23.5 23.7 26.3 81.5 25.2 21.8–30.1 25.9 1.4 24–30.1 26.8 1.6 Dorsal head length 27.2 25.4 28.2 84.4 26.1 23–31.1 27.3 1.4 26.2–31.1 28.3 1.4 Orbit diameter 2.8 3.6 2.7 14.6 4.5 3–4.8 3.8 0.4 3.4–4.8 3.8 0.5 Orbit and spiracle length 5.0 5.8 5.4 18.7 5.8 4.9–6.6 5.5 0.3 5.2–6.6 5.6 0.4 Spiracle length 2.7 2.1 2.7 7.4 2.3 1.7–3.1 2.5 0.3 1.9–3.0 2.6 0.4 Distance between orbits 6.4 6.4 6.6 16.1 5.0 4.6–6.8 5.6 0.5 5.7–6.8 6.2 0.4 Distance between spiracles 7.8 8.1 7.7 25.1 7.8 6.6–8.9 7.3 0.4 7.4–8.9 7.9 0.5 Distance snout to cloaca 61.6 61.7 64.9 183 56.5 49.1–69 59.4 3.3 60.3–69 63.4 2.5 Cloaca to tail tip 38.4 38.3 35.1 140.3 43.3 36.3–49.5 40.9 2.6 36.3–42 38.1 1.9 Preoral length 19.1 17.5 22 64.2 19.8 16.4–23.5 20.1 1.4 17.8–23.4 20.1 1.5 Direct prenasal length 17.9 16.7 20.7 60.7 18.7 15.1–21.7 18.5 1.2 16.5–21.7 18.7 1.4 Direct ventral head length 36.3 35.0 32.7 110.4 34.1 30–40.4 35.6 1.8 35.2–40.4 37.1 1.6 Mouth width 9.3 9.7 10.3 30.6 9.4 8.3–11 9.5 0.4 9.4–10.6 9.9 0.3 Distance between nostrils 9.4 9.6 9.7 30.4 9.4 7.6–10.6 9.5 0.5 9.3–10.6 9.8 0.4 Nasal curtain length 4.9 4.7 5.0 13.3 4.1 3.8–6 4.7 0.5 4.8–6 5.3 0.4 Nasal curtain maximum width 10.2 11.4 10.0 31.4 9.7 8.3–11.1 9.8 0.6 9.9–11.1 10.4 0.4 Width of first gill opening 2 1.5 2.1 4.5 1.4 0.9–2.5 1.7 0.2 1.4–2.1 1.7 0.2 Width of fifth gill opening 1.7 1.7 2.0 5.0 1.5 1.2–2.2 1.6 0.2 1.3–1.9 1.6 0.2 Distance between first gill openings 15 16.4 16 53.5 16.5 13.8–17.8 16.3 0.9 15.6–17.8 16.9 0.8 Distance between fifth gill openings 9.4 9.8 11.1 34.0 10.5 8.4–11.8 10.3 0.8 8.8–11.8 10.6 1.0 Length of anterior pelvic lobe 14.2 13.4 13.3 42.7 13.2 10.8–15.3 12.9 1.1 10.8–13.8 12.1 1.0 Length of posterior pelvic lobe 20.7 20.4 17.8 49 15.1 11.4–20.2 15.7 1.4 14.5–20.2 17.1 2.1 Pelvic base width 9.4 9.6 10.9 38.1 11.8 9.9–18.5 13.9 1.8 12–18.5 15.6 2.1 Tail width at axis of pelvic fin 4.1 4.0 3.9 13.5 4.2 2.7–5 4.0 0.5 3.2–4.6 4.0 0.5 Tail height at axis of pelvic fin 2.4 2.4 2.3 13.2 4.1 1–4.1 2.4 0.4 2.1–3.1 2.5 0.3 Tail width at tail midlength 2.6 3.0 2.7 7.6 2.3 1.4–3.2 2.4 0.4 1.6–3 2.5 0.5 Tail height at tail midlength 1.4 1.7 1.4 5.3 1.6 0.8–2.1 1.4 0.2 1.2–1.7 1.5 0.2 Tail width at base of D1 2.5 2.4 2.4 9.1 2.8 1.2–3 2.2 0.4 1.6–2.3 2.2 0.4 Tail height at base of D1 1.5 1.5 1.5 4.7 1.5 0.8–1.7 1.4 0.2 1.1–1.6 1.4 0.2 D1 base length 5.1 5.3 4.3 18.3 5.6 4.1–6.9 5.2 0.4 4.2–5.8 5.0 0.4 D1 height 4.3 3.2 3.9 11.4 3.5 2.8–4.8 3.8 0.4 3.4–4.7 4.0 0.5 Origin of D1 to tail tip 14.3 12.6 12.9 55.6 17.2 11.4–21.5 15.8 1.7 12.7–16.8 14.4 1.3 Origin of D2 to tail tip 6.6 6.9 7.1 33.4 10.3 5.1–14.2 9.2 1.5 5.7–9.7 8.1 1.4 Caudal fin length 2.3 2.4 2.3 13.2 4.1 1.7–7.6 4.0 1.0 1.7–5.2 3.3 1.1 Caudal fin height 0.7 0.6 0.2 2.7 0.8 0.1–1.2 0.7 0.2 0.5–1 0.6 0.2 Post cloaca clasper length 31.6 29.4 - - - - 29.1–30 - - Cloaca to clasper insertion 11.9 11.4 - - - - 12.1–12.3 - - SD: standard deviation; M: male, F: female, D1: first dorsal fin; D2: second dorsal fin. page 7 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan region coincides between both