Gene 626 (2017) 163–172

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Gene

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Research paper Characterization and phylogenetic analysis of complete mitochondrial MARK genomes for two desert cyprinodontoid fishes, latos and baileyi

⁎ Miguel Jimeneza,b, Shawn C. Goodchildc,d, Craig A. Stockwellc, Sean C. Lemab, a Alan Hancock College, Santa Maria, CA, USA b Biological Sciences Department, Center for Coastal Marine Sciences, California Polytechnic State University, San Luis Obispo, CA, USA c Department of Biological Sciences, North Dakota State University, Fargo, ND, USA d Environmental & Conservation Sciences Graduate Program, North Dakota State University, Fargo, ND, USA

ARTICLE INFO ABSTRACT

Keywords: The Pahrump poolfish () and White River springfish (Crenichthys baileyi) are small-bodied fishes (order Cyprinodontiformes) endemic to the arid Great Basin and Mojave Desert regions of western North America. These taxa survive as small, isolated populations in remote streams and springs and evolved to mtDNA tolerate extreme conditions of high temperature and low dissolved oxygen. Both have experienced severe Conservation population declines over the last 50–60 years that led to some subspecies being categorized with protected status Pahrump poolfish under the U.S. Act. Here we report the first sequencing of the complete mitochondrial DNA White River springfish Endangered species genomes for both E. l. latos and the moapae subspecies of C. baileyi. Complete mitogenomes of 16,546 bp nucleotides were obtained from two E. l. latos individuals collected from introduced populations at Spring Desert Mountain Ranch State Park and Shoshone Ponds Natural Area, , USA, while a single mitogenome of 16,537 bp was sequenced for C. b. moapae. The mitogenomes of both species contain 13 protein-encoding genes, twenty-two tRNAs, and two rRNAs (12S and 18S) following the syntenic arrangement typical of Actinopterygiian fish mitogenomes, as well as D-loop control regions of 858 bp for E. latos and 842 bp for C. baileyi moapae. The two E. latos individuals exhibited only 0.0181% nucleotide sequence divergence across the entire mitogenome, implying little intraspecific mtDNA genetic variation. Comparative phylogenetic analysis of the poolfish and springfish mitochondrial genomes to available mitogenomes of other Cyprinodontoid fishes confirmed the close relationship of these oviparous Empetrichthys and Crenichthys genera to the viviparous goodeid fishes of central , and showed the combined clade of these fishes to be a sister group to the Profundulidae killifishes. Despite several significant life history and morphological differences between the and Goodienae, estimates of evolutionary genetic distances using two partial regions of mtDNA point to inclusion of the Empetrichthys and Crenichthys genera within the family Goodeidae along with the goodeid fishes of central Mexico.

1. Introduction have experienced severe population declines over the past ~50 years due to the combined influences of habitat loss, habitat degradation, and The native fish fauna of the arid North American west includes invasive species (Deacon and Williams, 2010; Miller et al., 1989; several taxa of small-bodied killifishes (order Cyprinodontiformes) that Williams et al., 1985). Despite extensive study of the phylogenetic evolved within the harsh conditions of aquatic habitats within the Great organization of fishes within the order Cyprinodontiformes (e.g., Basin and Mojave Desert regions of the (Miller, 1948, Breden et al., 1999; Echelle, 2008; Martin et al., 2016; Meredith 1950; Minckley and Marsh, 2009; Minckley and Deacon, 1968). et al., 2010; Kang et al., 2013), there remains uncertainty about the Endemic to this region are the Pahrump poolfish (Empetrichthys latos) taxonomic classification of the Empetrichthys and Crenichthys genera and the White River springfish (Crenichthys baileyi), two species that within the Cyprinodontiformes (e.g., Grant and Riddle, 1995). These

