Six additional mitochondrial genomes for North American nightsnakes (Dipsadidae: Hypsiglena) and a novel gene feature for advanced snakes

Abstract The North American nightsnakes in the genus Hypsiglena is composed of nine named and at least two unnamed species. Here, we provide the first mt-genome of H. affinis, an additional mt-genome for H. sp. nov. 1, and four additional mt-genomes from the widespread H. jani. These mtDNA genomes were sequenced using both Illumina and Ion Torrent sequencing technologies. The resulting genomes contained the expected 13 protein coding genes, 22 tRNA genes, 2 rRNA genes, and 2 control regions typical of colubroid snakes. Two of the H. jani samples had partial tRNAIle genes upstream of CR2 which has not been previously documented in colubroid snakes. A maximum likelihood gene-tree based on these data combined with previously published sequence data recovers a well-supported phylogeny and is in concordance with previous estimates of evolutionary relationships in this group.

North American nightsnakes are small, nocturnal colubroid snakes widespread across the arid Nearctic (Mulcahy 2008). This genus contains nine known species, and two unnamed species awaiting formal recognition (Mulcahy 2008;Mulcahy et al. 2014). Mitochondrial genomes for nearly all species have previously been sequenced to better understand this group's historical biogeography (Mulcahy and Macey 2009 Mulcahy et al. (2014). For the others, DNA was extracted using Qiagen DNeasy Tissue Kits (Valencia, CA), genomic libraries were prepared using the Nextera XT kit, shearing DNA fragments to an average base pair length of 480, and were sequenced on an Illumina MiSeq with pairedend 250 base pair reads (Illumina, San Diego, CA). Sequences were initially mapped to a H. jani mitochondrial genome (EU728592) in Geneious v10.2.6 (Biomatters Ltd, 2005Ltd, -2017. After constructing a consensus sequence, all reads were mapped back to this consensus to construct the final assembly. All six newly assembled mitochondrial genomes contained 13 protein coding genes, 22 tRNA genes, 2 rRNA genes, and 2 control regions which is typical of colubroid snakes (Kumazawa et al. 1998). The control regions were difficult to assemble in C-rich regions of several samples, unobtainable in AMNH R-504774 and short three C's in AMNH R-504527, likely due to the difficulties of sequencing through homopolymers using Illumina sequencing. The recovered genome lengths and corresponding GenBank numbers of each specimen are: AMNH R-504522 (MT561495) 17,200; AMNH R-504524 (MT561496) 17,235; AMNH R-504527 (MT561497) 17,235; AMNH R-504774 (MT561500) 17,205; AMNH R-504773 (MT561498) 17,202; LSUMZ 39533 (MT561499) 17,190 base pairs. Mean depth of coverage ranged from 23.6-53.6x (average: 37.9). Two mt-genomes (AMNH R-504524 and AMNH R-504527) contained identical putative pseudogenes, partial tRNA Ile , on the 5 0 end of the second control region.
Our six new mt-genomes were aligned with data from 14 published Hypsiglena mt-genomes, $5kb of mtDNA data from H. tanzeri, and complete mt-genomes of Pseudoleptodeira latifaciata, Sibon nebulatus, Imantodes cenchoa, Leptodeira septentrionalis were included as outgroups (Mulcahy 2008;Mulcahy and Macey 2009;Mulcahy et al. 2014). A maximum likelihood phylogeny was generated with RAxML v8.2.3 using the simultaneous rapid-bootstrap (1000 replicates) and thorough ML search (Stamatakis 2014), with each gene in a separate partition and only one control region (CR2), with the GTRCAT substitution model. The phylogeny ( Figure 1) is well supported and in concordance with previous estimates of relationships within the genus Hypsiglena (Myers et al. 2013(Myers et al. , 2017Mulcahy et al. 2014).
A previous study of nightsnakes produced 12 complete mt-genomes (Mulcahy and Macey 2009) (Mulcahy and Macey 2009;Sun 2017), with the exception of lacking the partial tRNA Pro . Here, we provide the first mt-genome of H. affinis, an additional one for H. sp. nov. 1, and four additional mt-genomes from the widespread H. jani. Two individuals (AMNH R-504524 and AMNH R-504524) near the contact zone between H. jani and H. sp. nov. 1, show partial tRNA Ile genes in the same region as the pseudo tRNA Pro reported in Dinodon (NC 001945; Kumazawa et al. 1998). Both pseudo-genes, and their respective parent genes (tRNA Pro and tRNA Ile ), reside upstream of control regions. In Dinodon, the pseudo-tRNA Pro was found upstream of CR2, with the functional tRNA Pro upstream of CR1 (Kumazawa et al. 1998), as in most vertebrates. Viperids show two unique gene-orders in these regions, where both have functional tRNA Pro upstream of CR2 and one has a pseudo tRNA Pro upstream of CR1; the other lacks the pseudo tRNA Pro (Yan et al. 2008). The presence of two CRs in advanced snakes has been attributed to tandem duplication during replication or concerted evolution (Kumazawa et al. 1998;Dong and Kumazawa 2005). The repetitive nature of tRNAs bordering duplicate control regions are likely involved in, or are a product of, the evolutionary events driving this phenomenon. samples at AMNH. Lastly the authors thank J. R. Macey for assistance with assembling and annotating these data.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
EAM was supported through the Peter Buck and Rathbone Bacon Fellowship from the National Museum of Natural History.