species. Specimens of D. lamillai had three rows of tail thorns, as described for Z. brevicaudata. In the female paratype of D. lamillai a single lateral tail thorn above the lateral tail fold was found, and it was also observed in Z. brevicaudata. The female paratype of D. lamillai was described as having a continuous median row of thorns on trunk and tail, as well as parallel trunk thorns behind the shoulder girdle (Concha et al. 2019). Eight specimens, seven females and one male (688–1017 mm TL), of Z. brevicaudata were found to have a thorn pattern similar to that observed in the female paratype of D. lamillai. All of them presented parallel rows of thorns on the body, and two adult females presented median trunk thorns (Fig. 2). Moreover, the same sexual dimorphism was observed in parallel trunk thorns and lateral tail thorns, which had a wider range in females. With respect to malar thorns, adult males of Z. brevicaudata presented 3–6, while adult males of D. lamillai presented 6–10. The denticle pattern described for D. lamillai (Concha et al. 2019) matched the pattern of Z. brevicaudata (Gabbanelli et al. 2018). Individuals from both nominal species presented denticles on the head, dorsally from tip of snout to the posterior part of neurocranium, ventrally from tip of snout to about level of first gill slits, around cloaca in adults, on the anterior margin of the dorsal fins, and completely covering the caudal fin. In addition, both nominal species did not have denticles on the pectoral and pelvic fins, and on the tail, either ventrally or dorsally. Coloration Concha et al. (2019) indicated a coloration for D. lamillai different from the pattern described for Z. brevicaudata by Gabbanelli et al. (2018). Nevertheless, similarities in the descriptions were observed: both nominal species presented an ocellus on the base of each pectoral fin and the area flanking the rostral cartilage was translucent. Additionally, pale fuzzy circles were described for Z. brevicaudata, which were consistent with the light brown spots also described for D. lamillai as well. Moreover, new specimens of Z. brevicaudata were analyzed for the present study and some of them presented a coloration similar to that described for the female paratype of D. lamillai (Fig. 3). Clasper morphology Claspers of both nominal species did not differ in their shape and showed the same external components. However, two additional components of the claspers were described for D. lamillai: spur and slit (Concha et al. 2019). These components were not indicated for Z. brevicaudata (Gabbanelli et al. 2018). Nevertheless, after re-examination of the claspers of Z. brevicaudata the component spur and slit were both present. The spur is the external part of the dorsal terminal cartilage Table 3. Relationships between measurements used in the diagnosis and description of Dipturus lamillai, based on the holotype (FA-46) and paratypes (FA-39, FA-47) of D. lamillai and specimens of Zearaja brevicaudata (n = 69). D1: first dorsal fin Dipturus lamillai Zearaja brevicaudata