Abbreviations: ATP6 and ATP8, ATPase subunits 6 and 8; bp, Base pairs; CO1–CO3, Cytochrome c oxidase subunits I–III; ND1–ND6 and ND4L, NADH dehydrogenase subunits 1–6 and 4L; rRNA, Ribosomal RNA; tRNA, Transfer RNA ⁎ Corresponding author at: Biological Sciences Department, California Polytechnic State University, 1 Grand Avenue, San Luis Obispo, CA 93407, USA. E-mail address: [email protected] (S.C. Lema). http://dx.doi.org/10.1016/j.gene.2017.05.023 Received 1 January 2017; Received in revised form 4 April 2017; Accepted 9 May 2017 Available online 13 May 2017 0378-1119/ © 2017 Elsevier B.V. All rights reserved. M. Jimenez et al. Gene 626 (2017) 163–172 genera have sometimes been grouped as their own family Empe- on morphological differentiation (Williams and Wilde, 1981; see also trichthyidae (Miller and Smith, 1986; Miller et al., 2005; Minckley Minckley and Marsh, 2009), and C. baileyi has only one extant and Marsh, 2009) or alternatively been assigned to subfamily Empe- congener, the Railroad Valley springfish (C. nevadae), that occurs in trichthyinae within family Goodeidae (La Rivers, 1994; Parenti, 1981; isolated warm water springs in the Railroad Valley of Nevada (Hubbs, Webb, 1998). 1932). Physical environmental degradation and non-native species has The Pahrump poolfish (Empetrichthys latos) is the sole remaining led to severe population declines and listing of the C. b. baileyi and C. b. taxon of the Empetrichthys since its sister species – the Ash grandis subspecies as ‘endangered’ under the U.S. Endangered Species Meadows killifish E. merriami (Gilbert, 1893; Jordan and Evermann, Act. The moapae subspecies occurs in several warm headwater springs 1896) – went extinct in the early 1950′s(Miller et al., 1989; Pister, (~31–32 °C at the spring sources) and their outflows in the upper 1990; Soltz and Naiman, 1978). The only other known member of the Muddy River (Deacon and Bradley, 1972; Scoppettone et al., 1998). The genus is the extinct Empetrichthys erdisi, described from a fossil speci- introduction of nonnative species to these habitats has led to declines in men discovered in southern California, USA, from Pliocene Colorado the size of C. b. moapae populations (Cross, 1976; Scoppettone, 1993), River deposits (Uyeno and Miller, 1962). Miller (1948) first character- although recent conservation efforts (i.e., habitat restoration) to sustain ized E. latos as a distinct species comprised of three morphologically- populations of the cohabitating Moapa dace (Moapa coriacea) have also distinct subspecies that occurred in isolated groundwater-fed springs in enhanced C. b. moapae populations in some of these habitats (e.g., the Pahrump Valley, Nevada, USA. The combined effects of physical Syzdek, 2013). habitat alteration, groundwater withdrawal that reduced surface water In this study, we sequenced the complete mitochondrial genomes of flow, and the introduction of non-native species contributed to the two E. l. latos individuals, one each from Shoshone Ponds and from Lake of two of these subspecies (E. l. pahrump and E. l. concavus) Harriet at Spring Mountain Ranch. We also sequenced the full (Deacon and Williams, 2010; Miller et al., 1989; Minckley et al., 1991). mitogenome of a single C. b. moapae. For each fish, we describe the The only remaining subspecies, E. l. latos, was endemic to Manse Spring nucleotide composition of the complete mitogenome and identified the in Pahrump Valley, Nevada. Population surveys indicated that this genome organization, gene order and codon usage. We also analyzed population underwent dramatic declines to fewer than 50 fish in the molecular phylogenetic relationships of these Empetrichthys and 1962–63 and again in 1967–68 (Deacon and Williams, 2010). The Crenichthys fishes relative to other cyprinodontids in an effort to clarify precarious status of this species led to its listing as ‘endangered’ in 1967 the taxonomic relationships and status of these genera within the order under the U.S. Endangered Species Preservation Act. Individuals of this Cyprinodontiformes. sole remaining taxon of E. latos were translocated to refuge habitats in the 1970s prior to the complete failure of groundwater flow of Manse 2. Materials and methods Spring, in 1975 (Deacon and Williams, 2010; Goodchild, 2016; Minckley et al., 1991). Since the extirpation from its native range, E. 2.1. Specimen collection latos persists to this day entirely through ongoing active management of translocated refuge populations in Nevada, USA. Individual E. l. latos were collected from two populations: Spring E. latos from Manse Spring were originally transplanted into three Mountain Ranch State Park (36° 4′5.16″N, 115°27′41.92″W; Clark fishless locations in Nevada during the early 1970s: Los Latos Pools near County, Nevada, USA) on 23 August 2013, and Shoshone Ponds Lake Mohave in the Lake Mead National Recreation Area, Nevada, the Natural Area (38°56′22.43″N, 114°25′4.36″W; White Pine County, U.S. Bureau of Land Management Shoshone Ponds Natural Area south- Nevada, USA) on 8 August 2012. In addition, a single C. baileyi moapae east of Ely, Nevada, and Corn Creek on the Desert National Wildlife individual was collected from Plummer Stream in the upper Muddy Refuge, Nevada (Fig. 1). The refuge populations at Los Latos Pools and River complex (36°42′39.03″N, 114°42′42.81″W; Clark County, Shoshone Ponds later failed in ~1977 and 1974, respectively (U.S. Fish Nevada, USA) on 19 August 2012. Tissues from all specimens were and Wildlife Service, 2004), and all extant refuge populations of stored in 95% EtOH. Pahrump poolfish are descended from the population established with 29 poolfish at Corn Creek in 1971. In 1976, 66 poolfish were 2.2. Primer design, mitogenome amplification, and sequencing translocated from Corn Creek to re-establish a refuge population at the Shoshone Ponds Natural Area, where poolfish have been actively Genomic DNA was isolated from the skeletal muscle of each managed across a number of small ponds and a small stream since that specimen using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, time. In 1983, 426 poolfish were used to establish a refuge population CA, USA), and then quantified using a P300 NanoPhotometer (Implen, Lake Harriet in Spring Mountain Ranch State Park west of Las Vegas, Inc.). Overlapping regions of the mtDNA genome for each species were Nevada (Fig. 1; also see Goodchild, 2016). To date, however, the amplified in PCR reactions containing 25 μL of 2× GoTaq® Long PCR Pahrump poolfish continues to remain vulnerable to demographic Master Mix (Promega Corp., Madison, WI, USA), 1 μL each of forward effects, genetic drift, non-native species, and natural catastrophes and reverse primer (10 mM), 13 to 18 μL of nuclease-free H2O, and 5 to − (Deacon and Williams, 2010; Goodchild and Stockwell, 2016), as 10 μL of DNA template (85 to 298 ng·mL 1). Multiple pairs of oligo evidenced by the recent (2015–2016) compromise of the Lake Harriet primers were designed to partial mtDNA sequences for cytochrome b population by invasive species (U.S. Fish and Wildlife Service, 2016). (Cyt B), cytochrome c oxidase subunit-I (COI), or the D-loop region The White River springfish (Crenichthys baileyi) has also experienced available for each species on GenBank (http://www.ncbi.nlm.nih.gov/) severe declines in range and numbers. Since its original mistaken (E. latos: U09108, AY356573, AY356615; C. baileyi: U09102, description as a subspecies of the desert pupfish Cyprinodon macularius AF510819, AY356571, AY356614) to generate overlapping PCR in 1893 (Gilbert, 1893) and later redesignation as a separate species of products that spanned the entire mitogenomes of both taxa. Cyprinodon (Jordan and Evermann, 1896), it was subsequently reas- Nucleotide sequences for these primers are provided in Table 1. All signed to the new genus Crenichthys (Hubbs, 1932)bySumner and PCR products were examined on 0.8% ethidium bromide gels, and Sargent (1940). Similar to many fishes of the arid southwest of North products with bands of expected size were cleaned (QIAquick PCR America, the White River springfish is capable of surviving low oxygen, Purification Kit; Qiagen) before being Sanger sequenced with the same high temperature conditions (Hubbs and Hettler, 1964; Sumner and primers used for PCR (Molecular Cloning Laboratories, South San Sargent, 1940; Sumner and Lanham, 1942), and populations of this Francisco, CA, USA). The initial partial mtDNA sequences obtained springfish are found only in several warm water springs and their were then used to design additional gene-specific primers (see Supple- outflows scattered within the pluvial White River system of south- mentary materials, Tables S1 and S2) that were used to Sanger sequence eastern Nevada. Five subspecies of C. baileyi have been described based the complete mitogenomes of both species by primer walking.

