The Spanish Toothcarp (Apricaphanius iberus) is a small fish species endemic to the eastern coast of the Iberian Peninsula. Its area of distribution includes a variety of salt and freshwater habitats that experience large fluctuations in temperature and salinity throughout the year. The Spanish Toothcarp belongs to the Cyprinodontiformes order and to the family Aphaniidae. It is currently considered “Endangered” (category IUCN: EN), i.e., facing a very high risk of extinction in the wild. The genetics of A. iberus are not well known since most studies have only evaluated the genetic structure of the species under a conservation framework in order to identify its potential conservation units. In this study, the whole mitochondrial genome of A. iberus was obtained for the first time in the context of an A. iberus genome reference sequencing. The mitogenome was reconstructed and aligned against 83 other cyprinodontiformes and two outgroup taxa. From this, a phylogenetic reconstruction was created using PartitionFinder to test the best evolutionary model for both the 13 protein coding genes and two non-coding ribosomal genes. Following this, a phylogenetics analysis was conducted using two methods: Maximum-Likelihood approximation (IQTree) including bootstrap branching support and Bayesian inference (MrBayes) applying the partitions models obtained. By doing so, it was possible to find the molecular position of A. iberus within the Cyprinodontiformes order. The results showed A. iberus grouped together with Orestias ascotanensis and both species formed a sister group with North American and Caribbean cyprinodontiformes. These new data will be valuable for a better understanding of the evolution of A. iberus and will be highly useful for future genetic studies., This research was funded by Ministerio de Ciencia e Innovación (PID2019-103936GB-C22)., Peer reviewed

[Background]: The Spanish toothcarp (Aphanius iberus Valenciennes, 1846) is a small fish endemic to the eastern coastline of the Iberian Peninsula and is currently listed as "Endangered" (category IUCN: EN). It mainly inhabits brackish waters which can exhibit large fluctuations in temperature and salinity throughout the year. The genetics of A. iberus are not well-known since most studies have only evaluated the genetic structure of the species under a conservation framework in order to identify its potential conservation units. Different phylogenetic relationships of Aphanius have been published based on some particular genes. In the present study, the entire mitochondrial genome of A. iberus was obtained for the first time in the context of an A. iberus reference genome and a hypothesis regarding its phylogenetic position was considered., [Methods and results]: The mitogenome (a circular doble-stranded DNA sequence of 16,708 bp) was reconstructed and aligned against 83 Cyprinodontiformes and two outgroup taxa to identify the phylogenetic position of A. iberus. PartitionFinder was first used to test for the best evolutionary model and the phylogenetic analyses were performed using two methods: Maximun-Likelihood Approximation (IQ-Tree) and Bayesian inference (MrBayes). Our results show that A. iberus forms a sister group with Orestias ascotanensis, a cyprinodontiform species native to South America., [Conclusions]: The results were congruent with the traditional morphometric reconstructed trees and with a geological vicariant hypothesis involving Cyprinodontiformes where Aphaniidae is shown as a monophyletic family separated from the family Cyprinodontidae. The information gathered from this study is not only valuable for improving our understanding of the evolutionary history of A. iberus, but for future genomic studies involving the species., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study was funded by the Ministerio de Ciencia, Innovación y Universidades through a National Project titled “Conservation biology of endangered endemic cyprinodontiform fishes” (APHANIUS) (PID2019-103936 GB-C22). Alfonso López-Solano was granted a predoctoral contract by the same Ministry and project. Tessa Nester was granted a predoctoral contract by the same Ministry through the University Professor Training (FPU) Programme., Peer reviewed

