For many decades, livebearing fishes of the family Poeciliidae have been valuable models for research in evolutionary ecology and comparative biology. Specifically, the Guppy (Poecilia reticulata)a n dS o u t h e r nP l a t y f i s h (Xiphophorus maculatus) have served as indicator taxa and as models for behavioral ecology, life history evolution, and cancer biology (Schartl 2014;Reznick et al. 2017;Goldberg et al. 2019;Gomes-Silva et al. 2020). Poeciliids, including some invasive species, can use a wide range of habitats because they are successful colonizers and have high thermal and salinity tolerances (Meffe and Snelson 1989). Here, we present the mitochondrial genome of a lesser-known species with close phylogenetic affinity to P. reticulata, P. parae. We anticipate that the mitogenome presented here will aid future research in comparative biology and will be useful for noninvasive investigations of watersheds using environmental DNA (eDNA).
Poecilia parae (Eigenmann, 1894) occupies a geographic range from Guyana to northern Brazil (Figure 1). Poecilia parae is a novel model system for the study of sex chromosome evolution and sexual polymorphism (Metzger et al. 2021;Sandkam et al. 2021). The International Union for Conservation of Nature (IUCN) has not evaluated the conservation status of P. parae. Congeners of Poecilia in the genus Xiphophorus are important models for the study of sexual dimorphism, sex chromosome evolution, and carcinogenesis (Schartl 1990;Woolcock et al. 2006;Schartl and Walter 2016). While many studies have examined the evolutionary history of Poeciliids in the contexts of ornamentation and sexual selection, few have used complete mitochondrial data (Morris et al. 2001;Cui et al. 2013;Kang et al. 2013;Goldberg et al. 2019;M endez-Janovitz et al. 2019;Metzger et al. 2021;Sandkam et al. 2021).
An aquarium trade specimen of Poecilia parae (blue melanzona morph) was obtained from Suriname (5 51 0 36 00 N, 55 7 0 48 00 W). The preserved specimen was deposited in the University of West Alabama Zoological Collection (https:// www.uwa.edu/, kaylafast0@gmail.com) under voucher number AR20090201:03. Whole genomic DNA was extracted from the pectoral fin using the DNeasy Blood and Tissue Kit following the manufacturer's instructions (QIAGEN, Hilden, Germany). DNA quality was confirmed by gel electrophoresis using a 1.5% agarose gel stained with ethidium bromide. The quantity of DNA was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Purified DNA was stored at 4 C.
The sequencing library was prepared using the Oxford Nanopore Ligation Sequencing Kit and loaded onto a Flongle flow cell following the manufacturer's instructions (Oxford Nanopore, Oxford, UK). Sequencing was performed on a MinION device using the Flongle adapter and monitored with MinKNOW software v.22.08.9 (Figure S1; Oxford Nanopore, Oxford, UK). Basecalling was done in Guppy v.6.2.11 using the high-accuracy basecalling model and reads filtered to a minimum qscore ¼ 9. Reads were assembled using Geneious Prime v.2022.2.2 under the Medium/Fast sensitivity setting and iterative fine-tuning. The P. reticulata mitochondrial genome (KJ460033) was selected as a reference sequence. A consensus sequence was generated using a strict 50% threshold and then checked by eye and ambiguous base calls resolved in BioEdit v.7.2.5 (Hall 1999;H a l la n dA l z o h a i r y2011). The genome was Concatenated protein-coding sequences from the congener mitochondrial genomes and a Xenotoca eiseni outgroup were aligned using the MAFFT server v.7 before phylogenetic analysis (Katoh et al. 2002;Katoh and Standley 2013). Model selection and evolutionary analysis by the maximum-likelihood method were performed in MEGA11. A maximum-likelihood phylogenetic tree was reconstructed using the general time reversible model with gamma and invariable sites allowed and 1000 bootstrap replications.
The mitochondrial genome of P. parae is 16,559 bp long. The nucleotide composition of the P. parae mitochondrial genome is 29.70% A, 27.25% C, 14.80% G, and 28.26% T. The genome is circular, consisting of 13 protein-coding genes, two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and one control region (D-loop; Figure 2). The P. parae mitochondrial genome contains 29 forward genes and nine reverse genes; all protein-coding genes use the start codon ATG. Seven protein-coding genes in the P. parae mitochondrial genome (ND1, COI, ATP8, ND4L, ND5, ND6, and CYTB) end The following sequences were used: AP005982 (Miya et al. 2003), KT594624 (Zhang et al. 2016), MW934558 (Eastis et al. 2021), ON797008, FJ226476 (Bai et al. 2009), FJ234985 (Bai et al. 2009), CM021098 (van Kruistum et al. 2020), KJ013505 (Kong et al. 2016), KJ460033 K€ unstner et al. (2016), OP326603 (this study), KT166983 (Dang et al. 2016), KT175513, KT307617 (Sung et al. 2016), KT715811, MK263672, KT175514, KT175512, KT175511, LC026151 (Jiang et al. 2016), KX229692 (Jeon et al. 2016), MZ681841, MK860197 (Mateos et al. 2019), KP013108, MK860198 (Mateos et al. 2019), OL825609, OL457416, AP004422 (Miya et al. 2003), KP013085, KP013115, and AP006777 (Setiamarga et al. 2008). Numbers on nodes are bootstrap support values. The sequence generated in this study is written in bold font and marked with an asterisk.
with the complete TAA stop codon and six (ND2, COII, ATP6, COIII, ND3, and ND4) end with an incomplete stop codon which is completed by the addition of 3 0 A residues.
Phylogenetic analysis using the maximum-likelihood method places the genera Xiphophorus, Poecilia, Gambusia, and Poeciliopsis each as monophyletic groups (Figure 3). Our data place P. parae as the sister group to P. reticulata, the Guppy. The phylogenetic tree topology of poeciliid genera is consistent with recent phylogenetic studies performed on whole poeciliid mitochondrial genomes (Pollux et al. 2014;Jeon et al. 2016;Eastis et al. 2021) and one-to-one orthologs (Mateos et al. 2019). Previous phylogenetic studies conducted with a more exhaustive sampling of Poecilia support the placement of P. parae (Pollux et al. 2014;M endez-Janovitz et al. 2019;Metzger et al. 2021;Sandkam et al. 2021). A wider representation of Poecilia spp. in complete mitochondrial data will further resolve the positions of these taxa. The mitochondrial genome that we generated will be conducive to monitoring species presence using eDNA and aid in future research in comparative biology.