164 M. Jimenez et al. Gene 626 (2017) 163–172

Fig. 1. History of establishment and extirpation for refuge populations of the Pahrump poolfish, E. latos latos, from its sole native habitat of Manse Spring, Nevada (NV), USA. Three refuge populations were originally established with poolfish from Manse Spring: Latos Pools in 1970, Corn Creek in 1971, and Shoshone Ponds in 1972. Both the Latos Pools and Shoshone Ponds populations were subsequently extirpated in ~1977 and 1974, respectively. Later poolfish translocations reintroduced fish to Shoshone Ponds in 1976 and established of a refuge population in 1983 at Lake Harriet, Spring Mountain Ranch State Park, NV. The extirpation of the Corn Creek population in 1998 was followed by the re-establishment of a refuge population at this location in 2003 with poolfish from Lake Harriet. Adapted from Goodchild, 2016.

Table 1 Primer pairs used for amplification of E. latos and C. baileyi mtDNA.

Taxon Primer labela Nucleotide sequence (5′ to 3′) Primer labela Nucleotide sequence (5′ to 3′) Product size (bp)b

E. latos El_for5807 GCTCTCTCCTGGGTGATGACCAG El_rev14722 GGCAGCAAGGCAAAGCCCTAG 8915 El_for5980 TGGCTTCTCCCCCCATCATTCC El_rev14890 GCCAATGTGGTAGTAGATGCAGACA 8910 El_for14662 CCCCATCAACATCTCAGCCTGATG El_rev6339 CGGGTCAAAGAAGGTGGTGTTTAGG 8223 C. baileyi Cb_for5574 TGCAATCTAATGTGTAACACCTCAAGGC Cb_rev14979 AGGCTGTTATTATAGCGAGGAGGAAGAGG 9405 E. latos and C. baileyi ElCb_for14709 CTTTGCCTTGCTGCCCAAATCCTA ElCb_rev6310 GATCGGTTAGAAGCATGGTGATGCCA 8148c 8138d

a Primer label numbers indicate position within mitogenome of goodeid eiseni (acc. no. AP006777). b Product sizes represent bps of PCR product for E. latos and C. baileyi mitogenomes. c Indicates product size for E. latos mitogenome. d Indicates product size for C. baileyi mitogenome.

Table 2 Nucleotide composition (% A + T only) for specific coding regions and complete mitogenomes of E. latos latos and C. baileyi moapae.

Taxon (accession no.) Full mitogenome Protein-coding genes rRNAs tRNAs D loop

Length (bp) % A + T Length (bp) % A + T Length (bp) % A + T Length (bp) % A + T Length (bp) % A + T

E. l. latos 16,542 56.7% 11,429 56.5% 2646 55.8% 1548 56.4% 858 64.7% (KY014102) C. baileyi moapae 16,537 56.1% 11,429 55.8% 2650 55.1% 1549 56.5% 842 63.9% (KY014104)

2.3. Gene identification and comparative sequence analysis generate complete mitogenome contigs for each specimen. The result- ing complete mitogenomes were then annotated using MitoAnnotator, a Overlapping sequenced mtDNA fragments were assembled using web-based tool developed for fish mitochondrion genome annotation Sequencher v.5 software (Gene Codes Corp., Ann Arbor, MI, USA) to (Iwasaki et al., 2013). Annotations of protein-coding genes were

