Introduction
Kryptolebias is a killifish genus (family Rivulidae) composed of seven currently valid species (Berbel-Filho et al. 2022), although the number of species in the genus is likely to change as some taxonomic debates are still ongoing (Berbel-Filho et al. 2022; Huber 2016). Phylogenetic analyses have indicated the presence of two distinct monophyletic clades within Kryptolebias, one of them composed of narrowly distributed freshwater species living in temporary streams and pools in South America: K. campelloi (Costa 1990) from North Brazil; K. sepia Vermeulen & Hrbek 2005 from Suriname; K. gracilis Costa 2007, and K. brasiliensis (Valenciennes 1821) from Southeast Brazil. The other clade is composed of three species living in mangrove forests along the tropical and subtropical western Atlantic basin, the ‘mangrove killifish clade’: K. marmoratus (Poey 1880), K. hermaphroditus sensu Costa 2011, and K. ocellatus (sensu Costa 2011) (Berbel-Filho et al. 2022; Costa, Lima, and Bartolette 2010; Murphy, Thomerson, and Collier 1999; Tatarenkov et al. 2009, 2017).
Kryptolebias is a remarkable genus in many aspects. For instance, K. marmoratus and K. hermaphroditus sensu Costa 2011 are the only two vertebrates known to be capable of self-fertilization (Berbel-Filho et al. 2022), whereas K. ocellatus (sensu Costa 2011) is a hermaphroditic but obligate outcrossing species (Berbel-Filho et al. 2020). This variation in mating systems makes Kryptolebias a unique vertebrate system for investigating the genomic, physiological, and behavioral changes involved in the transition from outcrossing to selfing. In addition, K. marmoratus, the most well-studied Kryptolebias species, is considered a highly amphibious fish (Turko, Rossi, and Wright 2021), with extreme physiological and behavioral adaptations to live out of water, in some cases for months (Taylor 1990). The amphibious nature of K. marmoratus is also likely to be valid for other Kryptolebias species, providing unique opportunities for studying the phenotypic and genomic changes involved in the transition from aquatic to terrestrial habitats.
To avoid long-term taxonomic confusion, we would like to provide some background on the taxonomic status of K. ocellatus (Sensu Costa 2011), whose genome was sequenced here. Due to morphological similarities and syntopy between species, the taxonomic status of the mangrove killifish clade has been historically confusing, particularly in Southeast Brazil. Briefly, Rivulus ocellatus was initially described by Hensel (1868) using a single specimen from Rio de Janeiro, Brazil. Later, Seegers (1984) suggested the existence of two syntopic species in Rio de Janeiro: the hermaphroditic R. ocellatus as in Hensel (1868), and a yet undescribed species composed of hermaphrodites and males, named R. caudomarginatus. After taxonomic revision of the family Rivulidae, Costa (2004) reclassified some previously known Rivulus species (Rivulus brasiliensis, R. campelloi, R. caudomarginatus, R. ocellatus, and R. marmoratus) into a new genus called Kryptolebias. After morphological evaluation of the K. ocellatus holotype by Costa (2011) argued that the species originally described by Hensel as K. ocellatus was in fact K. caudomarginatus (as in Seegers (1984)). Therefore, K. caudomarginatus has become a junior synonym for K. ocellatus. The other syntopic species composed of selfing hermaphrodites was then named as K. hermaphroditus (Costa 2011). However, discussions on the taxonomic nomenclature of these mangrove killifish species are still ongoing (Huber 2016). This taxonomic connudrum is likely to be fully resolved only when the genetic data of the formalin-fixed K. ocellatus holotype, initially described by Hensel (1868), is available. For the genome generated here, we used the currently valid taxonomic classification, with the selfing species occurring from the Caribbean to Southeast Brazil, named K. hermaphroditus sensu Costa 2011, and the androdioceous outcrossing from South and Southeast Brazil, K. ocellatus (sensu Costa 2011) (Berbel-Filho et al. 2020, 2022).
Here we provide whole genome sequencing data for the mangrove killifish K. ocellatus (sensu Costa 2011) (Fig. 1a), and two freshwater Kryptolebias species: K. brasiliensis and K. gracilis (Fig. 1b and c, respectively). Although Kryptolebias ocellatus has no current classification of its conservation status, K. brasiliensis and K. gracilis are categorized as endangered and critically endangered species, respectively, by the Brazilian list of threatened fish species (MMA 2022).
_*kryptolebias_ocellatus*_(*sensu*__b_211146_)__(*b*)_*k._brasiliensis*__a.png)
Methods
Tissues from a single wild-collected individual per species were used in the present study. Kryptolebias ocellatus (sensu Costa 2011) was collected in the Piracão mangrove, Guaratiba, Rio de Janeiro, Brazil (23°0′1.90″S 43° 34′51.50″W). Kryptolebias brasiliensis was sampled in puddles close to the RPPN Campo dos Escoteiros, Magé, RJ, Brazil (22°34’21.06″ “S 43°02’5.16”″ W). Kryptolebias gracilis was sampled at São Lourenço stream, Saquarema, RJ, Brazil (22°52′46.90″S 42°37′58.40″W). All sampling locations were either the type locality or within the type locality area for the species. All sampling was performed under the license ICMBio/SISBIO 57145-4.
DNA extraction was performed with the Qiagen DNAeasy Genomic Extraction Kit using a standard process. A paired-end sequencing library was constructed using the Illumina TruSeq kit, according to the manufacturer’s instructions. The library was sequenced on an Illumina Hi-Seq platform in a paired-end, 2 × 150bp format. The resulting fastq files were trimmed of adapter/primer sequences and low-quality regions using Trimmomatic v0.33 (Bolger, Lohse, and Usadel 2014). The trimmed sequence was assembled using SPAdes v2.5 (Bankevich et al. 2012) followed by a final step using Zanfona (Kieras, O’Neill, and Pirro 2021).
Results and Data Availability
All raw read data and assembled genomes are available in the GenBank database.
Funding
Funding was provided by the Iridian Genomes, grant# IRGEN_RG_2021-1345 Genomic Studies of Eukaryotic Taxa. The fieldwork component of this study was supported by the National Geographic/Waitt Program [W461-16], Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [233161/2014-7], and a small research Grant from the Fisheries Society of the British Isles (FSBI).
Acknowledgments
We are grateful to ICMbio for providing help with accommodation and facilities, especially the teams working at Parque Estadual Serra do Mar: Núcleo Picinguaba and Parque Estadual Serra do Mar: Estação Ecológica Juréia-Itatins. We are grateful to Helder M. V. Espírito-Santo, Mateus Lira, and Anna Bennemann for their support during the sample collection.