Mitochondrial DNA Variation in Pupfishes Assigned to the Species

Mitochondrial DNA Variation in Pupfishes Assigned to the Species Cyprinodon macularius (Atherinomorpha: Cyprinodontidae): Taxonomic Implications and Conservation Genetics Author(s): Anthony A. Echelle, Ronald A. van den Bussche, Terrence P. Malloy, Jr., Michelle L. Haynie and C. O. Minckley Source: Copeia, Vol. 2000, No. 2 (May 8, 2000), pp. 353-364 Published by: American Society of Ichthyologists and Herpetologists (ASIH) Stable URL: http://www.jstor.org/stable/1448183 . Accessed: 22/07/2014 12:05

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Mitochondrial DNA Variation in Pupfishes Assigned to the Species Cyprinodonmacularius (Atherinomorpha: Cyprinodontidae): Taxonomic Implications and Conservation Genetics

ANTHONY A. ECHELLE, RONALD A. VAN DEN BUSSCHE, TERRENCE P. MALLOYJR., MICHELLE L. HAYNIE, AND C. O. MINCKLEY

Variationin mitochondrialDNA (mtDNA)was assessed in a captive stock and 11 wild populations (n = 259) from throughout the native range of Desert Pupfish Cyprinodonmacularius as traditionallyunderstood. Using PCR-SSCP,18 composite haplotypes were identified from a 333-bpsegment of the mitochondrialD-loop and two segments of the ND2 gene (333 and 325 bp). Representativesof each haplotype were sequenced for the entire ND2 gene and the 337-bp segment of the D-loop. Phylogeneticanalyses revealed that haplotypes form two monophyleticgroups, one in the Rio Sonoyta/QuitobaquitoSprings area and one in the Salton Sea/Colorado River Delta. This, with previous observations on morphology, color pattern, and geological history, supports recognition of the Rio Sonoyta/Quitobaquitopopula- tions as a separate species, the QuitobaquitoPupfish C. eremusMiller and Fuiman, from the more widespread desert pupfish C. maculariusBaird and Girard. More than 70% of mtDNA diversityacross all populationswas attributableto differences between the two species. Withinspecies, the averagelocal population contains94% and 97% of the diversityin, respectively,C. eremusand C. macularius.Differences between the Salton Sea and Colorado River Delta populations of C. maculariusex- plain a small (3.7%), but statisticallysignificant, portion of mtDNA diversityin this species. This and the history of connections between Salton Sea and the delta sug- gest that the two regions should be managed separately with no intermixing of pupfish other than what occurs when the present, human-regulatedhydrology is overcome by natural flooding. Haplotype frequencies in C. eremusfrom Quitoba- quito Springsand Rio Sonoytawere not significantlydifferent. However,the potentially long history of isolation between these two populationsand evidence of some degree of morphologicaldivergence indicate a need for conservativemanagement with no intermixing.The captive stock exhibited reduced mtDNAvariation relative to its wild parent population from a locality on the delta.

Variaci6nde ADNmt fue examinado por una cepa de cautivo y 11 poblaciones silvestres (n = 259) de todas partes del rango natural del cachorrito del desierto Cyprinodonmacularius como entendido tradicionalmente.Utilizando PCR-SSCP,18 haplotipos compuestos fueron identificados desde un segmento de 337-pb del D- loop mitocondrialy dos segmentos del gene DN2 (333 pb y 325 pb). Representantes de cada haplotipo fueron sequenciados por el gene DN2 entero y el segmento de 337-pb del D-loop. Anilisis filogenetico mostr6 una relaci6n monofileticareciproca entre poblaciones de dos regiones generales, Rio Sonoyta/Quitobaquitoy Salton Sea/Delta del Rio Colorado. Esto, con observacionesprevias de morfologia,patr6n de color, y la hist6ria geol6gica, sostiene el reconocimiento de las poblaciones de Rio Sonoyta/Quitobaquitocomo una especie distinta,el cachorritode Quitobaquito C. eremus Miller and Fuiman, de la especie con una distribuci6nmis amplia, el cachorritodel desierto C. maculariusBaird and Girard.Mis de 70%de la diversidad ADNmt de todas las poblaciones fue atribuidaa diferencias entre las dos especies. Dentro de cada especie, la poblaci6n promedia local contiene 94% y 97% de la diversidadde, respectivamente,C. eremusy C. macularius.Las diferencias entre las poblaciones de C. maculariusdel Salton Sea y las de la Delta del Rio Colorado explican una pequefia (3.7%) pero una porci6n estadisticamentesignificativa de la diversidadADNmt de esta especie. Esto y la historia de conecciones entre el Salton Sea y la delta sugieren que los dos regiones deben ser manejadasaparte sin mezclar a los cachorritosmis que lo que ya ocurre cuando la hidrologia actual controlada por los humanos, esti conquistadapor inundacionesnaturales. Frequencias de los haplotipos en C. eremusdel Quitobaquito Springs y el Rio Sonoyta no fueron sig-