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Fig. 2. Gene localization for E. latos latos and C. baileyi moapae mitogenomes. Protein-coding genes, rRNAs, tRNAs, and the D loop non-coding region are illustrated in different colors. Gene or RNAs positioned on the heavy (H) strand of the mitogenome are shown in the outer ring, while features on the light (L) strand are illustrated on the inner ring. Arrow direction for each gene or RNA feature indicates the coding orientation of that feature. Black ring shows the GC content of that region, with outer peaks indicating higher GC content and inner rings indicating lower GC content. The inner purple-green ring illustrates GC skew within the mitogenome calculated as follows: [(G − C)/(G + C)]; purple regions indicate values between −1 and 0 (‘C’ nucleotide skew), and green regions indicate values between 0 and +1 (‘G’ nucleotide skew). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) confirmed using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/ several Goodeid fishes and other members of order Cyprinodonti- ) as well as NCBI-BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast. formes. A second alignment was made using a ~1190 bp nucleotide cgi). Annotations of RNA positions within the mitogenomes of both mtDNA region comprised of Cyt B and the tRNAGlu and tRNAThr genes species were likewise confirmed using RNAmmer (Lagesen et al., 2007). (C. nevadae: KJ696794). Nucleotide sequences for these ND2 and Cyt B Mitogenome maps were generated using the CGView server application partial mtDNA regions were used to construct maximum likelihood (Grant and Stothard, 2008; Grant et al., 2012). phylogenetic trees in MEGA v7 (Kumar et al., 2016). Trees were constructed by nearest-neighbor-interchange (NNI) using Tamura-Nei 2.4. Phylogenetic analyses models. Uniform substitution rates were assumed for all sites, and node confidence was calculated as bootstrap values from 1000 replicates. Complete mitogenome nucleotide sequences were aligned using Both the ND2 and Cyt B trees were rooted using the Turquoise killifish ClustalX (Larkin et al., 2007) to available complete mitogenomes for Nothobranchius furzeri (Nothobranchiidae, Cyprinodontiformes) as a other fishes of order Cyprinodontiformes – including the only other basal taxon. Pairwise estimates of evolutionary distance were subse- member of family Goodeidae with a complete mitochondrial genome: quently calculated as the number of nucleotide basepair differences per the redtail splitfin Xenotoca eiseni (AP006777)(Setiamarga et al., site across all codon positions (p distance method) between taxon 2008). Select fishes of orders Beloniformes and Atheriniformes were sequence pairs (MEGA v7 software), and then average distance values also included in these phylogenetic analyses because these groups are both within and between cyprinodontoid families were calculated for considered the most closely related orders to the Cyprinodontiformes comparison. within the teleost series Atherinomorpha (e.g., Hertwig, 2008; Setiamarga et al., 2008). Phylogenetic trees were subsequently con- 3. Results and discussion structed with MEGA v.7 software (Kumar et al., 2016) using both the neighbor-joining method (Tamura-Nei model with uniform rates among 3.1. Characteristics and annotations of the E. latos and C. baileyi sites and pairwise deletion of gaps) and the maximum parsimony mitogenomes method (using all sites). All trees were bootstrapped by 1000 replicates. The complete mitogenome of coho salmon, Oncorhynchus kisutch (order The complete mitogenomes for the E. latos individuals from Lake Salmoniformes; GenBank accession no. EF126369), was used to root Harriet in Spring Mountain Ranch State Park (accession no. KY014102) the mitogenome trees. and Shoshone Ponds Natural Area (KY014103) were deposited into We performed separated nucleotide sequence alignments and GenBank, as was the complete mitogenome for the C. baileyi moapae phylogenetic analyses using partial sequences from the complete individual (KY014104). The complete mitogenomes for the two E. latos mitogenomes of E. l. latos and C. b. moapae, as well as previously fish were each 16,546 bps in length and differed at only three published partial mtDNA sequences from Crenichthys nevadae, the sole nucleotide sites, representing only 0.0181% nucleotide divergence extant congener to C. baileyi (Miller and Smith, 1986; Miller et al., across the entire mitogenomes. Base composition analysis indicated 2005; Minckley and Marsh, 2009). Specifically, we identified a partial that the E. latos mitogenomes have an A + T content of 56.7%, mtDNA sequence from C. nevadae (GenBank acc. no. KJ697205) that reflecting a similar nucleotide composition of 28.4% A, 28.3% T, spanned a ~ 1320 bp region containing ND2 and the following tRNAs: 16.2% G, and 27.2% C in both sequenced individual's mitogenomes. tRNAGln, tRNAMet, tRNATrp, tRNAAla, and tRNAAsn. This partial mtDNA The complete mitogenome for C. baileyi moapae was 16,537 bps in sequence was aligned to homologous mtDNA regions from the E. l. latos length and had an A + T content of 56.1% (28.4% A, 27.7% T, 16.0% and C. b. moapae mitogenomes, as well as to mtDNA sequences from G, and 27.9% C). Nucleotide composition analyses for regions contain-

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Table 3 Location and arrangement of genes on the mitogenomes of E. latos latos and C. baileyi moapae.

Gene Stranda Empethrichthys latos latos Crenichthys baileyi moapae Positionb Size (bp) Codon Positionb Size (bp) Codon From (bp) To (bp) Start Stopc #of Intergenic From (bp) To (bp) Start Stopc #of Intergenic amino spacer (bp)d amino spacer (bp)d acids acids

tRNAPhe H 1 68 68 1 68 68 12S rRNA H 69 1015 947 69 1016 948 tRNAVal H 1016 1087 72 1017 1088 72 16S rRNA H 1088 2786 1699 1089 2790 1702 tRNALeu H 2787 2860 74 2791 2864 74 ND1 H 2861 3835 975 ATG TAA 324 4 2865 3839 975 ATG TAG 324 5 tRNAIle H 3840 3909 70 3845 3914 70 tRNAGln L (3909) (3979) 71 (3914) (3984) 71 tRNAMet H 3979 4047 69 3984 4052 69 ND2 H 4048 5094 1047 ATG TAG 348 0 4053 5097 1047 ATG TAG 348 -2 tRNATrp H 5095 5165 71 5098 5170 71 tRNAAla L (5169) (5237) 69 (5174) (5242) 69 tRNAAsn L (5240) (5312) 73 (5245) (5317) 73 tRNACys L (5350) (5414) 65 (5355) (5419) 65 tRNATyr L (5418) (5487) 70 (5423) (5492) 70 COI H 5489 7039 1551 GTG TAA 516 15 5494 7044 1551 GTG TAA 516 15 tRNASer L (7054) (7125) 72 (7060) (7131) 72 tRNAAsp H 7129 7199 71 7135 7205 71 COII H 7207 7897 691 ATG T- 230 0 7213 7903 691 ATG T- 230 0 tRNALys H 7898 7971 74 7904 7977 74 ATPase8 H 7973 8140 168 ATG TAA 55 -10 7979 8146 168 ATG TAA 55 -9 ATPase6 H 8131 8813 683 ATG TA- 227 0 8137 8819 683 ATG TA- 227 0 COIII H 8814 9598 785 ATG TA- 261 0 8820 9604 785 ATG TA- 261 0 tRNAGly H 9599 9670 72 9605 9676 72 ND3 H 9671 10,019 349 ATG T- 116 0 9677 10,025 349 ATG T- 116 0 tRNAArg H 10,020 10,088 69 10,026 10,094 69 ND4L H 10,089 10,385 297 ATG TAA 98 -7 10,095 10,391 297 ATG TAA 98 -7 ND4 H 10,379 11,759 1381 ATG T- 460 0 10,385 11,765 1381 ATG T- 460 0 tRNAHis H 11,760 11,828 69 11,766 11,834 69 tRNASer H 11,829 11,896 68 11,835 11,902 68 tRNALeu H 11,907 11,979 73 11,913 11,985 73 ND5 H 11,980 13,818 1839 ATG TAG 612 -4 11,986 13,824 1839 ATG TAA 612 -4 ND6 L (13815) (14336) 522 ATG TAG 173 0 (13821) (14342) 522 ATG TAG 173 0 tRNAGlu L (14337) (14404) 68 (14343) (14410) 68 Cyt b H 14,409 15,549 1141 ATG T- 380 0 14,415 15,555 1141 ATG T- 380 0 tRNAThr H 15,550 15,620 71 15,556 15,626 71 tRNAPro L (15620) (15689) 69 (15626) (15696) 70 D-loop – 15,690 16,546 858 15,697 16,537 842