[EN] Three new species, Achondrostoma garzonorum sp. nov., Achondrostoma asturicense sp. nov. and
Achondrotoma numantinum sp. nov. are described on the basis of morphological and genetic characters.
Achondrostoma garzonorum sp. nov. is restricted to the Cuerpo de Hombre and Alagón rivers (Tajo Drainage,
Alagón sub-basin) and Corneja river (Duero Drainage, Tormes sub-basin) in western Spain. Achondrostoma
garzonorum sp. nov. can be distinguished from other Achondrostoma species through a combination of
morphometric, meristic and genetic characters: 43-49 ( ¯x = 45.9; Md = 46) canaliculate scales on the lateral line; 6-7 ( ¯x = 6.8; Md = 7) scales above the lateral line; 4-5 ( ¯x = 4.4; Md = 4) scales below the lateral line; a high caudal peduncle in proportion to the body depth; 5-5 pharyngeal teeth, narrow and high coronoid process of dentary and 3 autapomorphies, none of them transversions, in the mitochondrial cytochrome b gene. Achondrostoma asturicense sp. nov. inhabits the sub-basin of the Esla river in the Duero Drainage of Spain. Achondrostoma asturicense sp. nov. can be differentiated from other Achondrostoma species through a set of morphometric, meristic and genetic characters: 38-44 ( ¯x = 40.9, Md = 41) canaliculate scales on the lateral line; 5-6 ( ¯x = 5.8,Md = 6) scales above the lateral line; 3-4 ( ¯x = 3.1, Md = 3) scales below the lateral line; a narrow skull; 5-5 pharyngeal teeth; and 13 autapomorphies in the mitochondrial cytochrome b gene, one of which is a transversion. Achondrostoma numantinum sp. nov. inhabits the Duero Drainage in Spain except for Esla and Sabor sub-basins and Corneja river within the Duero Drainage. Achondrostoma numantinum sp. nov. can be distinguished from other Achondrostoma species through a combination of morphometric, meristic and genetic characters: 43-48 ( ¯x = 45.1; Md = 45) canaliculate scales on the lateral line; 6-8 ( ¯x = 6.9; Md
= 7) scales above the lateral line; 4-5 ( ¯x = 4.3; Md = 4) scales below the lateral line; a wide skull; low caudal peduncle in proportion to the body depth; 5-5 pharyngeal teeth, and one autapomorphy, no transversion, in the mitochondrial cytochrome b gene., [ES] Se describen tres nuevas especies, Achondrostoma garzonorum sp. nov., Achondrostoma asturicense sp. nov. y Achondrotoma numantinum sp. nov., sobre la base de caracteres genéticos y morfológicos. La distribución de Achondrostoma garzonorum sp. nov. se restringe a los ríos Alagón y Cuerpo de Hombre (cuenca del Tajo, subcuenca del Alagón) y Corneja (cuenca del Duero, subcuenca del Tormes), oeste de España. Achondrostoma garzonorum sp. nov. se diferencia de otras especies del género Achondrostoma que viven en la Península Ibérica por una combinación de caracteres morfométricos, merísticos y genéticos, como 43-49 ( ¯x = 45.9; Md = 46) escamas canaliculadas en la línea lateral; 6-7 ( ¯x = 6.8; Md = 7) escamas por encima de la línea lateral; 4-5 ( ¯x = 4.4; Md = 4) escamas por debajo de la línea lateral, un pedúnculo caudal alto en comparación a la anchura del cuerpo; un alto y estrecho proceso coronoideo del dentario; 5-5 dientes faríngeos y 3 autapomorfias, ninguna de ellas transversiones, para el gen mitocondrial citocromo b. Achondrostoma asturicense sp. nov. vive en la subcuenca del río Esla dentro de la cuenca del Duero. Achondrostoma asturicense sp. nov. se diferencia de otras especies del género Achondrostoma que viven en la Península Ibérica por una combinación de caracteres morfométricos, merísticos y genéticos, como 38-44 ( ¯x = 40.9, Md = 41) escamas canaliculadas en la línea lateral;5-6 ( ¯x = 5.8, Md = 6) escamas por encima de la línea lateral; 3-4 ( ¯x = 3.1, Md = 3) escamas por debajo de la línea lateral, un cráneo estrecho, 5-5 dientes faríngeos y 13 autapomorfias, una de ellas una transversión para el gen mitocondrial citocromo b. Achondrostoma numantinum sp. nov. vive en los ríos de la cuenca del Duero excepto en las subcuencas de los ríos Esla y Sabor y en el río Corneja. Achondrostoma numantinum sp. nov. se diferencia de otras especies del género Achondrostoma que viven en la Península Ibérica por una combinación de caracteres morfométricos, merísticos y genéticos, como 43-48 (x¯ = 45.1; Md = 45) escamas canaliculadas en
la línea lateral; 6-8 (x¯ = 6.9; Md = 7) escamas por encima de la línea lateral; 4-5 (x¯ = 4.3; Md
= 4) escamas por debajo de la línea lateral; cráneo ancho; pedúnculo caudal estrecho en comparación con la anchura del cuerpo; 5-5 dientes faríngeos, y una autapomorfia, no transversión, para el gen mitocondrial citocromo b., This research study was funded by the Spanish Ministry of Science and Innovation and the State Agency of Investigation (MCIN/ AEI/ 10.13039/501100011033) as a part of the Project Aphanius (PID2019-103936GB-C22)., Peer reviewed