O2000 by the American Society of Ichthyologists and Herpetologists

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nificativamentediferentes. No obstante, la historia probablementelarga del aisla- miento de estas dos poblaciones y la evidencia de alg6n grado de divergenciamor- fol6gica indican la necesidad de un manejo conservativosin mezclarlas formas. La cepa a cautivamostr6 variaci6nreducida de ADNmt en relaci6n a la de su poblaci6n silvestre de origin de una localidad en la delta.

Desert Pupfish Cyprinodonmacularius is aimed primarily at resolving evolutionary rela- THElisted as an endangered species by the In- tionships among western pupfishes and includ- ternational Union for Conservation of Nature ed samples from only one or two localities and Natural Resources (Miller, 1979) and by the (Turner, 1974; Echelle and Dowling, 1992; United States government (U.S. Department of Echelle and Echelle, 1993). the Interior, 1986). In this paper, we use mitochondrial DNA (mtDNA) variation to describe MATERIALS AND METHODS the genetic structure of populations tradition- ally classified as C. macularius and to provide a In 1997-1998, collections of the desert pup- basis for recommendations regarding conser- fish complex were made at 11 sites (n = 19-25) vation genetics of the species. The results, to- representing all major populations (Fig. 1; Ap- gether with geological history and color pat- pendix). We also included 33 specimens from a terns in nuptial males, indicate that populations captive population at Dexter National Fish grouped as C. macularius represent two evolu- Hatchery and Technology Center (DNFH) in tionarily divergent entities that should be rec- New Mexico. Specimens were initially frozen in ognized as separate species. We hereafter refer liquid nitrogen or on dry ice and stored in the to the two species as the "desert pupfish com- lab at -75 C until processed for analysis. plex." DNA was extracted from muscle tissue follow- The historic range of the desert pupfish com- ing the method of Longmire et al. (1997), and plex once extended from Gila River tributaries the polymerase chain reaction (PCR) and sin- in southeastern Arizona and northern Sonora, gle-stranded conformational polymorphism westward to the Salton Sea area of southern Cal- (SSCP) analysis (Orita et al., 1989; Fan et al., ifornia and southward into the Colorado River Delta region in Sonora and Baja California (Miller, 1943). Across this region, Miller and Fuiman (1987) recognized one wide-ranging California 114• C. m. macularius, and a local endem- Salton Arizona subspecies, SSea ic, C. m. eremus, restricted to Quitobaquito 2 3 o ef Springs, Organ Pipe Cactus National Monu- GO ment, southern Arizona. The wide-ranging subspecies has disappeared from the Gila and lower Colorado River areas of Arizona/California Baja 1973; and the remain- 467 6 (Minckley, Moyle, 1976), California 8 *10320 natural are much smaller and ing populations S9 onora 11 more fragmented than in earlier times (Hen- 0 50 100 drickson and Varela, 1989; Dunham and Minck- Km -A ley, 1998). This decline is a result of interactions with introduced and habitat losses re- species 1. Localitiessampled for mtDNAvariation in from reservoir surface-wa- Fig. sulting construction, the desert pupfish complex. Sample sites represent ter diversion projects, groundwater depletion, the three majorareas occupied by extant populations: and other factors (Miller and Fuiman, 1987; the Salton Sea area (sites 1-3), the Colorado River Shoenherr, 1988; Hendrickson and Varela, Delta (sites 4-9), and the Rio Sonoyta/Quitobaquito 1989). Springs area. (sites 10 and 11). Now-extirpatedpop- Previous studies of variation in the ulations are known to have occurred in waters asso- genetic River in Arizona and desert have been restricted to ciated with the lower Colorado pupfish complex from the Gila River Basin at scattered localities from populations in the United States (Turner, near the confluence with the Colorado River to the 1984), or they included only one or two samples San Pedro River (= Rio San Pedro) in southeastern of transplanted Mexican populations (Turner, Arizona and northern Sonora, somewhat beyond the 1983; Dunham and Minckley, 1998). Other ge- eastern limit of this map (Minckley, 1973; Miller and netic studies that included desert pupfish were Fuiman, 1987).