a H and L indicate position on the heavy (H) or light (L) strand. b Nucleotide (bp) positions in parentheses indicate encoded on complementary, light (L) strand. c Codons containing “-“symbols indicate an incomplete stop codon. d Values correspond to the number of nucleotides separating this and the next gene or RNA. Negative numbers denote overlapping positions. ing the protein-coding genes, rRNAs, tRNAs, and non-coding D-loops of Overall, however, the E. latos and C. baileyi mitogenomes exhibited the E. latos and C. baileyi moapae mitogenomes are provided in Table 2. similar gene arrangements (Fig. 2) and lengths, and the two species Annotation of the mitogenomes revealed that both species contain exhibited identical gene lengths for all protein-coding genes, rRNAs, 13 protein-coding genes, a 12S and 16S rRNA gene, and 22 tRNA genes and tRNAs in the mitogenomes (Table 3). following the typical positional arrangement observed in The mtDNA genomes of E. latos and C. baileyi exhibited 92.31% Cyprinodontiform fishes (e.g., Keepers et al., 2016; Lema et al., 2016) nucleotide identity across 15,289 of the 16,562 aligned nucleotides. and most other Actinopterygiian species (Fig. 2) (e.g., Satoh et al., More detailed comparisons of nucleotide and deduced amino acid 2016). In both species, the ND6 protein-coding gene and eight of the variation between the two species' mitogenomes revealed that rRNA tRNA genes (tRNAPro, tRNAGlu, tRNASer, tRNATyr, tRNACys, tRNAAsn, genes shared an average of 97.01% nucleotide identity and tRNAs tRNAAla, and tRNAGln) were positioned on the light (L) stand of the 97.61% identity (Table 4). Protein-coding genes in the E. latos and C. mitogenome; all other protein-coding genes, rRNAs and tRNAs were baileyi mtDNA genomes averaged 91.21% identity in nucleotide encoded on the heavy (H) strand (Table 3). Putative control regions (D sequence but 96.23% similarity in deduced amino acid sequence. As loop) of 858 bps for E. latos and 842 bps for C. baileyi moapae were is typical for the mitogenomes of vertebrates including fishes (e.g., Lee localized between the tRNAPro and tRNAPhe genes. Further analysis of et al., 1995; Satoh et al., 2016), the highest nucleotide divergence (only the structure of the protein-coding genes showed that all genes had the 85.82% identity) between the E. latos and C. baileyi mitogenomes was usual ATG start codon except the COI gene, which starts with a GTG observed in the D loop control region (Table 4). The ND2 gene was codon. Four protein-coding genes in each species used the TAA stop identified as the region showing the second greatest evolutionary codon (E. latos: ND1, COI, ATPase8, and ND4L; C. baileyi: COI, ATPase8, divergence with 86.72% nucleotide identity and 92.82% deduced ND4L, and ND5). However, nucleotide variation generated two stop amino acid identity between E. latos and C. baileyi. This nucleotide codon differences between the species: 1) a change from a TAA stop divergence between the ND2 genes is illustrated by the higher propor- codon for COI in E. latos to a TAG stop codon in C. baileyi, and 2) a tion of A-T nucleotides (i.e., negative G-C skew) for the ND2 region of change from a TAG stop codon for ND5 in E. latos to a TAA in C. baileyi. the C. baileyi mitogenome, compared to the E. latos mitogenome

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Table 4 Table 5 Comparison of nucleotide and deduced amino acid (AA) sequence identities (%) and Nucleotide variation in the mitogenomes from the E. l. latos individuals collected from the number of variable nucleotide sites or AA residues between the E. latos latos and C. baileyi Spring Mountain Ranch State Park and Shoshone Ponds Natural Area populations. moapae mitogenomes. Nucleotide Population Consequence for Locus Nucleotide Deduced Protein position (bp) deduced amino acid % identity # variable sites % identity # variable sites Spring Mountain Shoshone Ponds Ranch State Park Natural Area Protein-coding genes ND1 90.35% 94 95.68% 14 Basepair 4269 ‘T’ nucleotide ‘C’ nucleotide Silent mutation in ND2 86.72% 139 92.82% 24 ND2 gene COI 94.26% 89 99.81% 1 Basepair 5079 ‘T’ nucleotide ‘C’ nucleotide Silent mutation in COII 93.92% 42 99.57% 1 ND2 gene ATPase8 91.07% 15 90.90% 5 Basepair 12,655 ‘G’ nucleotide ‘A’ nucleotide Changes deduced ATPase6 92.09% 54 97.80% 5 amino acid in ND5 COIII 92.10% 62 97.70% 6 protein ND3 89.40% 37 93.97% 7 ND4L 93.60% 19 97.96% 2 ND4 88.70% 156 95.22% 22 structures for the deduced ND2 proteins (SWISS-MODEL https:// ND5 89.94% 185 94.28% 35 swissmodel.expasy.org)(Biasini et al., 2014) suggested that the residue ND6 91.00% 47 97.11% 5 ff Cyt B 92.55% 85 98.16% 7 di erences between the species may not have a major impact on protein structure. However, additional physiological studies are necessary to rRNAs 12S 97.37% 21 fully identify any functional consequences of this ND2 structural 16S 96.65% 54 variation between E. latos and C. baileyi.