FA-46 (FA-39:FA-47) Total range Adults Diagnosis Preorbital snout length/distance between orbits 3.2(3.1:3.5) 2.9–4.3 3.1–3.6 Orbit diameter/interorbital distance 0.4(0.6:0.4) 0.5–0.9 0.5–0.7 Description Disc width/disc length 1.3(1.4:1.3) 1.2–1.4 1.2–1.3 Preorbital snout length/orbit length 7.2(5.5:8.7) 1.1–6.6 4.5–6.6 Preorbital snout length/interorbital distance 3.2(3.1:3.5) 2.9–4.3 3.1–3.6 Spiracles/orbit diameter 1.1(1.7:1) 1.1–2.2 1.2–1.8 Distance 1st gills/internasal distance 1.6(1.7:1.7) 1.4–2 1.6–1.9 Distance 5th gills/internasal distance 1(1:1.1) 0.6–1.3 0.9–1.2 Anterior lobe/posterior lobe 0.7(0.7:0.8) 0.6–1.1 0.6–0.8 Cloaca to caudal fin-snout to cloaca 0.6(0.6:0.5) 0.6–2.2 0.6–0.7 Width at pelvic fin axils/width at midlength tail 1.6(1.4:1.4) 1.1–2.8 1.3–2 Width at pelvic fin axils/width at D1 1.6(1.7:1.6) 1.1–3.7 1.4–2.6 Width at pelvic fin axils/height at pelvic fins axils 1.7(1.7:1.7) 1–4 1.2–1.8 Width at pelvic fin axils/height at 1/2 tail 2.8(2.3:2.8) 1.4–3.8 2.5–2.9 Width at pelvic fins axils/height D1 2.8(2.6:2.6) 2.1–4 2.3–3.4 D1 height/base length 1.2(1.1:1.1) 0.5–1.1 0.7–1 page 8 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan Table 4. Variable loadings for the first three axes of a principal component analysis for standardized measurements of specimens of Zearaja brevicaudata and Dipturus lamillai. The highest loadings are indicated in bold. D1: first dorsal fin, D2: second dorsal fin PC1 PC2 PC3 Disc width 0.6 0.4 0.0 Direct disc length 0.6 0.2 0.2 Snout to maximum width 0.2 0.1 0.1 Direct preorbital length 0.9 0.0 0.1 Snout to spiracle 0.9 0.1 0.2 Dorsal head length 0.9 0.1 0.2 Orbit diameter -0.2 0.3 -0.2 Orbit and spiracle length -0.3 0.6 -0.1 Spiracle length -0.3 0.5 0.2 Distance between orbits 0.2 0.3 -0.2 Distance between spiracles 0.3 0.4 -0.6 Snout to cloaca 0.9 0.2 0.0 Cloaca to tail tip -0.9 0.0 0.2 Preoral length 0.9 0.0 0.1 Direct prenasal length 0.9 0.0 0.1 Direct ventral head length 0.3 0.6 0.5 Mouth width -0.1 0.5 0.2 Distance between nostrils 0.2 0.6 0.1 Nasal curtain length 0.0 0.2 0.0 Nasal curtain maximum width -0.3 0.5 0.1 Width of first gill opening -0.3 0.3 0.2 Width of fifth gill opening -0.2 0.4 0.0 Distance between first gill openings -0.5 0.5 0.4 Distance between fifth gill openings 0.4 0.4 0.3 Length of anterior pelvic lobe 0.4 0.2 -0.1 Length of posterior pelvic lobe -0.1 0.3 -0.3 Pelvic base width 0.5 0.3 0.0 Tail width at axis of pelvic fin 0.3 0.4 -0.3 Tail height at axis of pelvic fin -0.1 0.4 -0.1 Tail width at tail midlength -0.1 0.3 -0.6 Tail height at tail midlength -0.2 0.5 -0.2 Tail width at base of D1 fin -0.2 0.3 -0.6 Tail height at base of D1 fin 0.2 0.4 -0.5 D1 base length -0.6 0.5 -0.1 D1 height -0.1 0.5 0.1 Origin of D1 to tail tip -0.7 0.2 0.3 Origin of D2 to tail tip -0.6 0.2 0.3 Caudal fin length -0.6 0.2 0.3 Fig. 2. Dorsal spinulation pattern of an adult female of Zearaja brevicaudata (RM 170, 943 mm TL). White arrows point the dorsal thorns. Scale bar = 50 mm. page 9 of 19Zoological Studies 61:76 (2022) © 2022 Academia Sinica, Taiwan