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1993) were used to screen all specimens for var- gene, and their D-loop sequences are based on iation in portions of the mitochondrial D-loop the primers we used. and ND2 gene. For ND2, we used primers Sequences for each composite haplotype ND2B-L and ND2E-H (T. E. Dowling, pers. identified by SSCP and those extracted from comm.) to amplify and sequence the entire GenBank were aligned with CLUSTAL W gene. Using the ND2 sequence, we developed (Thompson et al., 1994) and subjected to phy- internal primers for a 333-bp segment (5'- logenetic analysis with PAUP (vers. 3.05, D. L. CCTTCCTTTGCTAATGAACC-3'and 5'-CCAA- Swofford, Champaign, IL, 1991, unpubl.). Se- TTTTAAGTGCCAGGG-3') at the 5' end (posi- quence data were coded as discrete, unordered tions 8 to 340) and a 325-bp segment (5'-ATA- characters, and we used PAUP's heuristic search CCTCGCCACCTCTTG-3' and 5'-AGCCAGA- algorithm with 10 iterations of random input TTGTTGCGGAG-3') at the 3' end (positions order of taxa to find the most parsimonious 661 to 986). For SSCP analysis of the D-loop, we trees. To evaluate robustness of the results, we used primers L15926 (Kocher et al., 1989) and performed a heuristic bootstrap analysis (Fel- H16498 (Meyer et al., 1990) to amplify a por- senstein, 1985) of 100 iterations with 10 repli- tion of mtDNA extending from the threonine cations of random input order and tree-bisec- tRNA gene through 337 bp of the D-loop; these tion-reconnection for each iteration. Gaps rep- are primers E and K in Lee et al. (1995). To resenting four indels in the control region were screen for SSCP variation, the three mtDNA seg- treated as missing data. ments from each specimen were PCR-amplified Haplotype frequencies and sequences for the from genomic DNA in separate reactions and three mtDNA segments assayed for SSCP varia- in the presence of t-32P-dCTP,2.0 p1 of each tion were used in analyses of population genetic diluted PCR product was electrophoresed in a structure. We used Arlequin (vers. 1.1 S. Schnei- 5% polyacrylamide gel (40:1 acrylamide:bis ac- der, J.-M. Kueffer, D. Roessli, and L. Excoffier, rylamide; 10% glycerol) at 300 V (3 W) for 22 Geneva, Switzerland, 1997, unpubl.) to com- h, and bands were visualized by autoradiogra- pute within-sample estimates of haplotype diver- phy. sity (h) and nucleotide diversity (wr;from Taji- For phylogenetic analysis, we PCR-amplified ma-Nei distances, no gamma correction), pair- the entire ND2 gene (1047 bases; primers wise FST and (sT statistics, exact tests (Raymond ND2B-L and ND2E-H) and the 337-bp fragment and Rousset, 1995) of difference in haplotype of the mtDNA D-loop (primers L15926 and frequencies, and an analysis of molecular vari- H16498) in 35 specimens representing all com- ance (AMOVA; Excoffier et al., 1992). The posite haplotypes detected by SSCP analysis. AMOVA partitioned molecular variance into The amplicons were sequenced directly using proportions resulting from within-sample varia- either an ABI 373 or 377 automated sequencer. tion, differences among samples within regions, All haplotypes occurring at more than one lo- and differences among regions. We designated cality were sequenced in representatives from three regions for this analysis: the Salton Sea two to six localities, depending on distribution area (sites 1-3, Fig. 1), the Colorado River Delta of the haplotype. One haplotype occurred in (sites 4-9), and the Rio Sonoyta/Quitobaquito nine of the 11 samples and was sequenced in area (sites 10-11). The AMOVA was performed nine specimens from six different localities. To on haplotype frequencies alone (producing F- test the monophyly of haplotypes in desert pup- statistics) and on frequencies plus sequence di- fish, we included GenBank sequences provided vergences (producing ()-statistics; Excoffier et by Duvernell and Turner (1998) for three other al., 1992). Also using Arlequin, we obtained sig- pupfishes, two species grouped with the desert nificance tests for individual, pairwise FST and pupfish complex in the "western pupfish clade" (DST values and the variance components in the (Echelle and Dowling, 1992), the Amargosa AMOVAs [nonparametric permutation method Pupfish, C. nevadensis (GenBank accession num- described by Excoffier et al. (1992)]. For mul- bers: ND2, AF028308; D-loop, AF028290), and tiple pairwise tests, we used the stepwise Bon- Owens Pupfish, C. radiosus (ND2, AF028311; D- ferroni correction (Rice, 1989) to obtain a Type loop AF028293), and a rather distantly related I error less than 0.05. (Echelle and Echelle, 1998) outgroup species, the Sheepshead Minnow, C. variegatus (ND2, RESULTS AF028313; D-loop, AF028299). The mtDNA of C. radiosus is the sister group to the mtDNA lin- SSCP analysis of haplotype variation among eage in the desert pupfish complex (Echelle 258 specimens of desert pupfish identified 10 and Dowling, 1992). Duvernell and Turner's variants for the 5' end of the ND2 gene and 5 (1998) sequences included the entire ND2 for the 3' end of the gene, producing 13 com-