tRNAs Comparison of the nucleotide sequences for the two E. latos tRNAPhe 97.06% 2 mitogenomes from the different populations revealed only three tRNAVal 97.22% 2 nucleotide point mutations across within the entire 16,546 bp mtDNA Leu tRNA 94.59% 4 genomes (Table 5). Two of those identified nucleotide differences tRNAIle 92.86% 5 Gln occurred within the ND2 gene and are silent mutations. The third tRNA 97.18% 2 ff tRNAMet 97.10% 2 nucleotide di erence occurred at basepair 12,655 in the coding region tRNATrp 98.59% 1 of the ND5 gene; the poolfish from the Shoshone Ponds population had tRNAAla 98.55% 1 a ‘C’ nucleotide at this 12,655 bp position while the fish from the Spring tRNAAsn 100.00% 0 ‘ ’ Cys Mountain Ranch population had an A nucleotide. This nucleotide tRNA 100.00% 0 ff ‘ ’ tRNATyr 95.71% 4 di erence results in a change in codon from ACC encoding a deduced tRNASer 98.61% 1 threonine (Thr) residue in the Shoshone Ponds fish to a codon of ‘GCC’ tRNAAsp 98.59% 1 encoding an alanine (Ala) in the poolfish from Spring Mountain Ranch. Lys tRNA 100.00% 0 Even including the silent mutations, the complete mitogenomes of E. Gly tRNA 98.61% 1 – Arg latos exhibited only 0.0181% nucleotide sequence divergence, which tRNA 97.10% 2 – tRNAHis 95.65% 3 if characteristic of these E. latos populations broadly would signify a tRNASer 98.53% 1 level of intraspecific genetic variation nearly 40-fold lower than the tRNALeu 98.63% 1 0.73% mean intraspecific variation estimated by April et al. (2011) tRNAGlu 97.06% 2 Thr using a ~615 bp region of the COI gene in 690 species of North tRNA 98.59% 1 fi tRNAPro 97.14% 2 (& 1 nt gap) American freshwater shes. The low mitochondrial genetic variation observed here likely Control region reflects historic bottlenecks within the native Manse Spring population D-loop 85.28% 127 (& 26 nt gaps) as well as subsequent bottlenecks associated with the complicated translocation history of the refuge populations. Both the population of (Fig. 2). Alignment of the deduced ND2 protein sequences from the two E. latos in the Shoshone Ponds Natural Area habitat and the population taxa revealed that the majority of divergent residues occurred in at Spring Mountain Ranch were derived from another refuge population regions of the protein that did not impact protein secondary structure of poolfish at Corn Creek, which itself was founded with only 29 (e.g., α-helix structures) (Fig. 3). A further comparison of tertiary individuals from Manse Spring (Fig. 1), a habitat that supported the sole

Fig. 3. Sequence alignment for deduced ND2 amino acid residues from E. latos latos and C. baileyi moapae. Consensus residues between the sequences are indicated by asterisks (*), and the degree of amino acid similarity is blocked by background color. Putative alpha helical secondary structure regions are denoted by lines (=) above the residues.