will confound our assessments of the population status of Z. brevicaudata which has recently been assessed as “Vulnerable” (Pollom et al. 2021). Unfortunately, the Covid-19 pandemic prevented our examination of the type material for the current work. This is a task that must be done in the future when safe travel becomes possible again. However, considering the importance of taxonomy in properly assessing the conservation status of Z. brevicaudata, the most exploited skate species in the Southwest Atlantic, the synonymization of D. lamillai with Z. brevicaudata is timely. CONCLUSIONS The present study combined morphological and molecular analyses, with the implementation of both COI and NADH2 genes, that allowed the integrative comparison between Z. brevicaudata and D. lamillai, and consequently, their synonymization. It demonstrated the importance of accurate taxonomic research and the impact it may have on species conservation. In this sense, this research contributes baseline information to the conservation of a fishery resource by clarifying taxonomic issues within the longnose Zearaja skates occurring off the coasts of South America. Acknowledgments: This research is part of V. Gabbanelli Ph.D at Universidad Nacional de Mar del Plata and CONICET scholarship. This work was supported by CONICET (PIP No. 11220200101475CO), MINCYT (PICT-2018-0790, PICT-2018-2974) and Universidad Nacional de Mar del Plata (EXA 970/20). We thank Ph. D Flávia Petean for external advice. We thank to the anonymous reviewers and Editors for offering helpful comments that improved the previous versions of the manuscript. Authors’ contributions: Specimen collection and identification: VG, EM. Morphological description: VG, EM, SW. Molecular analyzes: VG, EM, GN, LY. Language revision: PL, GN, SW. Manuscript writing: VG, GN, SW, EM, JMDA, PL, DMV. Funding: EM, JMDA, GN. All authors approved the final manuscript and consent to publication. Competing interests: All authors declare that they have no conflict of interests. Availability of data and materials: Specimens are stored at the Universidad de Mar del Plata Fish Collection, sequences were deposited into BOLD Data System and GenBank. Consent for publication: The authors give their consent to publish. Ethics approval consent to participate: Ethics approval does not apply. REFERENCES Agnew DJ, Nolan CP, Beddington JR, Baranowski R. 2000. 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Sequences information of the Cytochrome oxidase subunit I mitochondrial gene used in the present paper, corresponding to the following species Dipturus argentinensis, Z. brevicaudata, Z. chilensis, Z. nasuta, and Amblyraja doellojuradoi. BOLD: Barcode of Life Data System, PN: pending number. (download) Table S3. Sequence information of the NADH dehydrogenase subunit 2 mitochondrial gene, used in the present paper, corresponding to the following species: Dipturus argentinensis, Z. brevicaudata, Z. chilensis, Z. nasuta y D. olseni. PN: pending Number. (download) page 19 of 19Zoological Studies 61:76 (2022)