[Background]: Ictalurus is one of the most representative groups of North American freshwater fishes. Although this group has a well-studied fossil record and has been the subject of several morphological and molecular phylogenetic studies, incomplete taxonomic sampling and insufficient taxonomic studies have produced a rather complex classification, along with intricate patterns of evolutionary history in the genus that are considered unresolved and remain under debate., [Results]: Based on four loci and the most comprehensive taxonomic sampling analyzed to date, including currently recognized species, previously synonymized species, undescribed taxa, and poorly studied populations, this study produced a resolved phylogenetic framework that provided plausible species delimitation and an evolutionary time framework for the genus Ictalurus., [Conclusions]: Our phylogenetic hypothesis revealed that Ictalurus comprises at least 13 evolutionary units, partially corroborating the current classification and identifying populations that emerge as putative undescribed taxa. The divergence times of the species indicate that the diversification of Ictalurus dates to the early Oligocene, confirming its status as one of the oldest genera within the family Ictaluridae., This study was funded by the projects “Sistemática y taxonomía del género Ictalurus (Siluriformes:Pisces), en México (PRODEP-SEP2016/UMSNH-PTC-384)” and “Delimitación de especies dentro del género de peces dulceacuícolas Ictalurus (Siluriformes:Pisces) (CIC-UMSNH-2016-2017)”. This study was partly supported by the Spanish Ministry of Science and Innovation (PID2019-103936GB-C22). The funding body played no role in the design of the study and collection, analysis, interpretation of data, and in writing the manuscript. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature., Peer reviewed

Nemacheilid fishes in the genus Turcinoemacheilus are physically small members of the ichthyofauna communities of high-altitude and mountainous freshwater ecosystems. They are all distributed in Western Asia apart from a single species, described in the Himalayas. They are usually very similar in appearance, which complicates their proper identification and/or description. This is why it is important to use multidisciplinary and integrative taxonomical approaches in order to study their true diversity. In this study, three new species of Turcinoemacheilus are described from Iran, raising the total number of valid species to nine. Turcinoemacheilus ansari new species, is distinguished by the anus being situated behind the midpoint of the pelvic-fin and anal-fin origins and the short anal-fin base length. Turcinoemacheilus christofferi new species, differs by the anus being situated behind the midpoint of the pelvic-fin and anal-fin origins, with a complete lateral line reaching to the anterior part of the caudal fin. Turcinoemacheilus moghbeli new species, is distinguished by the anus being situated at or in front of the midpoint of the pelvic-fin and anal-fin origins, with a great pre-pelvic distance and a caudal peduncle length 1.5–2.3 times its length. In Western Asia, all Turcinoemacheilus species are well separated by molecular characters, showing between 3.6 and 14.1% uncorrected p genetic distances in the COI barcode region. This work shows the importance of studying the hidden diversity of under-sampled and understudied groups of organisms to have a clear image of true biodiversity in order to effectively conserve and protect it., This research was funded by a grant from the Royal Physiographic Society of Lund to HRG and the Spanish Ministry of Science and Innovation and the State Agency of Investigation project PID2019-103936GB-C22 to ID., Peer reviewed

Biodiversity conservation entails not only the preservation of specific taxa but also genetic diversity. Despite the crucial role of molecular data in freshwater fish conservation management, there is a scarcity of information regarding the genetic diversity of Luciobarbus Heckel, 1843 (Actinopterygii, Cyprinidae) populations in the Aral system. Therefore, the primary aim of this study was to provide genetic information on two native species of the Luciobarbus genus found in the Aral system: L. conocephalus (Kessler, 1872) and L. brachycephalus (Kessler, 1872). These species, like many others in the Aral system, confront the imminent threat of extinction due to system alterations. However, genetic studies on these species at the nuclear level are challenging because Luciobarbus is an allotetraploid genus. Consequently, genetic investigations thus far have focused mainly on sequencing mitochondrial genes due to their haploid nature. This study has successfully developed fifteen new polymorphic microsatellite loci, which can prove to be valuable for population genetics, conservation, and other pertinent research on these species, Ministerio de Ciencia e Innovación: PID2019-103936GB-C22., Peer reviewed

[Description of methods used for collection/generation of data] A hybrid assembly approach was employed, integrating PacBio long-read sequencing with Illumina short-read data, an extensive functional annotation of the genome is provided by using AUGUSTUS with InterProScan and Sma3s., [Methods for processing the data] Software MaSuRCA and HASLR. Also Transdecoder to predict candidate coding regions., The sequencing of non-model species has increased exponentially in recent years, largely due to the advent of novel sequencing technologies. In this study, we construct the reference genome of the Spanish toothcarp (Aphanius iberus (Valenciennes, 1846)), a renowned freshwater fish species. A hybrid assembly approach was employed, integrating PacBio long-read sequencing with Illumina short-read data. In addition to the assembly, an extensive functional annotation of the genome is provided by using AUGUSTUS, and two different approaches (InterProScan and Sma3s). The integration of genomic data with predicted genes presents future research opportunities across multiple disciplines, such us physiology, reproduction, disease, and opens up new avenues for future studies in comparative genomic studies., PID2019-103936GB-C22, Project Aphanius., Annotation_Aphanius_iberus.xlxs, Peer reviewed