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Fig. 4. Neighbor-joining phylogenetic tree generated by alignment of complete mitogenome nucleotide sequences for E. l. latos, C. baileyi moapae, and fishes of the orders Cyprinodontiformes, Beloniformes and Atheriniformes. The complete mitogenome from Coho salmon, Oncorhynchus kisutch (GenBank accession no. EF126369; order Salmoniformes) was used as an outgroup. Note that mitogenomes used were complete for all taxa except Jordanella floridae, which lacked a D-loop region sequence. GenBank accession numbers accompany each taxon. Provided bootstrap values (1000 replicates) represent node recovery for both the neighbor-joining (NJ) reconstruction method, as well as the maximum parsimony (MP) method: NJ value/MP value. Note that phylogenies generated both methods showed nearly full agreement in evolutionary relationships, with the sole exception of the placement of the Oryzias spp. clade, which was basal to the O. kisutch outgroup in the MP tree, and thus still not monophyletic with the Beloniformes. natural population of the E. l. latos subspecies. Further, the Manse with C. baileyi. This comparative full mitogenome analysis also pointed Spring population experienced two separate populations bottlenecks to to the Fundulidae as exhibiting a close phylogenetic relationship to E. fewer than 50 individuals in 1962–1963 and 1967 (Deacon et al., 1964; latos, C. baileyi, and the goodeids of central Mexico. Deacon and Williams, 2010), before the complete loss of groundwater Uncertainty concerning the extent of evolutionary differentiation discharge at Manse Spring – and loss of this subspecies' only natural between the Empetrichthys/Crenichthys genera and the central Mexican habitat – in 1975 (Deacon, 1979). goodeids – combined with a geographic distance of nearly 1500 km separating these groups – has led to discrepancies in the assignment of 3.2. Comparison with other Cyprinodontiform fishes taxonomic status with the Empetrichthys and Crenichthys genera at times being assigned to their own family (Empetrichthyidae: Miller and Alignment and phylogenetic analyses using the complete mitogen- Smith, 1986; Miller et al., 2005; Minckley and Marsh, 2009) and at ome nucleotide sequences of E. latos, C. b. moapae and other fishes of other times being assigned subfamily status (Empetrichthyinae) within the order Cyprinodontiformes confirmed the sister relationship of these the Goodeidae (La Rivers, 1994; Parenti, 1981; Webb, 1998). In an ff two genera. Previous cladistic analyses have indicated that the closest e ort to resolve the family/subfamily taxonomic status of the Empe- living relatives of the Empetrichthys/Crenichthys genera are the thirty-six trichthys and Crenichthys genera, Grant and Riddle (1995) aligned described taxa of viviparous Goodeid (family Goodeidae) fishes that 314 bp of the Cyt B gene and observed 96.6% nucleotide identity occur in the central plateau of Mexico (Parenti, 1981; Webb, 1998; across this partial Cyt B sequence between C. baileyi and its sister Webb et al., 2004). Phylogenetic analysis using complete mitogenome species C. nevadae, and 91.2% nucleotide identity between Crenichthys sequences supported that previously proposed evolutionary relation- (baileyi and nevadae) and Empetrichthys latos. The Cyt B analyses by ship to Mexico's goodeid fishes, which are represented in the mitogen- Grant and Riddle (1995) supported the Empetrichthys/Crenichthys clade – ome phylogenetic analysis by Xenotoca eiseni – the sole Mexican to be most closely related to the family Goodeidae rather than the – goodeid with a fully sequenced mitochondrial genome (Fig. 4). Align- Fundulidae, Poeciliidae, or Cyprinodontidae families within the ment of complete mitogenomes evinced 85.75% nucleotide sequence Cyprinodontiformes. Grant and Riddle (1995) were unable, however, identity between X. eiseni and E. latos, and 85.86% identity of X. eiseni to clarify the relationship between the Empetrichthys/Crenichthys clade

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Fig. 5. Maximum likelihood phylogenetic trees based on aligned partial mtDNA nucleotide sequences comprised of either (a) an approximately 1320 bp nucleotide partial sequence (ND2- containing region) of mtDNA containing genes for ND2, tRNAGln, tRNAMet, tRNATrp, tRNAAla, and tRNAAsn, or (b) an approximately 1190 bp partial mtDNA sequence encompassing the genic region with the genes for Cyt B, tRNAGlu and tRNAThr. Trees were constructed using the Tamura-Nei model with all sites. Node confidence is indicated by bootstrap values (1000 replicates). GenBank accession numbers accompany each taxon. and the only two Goodeid fishes examined in that study: the Spotted geographic representation in those wide-ranging taxa. For instance, skiffia(Skiffia multipunctata) and the Goldbreast splitfin( within the Cyprinodontidae, fishes in the Cyprinodon pupfish genus furcidens). North and Central America (e.g., Echelle, 2008) formed a distinct clade In light of this unresolved question about family/subfamily status in both the ND2-containing and Cyt B-containing trees, while the for the Empetrichthys/Crenichthys clade, we conducted additional phy- relationships of the Cyprinodon fishes to the Orestias pupfishes of the logenetic analyses using partial mtDNA regions that were available for Andes Mountains of South America (Parenti, 1984), as well as to the at least ten representative taxa from the order Goodeidae, in an effort to Aphanius fishes of northern Africa and southwestern Asia (e.g., Hrbek provide insight into the extent of genetic divergence between the et al., 2002), were poorly resolved. Empetrichthys/Crenichthys clade and the Goodeid fishes of central Estimates of evolutionary genetic distance among species within Mexico. We conducted these phylogenetic analyses using a ~1320 bp each taxonomic family showed that the average nucleotide divergence nucleotide region of mitochondrial DNA containing the ND2 gene and a within the Profundulidae, Fundulidae, Cyprinodontidae, and ~1190 bp mtDNA region inclusive of the Cyt B gene. Maximum Poeciliidae families was 0.19002 base differences per site for the Cyt likelihood trees for the aligned 1320 bp ND2-containing sequences B-containing region and 0.15431 base differences per site for the ND2- (Fig. 5a) and 1190 bp Cyt B-containing sequences (Fig. 5b) again containing region, for a combined average nucleotide divergence of revealed the Empetrichthys and Crenichthys genera to be basal within a 0.16216 base differences per site within a family taxonomic level clade containing the viviparous goodeids of central Mexico. The designation (Table 6). Kartavtsev (2011a, 2011b) calculated genetic Profundulidae fishes were observed to be the sister taxa to this clade divergence estimates for the mtDNA Cyt B and COI genes from 20,731 of Empetrichthys/Crenichthys and Mexican goodeids in both phyloge- species of vertebrates including fishes, and observed mean unweighted netic analyses, as has been observed previously by Pollux et al. (2014). scores of p-distances (%) for these five groups are as follows: popula- Relationships among other families including the Fundulidae, Cyprino- tions within a species (Cyt B: 1.38 ± 0.30%; COI: 0.89 ± 0.16%; dontidae, and Poeciliidae were less well resolved in both analyses mean ± SD); subspecies or semi-species (Cyt B: 5.10 ± 0.91%; COI: (Fig. 5a,b). That poor resolution may be in part related to uneven 3.78 ± 1.18%); species within a genus (Cyt B: 10.31 ± 0.93%; COI:

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Table 6 Estimates of evolutionary divergence (avg. number of nucleotide distances per site) for a ~1320 bp partial sequence (ND2-containing region) of mtDNA containing genes for ND2, tRNAGln, tRNAMet, tRNATrp, tRNAAla, and tRNAAsn, or a ~1190 bp partial mtDNA sequence encompassing the genic region with the genes for Cyt B, tRNAGlu and tRNAThr. Within family genetic variation is illustrated in red text.