This content downloaded from 128.123.44.23 on Tue, 22 Jul 2014 12:05:11 PM All use subject to JSTOR Terms and Conditions 356 COPEIA, 2000, NO. 2 posite ND2 haplotypes; nine variants were de- the three regions (Rio Sonoyta/Quitobaquito, tected for the D-loop, giving 18 composite hap- Salton Sea, and Colorado River Delta), 64.8% lotypes across the three segments (Table 1). All to variation within samples, and only 0.9% to SSCP-identified haplotypes for each of the three differences between samples within regions. segments had at least one unique base substi- The corresponding values for D-statistics ("total tution. In three instances, haplotypes identical diversity"; i.e., haplotype frequencies plus se- on the basis of SSCP differed by one base sub- quence divergences) were, respectively, 75.8%, stitution in the midportion of the ND2 gene 23.6%, and 0.6%, indicating that a large pro- that was not surveyed for SSCP variation; these portion of the among-region genetic variance variants were ignored in the analysis because ad- reflects sequence differences and not just hap- ditional sequencing would have revealed other lotype frequencies. For both analyses, there base substitutions for other portions of the were highly significant differences among re- mtDNA. All ND2 and D-loop sequences have gions (P < 0.00001) but not among samples been deposited in GenBank (accession num- within regions (P = 0.11-0.15). More than 70% bers AF198967-AF199002). of the total diversity reflects differences between samples from the Rio Sonoyta/Quitobaquito re- Genetic structure.-The 18 haplotypes detected gion and those from the Salton Sea and Colo- by SSCP analysis varied at a total of 18 sites (15 rado River Delta regions. When the Rio Son- transitions, three transversions) across the two oyta/Quitobaquito populations were excluded ND2 segments surveyed: 11 in the 333-bp seg- from the analysis, only 3.7% of total diversity ment at the 5' end and seven in the 325-bp seg- was attributable to differences between the Sal- ment at the 3' end. Five variable sites (all with ton Sea and Colorado River Delta regions (Ta- transitions) were detected in the ND2 segments ble 2). The corresponding value for differences not included in the SSCP analysis (389 bp). For between the samples from Rio Sonoyta and Qui- the 337-bp D-loop fragment, there were 12 var- tobaquito Springs was 2.9%. iable sites and 13 substitutions (eight transi- Considering only haplotype frequencies, tions, five transversions). Individual haplotypes roughly equivalent proportions of gene diversity differed at one to 10 sites. The measures of hap- were attributable to differences among regions lotype and nucleotide diversities showed no for the ND2 gene and the D-loop sequences geographic pattern of variation and no consis- (Table 2). However, when sequence divergence tent pattern of variation between ND2 and the was included in the AMOVA, the among-region D-loop (Table 1). component of diversity was much greater for All populations had two to five SSCP variants ND2 than for the D-loop (87% vs 11%; Table except the DNFH sample, which was monomor- 2). Within regions, the two sequences are phic for haplotype A. The Rio Sonoyta and Qui- roughly equivalent in proportion of genetic di- tobaquito populations exhibited no significant versity attributable to differences among sam- difference in haplotype frequencies (Tables 1- ples. 2). These populations shared no haplotypes A small (3.7%), but statistically significant, with samples from the Salton Sea and the Col- among-region component of variation was de- orado River Delta areas. Thus, all pairwise tests tected in the combined ND2/D-loop data for of FsTand OsTand the corresponding exact tests the Salton Sea and Colorado River Delta when of differences in haplotype frequency were sequence data were included in the analysis but highly significant (P < 0.0001) in comparisons not when sequence data were excluded (Table of Rio Sonoyta/Quitobaquito samples with Sal- 2). The two regions share the common haplo- ton Sea and Colorado River Delta samples. All type (A), and the difference in sequence infor- Salton Sea and Colorado River Delta samples mation is attributable to the remaining, relative- had high frequencies (60-89%) of haplotype A, ly uncommon, haplotypes in the two regions and, with the Bonferroni correction, there were (Table 1). The geographically intermediate no significant differences among paired sam- population at Cerro Prieto (Site 4, Fig. 1) ples within or between the two regions. There shared one uncommon haplotype (E) with Sal- was, however, evidence of significant divergence ton Sea populations and none with the other between the captive stock (DNFH) we exam- populations on the delta. ined and its parent population (site 6, Fig. 1; FsT = 0.31, P < 0.00001; csT = 0.21, P < Phylogenetic analysis.-The 1384 sites available 0.00001). for phylogenetic analysis of the ND2/D-loop se- The AMOVA based on F-statistics indicated quences included 191 variable sites, 54 of which that 34.3% of the variation in haplotype fre- were parsimony-informative (40 ND2 sites, 14 D- quencies is attributable to differences among loop sites); that is, two or more (but not all)