ND2-containing region E. and C. Goodeidaeb Profundulidae Fundulidae Cyprinodontidae Poeciliidae generaa Empetrichthys and 0.092956 Crenichthys genera (E. and C. genera) Goodeidae 0.169491 0.106940 (w/o E. and C. genera)b Profundulidae 0.248329 0.248163 0.127030 Fundulidae 0.262301 0.262792 0.319771 0.184373 Cyprinodontidae 0.251429 0.254785 0.308300 0.269527 0.199769 Poeciliidae 0.293646 0.295314 0.330755 0.308142 0.290878 0.168915 Cyt B-containing region E. and C. Goodeidaeb Profundulidae Fundulidae Cyprinodontidae Poeciliidae generaa E. and C. genera 0.070009 Goodeidaeb 0.141172 0.102655 Profundulidae 0.195675 0.191118 0.133360 Fundulidae 0.214520 0.215502 0.207542 0.148597 Cyprinodontidae 0.224528 0.223805 0.220190 0.211443 0.178486 Poeciliidae 0.216295 0.217498 0.220101 0.215416 0.220149 0.156799 Notes. a “E. and C. genera” indicates the three taxa Empetrichthysand Crenichthysspecies: E. latos latos, C. baileyi moapae, and C. nevadae. b The Goodeidae family category here includes the central Mexican viviparous goodeid only, and not representatives from the Empetrichthysand Crenichthys genera.

11.06 ± 0.53%); species from different genera within a family (Cyt B: Even with those considerable life history and phenotypic distinctions, 17.86 ± 1.36%; COI: 16.60 ± 0.69%); and separate families within however, previous evolutionary reconstructions of the Cyprinodonti- an order (Cyt-b: 26.36 ± 3.88%; COI: 120.57 ± 0.40%). Based on our formes using molecular genetics or morphology have grouped the data, the average nucleotide divergence of the family Goodeidae is Empetrichthys and Crenichthys genera as part of family Goodeidae 14.12 ± 1.74% base differences per site for the Cyt B-containing (Doadrio and Domínguez, 2004; Hertwig, 2008). Based on our results mtDNA region, and 16.95 ± 2.67% for the ND2-containing region, here comparing evolutionary genetic distances across cyprinodontoid when the three Empetrichthys and Crenichthys species are grouped families, combined with the results of those prior morphological and within the family Goodeidae together with the central Mexican good- genetic studies, we recommend the inclusion of the oviparous Empe- eids (Table 6). Those estimates of genetic distance approach the range trichthys and Crenichthys genera within family Goodeidae along with the of genetic distances observed across different genera within a single viviparous central Mexican goodeid fishes. family by Kartavtsev (2011a, 2011b), and also resemble the range of genetic diversity values observed within other Cyprinodontiform families (i.e., Profundulidae, Fundulidae, Cyprinodontidae, and Poeci- Funding information liidae) (Table 6). In contrast, describing the clade of the Empetrichthys and Crenichthys genera as a separate family (Empetrichthyidae) (Miller This publication was supported by the National Institute of General and Smith, 1986; Miller et al., 2005; Minckley and Marsh, 2009) results Medical Sciences of the National Institutes of Health under award in a family taxon exhibiting genetic diversity estimates (7.01 ± 1.23% number 5R25GM086299-07, as part of the Bridges to the Baccalaureate for Cyt B and 9.30 ± 1.97% for ND2) lower than that typically Program. Additional support came from the California State University observed for family-level taxonomic comparisons (Kartavtsev, 2011a, Program for Education and Research in Biotechnology (CSUPERB) to 2011b), including the other well established Cyprinodontiform families SCL. CAS and SCG were supported by funding from a Nevada State (Table 6). Similarly, grouping the central Mexican goodeids as their Wildlife Grant administered by Jon Sjoberg. Additional stipend funding own family, without the Empetrichthys and Crenichthys genera, results in was provided to SCG by the Environmental & Conservation Sciences a family with an average genetic distance of only 10.48% across both Graduate Program at North Dakota State University. the Cyt B and ND2 genes, which again represents a genetic distance lower than that typically observed in comparisons of genera within the same family (Kartavtsev, 2011a, 2011b). Acknowledgements These genetic distance estimates suggest that the Empetrichthys/ Crenichthys clade is best classified as the subfamily Empetrichthyinae We thank J. Harter (U.S. Fish and Wildlife Service) and K. within the family Goodidae. However, it is important to point out that Guadalupe (Nevada Department of Wildlife) for assistance in adminis- the Empetrichthyinae fishes may also exhibit low levels of genetic tration and tissue sample collection. The poolfish collections were diversity due to the limited number of taxa in this group. What is more, conducted under USFWS permit TE 126141-2, Nevada Scientific fishes within the Empetrichthys/Crenichthys clade exhibit several life Collecting Permit S-34628, and NDSU IACUC protocol #12027. The history and morphological characters distinct from the goodeids of Moapa Valley National Wildlife Refuge facilitated the collection of central Mexico. For example, fishes within the Empetrichthys/ springfish. The authors also thank two anonymous referees, whose Crenichthys clade are oviparous (La Rivers, 1994, Uribe et al., 2012), comments improved this manuscript. which represents a distinct difference in reproductive strategy from the viviparous reproduction of goodeid fishes in Mexico. They also have contact organs on the scales similar to cyprinodontoids, while all Appendix A. Supplementary data Mexican goodeid fishes lack this organ. Such life history and morpho- logical distinctions point to significant evolutionary divergence of the Supplementary data to this article can be found online at http://dx. Empetrichthys/Crenichthys clade from other members of the Goodeidae. doi.org/10.1016/j.gene.2017.05.023.

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