This content downloaded from 128.123.44.23 on Tue, 22 Jul 2014 12:05:11 PM All use subject to JSTOR Terms and Conditions TABLE 1. NUMBER OF OCCURRENCESFOR 18 HAPLOTYPESIN THE DESERT PUPFISH COMPLEX, WITH HAPLOTYPEDIVERSITY (h) AND NUCLEOTIDE DIVERSITY(IT) FOR THE ND2 GENE,THE D-LOOP, AND BOTH COMBINED. Locality numbers correspond with those in Figure 1 and the Appendix.

Haplotypes Area/locality A B C D E F G H I J K L M N O P Q R Salton Sea area 1. Shoreline Pool 18 1 2 3 1 2. San Felipe Creek 16 1 2 2 3. CountyLine Drain 17 3 Colorado River Delta 4. Cerro Prieto 16 2 1 5. LagunaSalada 14 3 3 6. Santa Clara Slough 12 4 3 1 7. Flor del Desierto 17 1 1 r• 8. El Doctor 1 17 1 1 1 9. El Doctor 2 13 3 1 1 1 1 r Rio Sonoyta/QuitobaquitoSpring 10. Rio Sonoyta 10 5 4 11. Quitobaquito 11 2 8 CaptiveDNFH populationa 33 h 7r

Area/locality ND2 D-loop Both ND2 D-loop Both Salton Sea area H 1. Shoreline Pool 0.48 0.42 0.48 0.00087 0.00179 0.00118 2. San Felipe Creek 0.42 0.27 0.42 0.00084 0.00057 0.00075 3. CountyLine Drain 0.27 0.27 0.27 0.00082 0.00161 0.00108 ,,4 Colorado RiverDelta 4. Cerro Prieto 0.29 0.20 0.29 0.00062 0.00000 0.00041 5. LagunaSalada 0.49 0.27 0.49 0.00123 0.00241 0.00163 6. Santa ClaraSlough 0.42 0.61 0.61 0.00118 0.00407 0.00215 7. Flor del Desierto 0.11 0.20 0.20 0.00032 0.00126 0.00064 8. El Doctor 1 0.19 0.19 0.28 0.00046 0.00120 0.00071 ,0o 9. El Doctor 2 0.51 0.35 0.57 0.00143 0.00271 0.00186 Rio Sonoyta/Quitobaquito 10. Rio Sonoyta 0.34 0.68 0.68 0.00051 0.00248 0.00118 11. Quitobaquitospring 0.50 0.60 0.60 0.00075 0.00202 0.00118 CaptiveDNFH populationa 0.00 0.00 0.00 0.00000 0.00000 0.00000

DNFH = Dexter National Fish Hatchery and Technology Center; population was founded by 280 specimens from locality 6 (Fig. 1; Appendix) in 1983 (Dunham and Minckley, 1998).

This content downloaded from 128.123.44.23 on Tue, 22 Jul 2014 12:05:11 PM All use subject to JSTOR Terms and Conditions TABLE PERCENTAGEOF GENETIC 2. VARIATIONEXPLAINED BY DIFFERENTLEVELS OF GEOGRAPHICSTRUCTURE IN DESERT PUPFISH. Results of hierarchical analyses of genetic diversity for different segments of mtDNA. Asterisks signify probability levels: *<0.05, **<0.01, ***<0.0001. 0 ND2 and D-loop Haplotype frequencies only Haplotype frequencies and sequences Salton Salton Rio Sonoyta/ Source of Sea/Colorado Rio Sonoyta/ All three Sea/Colorado Quitobaquito All three variation River Delta Quitobaquito regions River Delta Region regions Among regions 1.5 - 34.3** 3.7* - 78.5* Among populations n within regions 1.9 0.0 0.9 2.1 2.9 0.6 0 Variation within populations 96.6a 100.0 64.8 94.2 97.1 23.6 1', ND2 only Among regions 1.9*" 44.6** 3.5** - 86.8* ?) Among populations 1', within regions 1.3 2.9 0.8 2.0 2.9 0.3 Variation within populations 96.7 97.1 54.6 94.5 97.1 12.9 D-loop only Among regions 2.0 - 40.9* 3.8 - 10.6*** Among populations within regions 2.1 0.0 0.8 2.3 2.9 2.1 Variation within populations 95.9a 100.0 58.3 93.9a 97.1 87.4

A significant (P < 0.05) portion of the genetic variation was attributable to differences among samples, but the differences were not consistent among or within regions.

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four restricted to the Rio Son- 66 (65) 0 C Salton ing haplotypes 0 Sea area, and a second o 2 D oyta/Quitobaquito compris- o A ing 14 haplotypes restricted to the Salton Sea/ 1 E Colorado River Delta area (Fig. 2). The Salton 1 G Sea/Colorado River Delta clade included one 2 H subclade B (83) containing haplotypes (widespread 62 (70) 1 Colorado in Salton Sea and F in the 200 N River area) (widespread Delta Colorado River Delta) and one all 10 K containing 77(81) 1 L other Salton Sea/Colorado River Delta haplo- 0/02 Mm types. In the latter subclade of 12 haplotypes, 2 J the most widespread member (Haplotype A; Ta- 100 59(0) ble was also the most with no (100) E 1) plesiomorphic, F-'a-2 B salton sea evidence of divergence from the hypothetical 1 R Rio Sonoyta 7072) common ancestor of the group, whereas the 72(74) 1/0 0 0 8/3 72(84) 0/0/0 p other members of the group showed 1-3 de- 81/3 3/1( 1 QuitobaquitoSprings 1/0 rived mutations (Fig. 2). These 12 haplotypes formed a with no 67/10 28/4 C.radiosus large polytomy phylogenetic 16/2 19/0 C. nevadensis resolution except that haplotypes C and D from C. variegatus the Salton Sea area formed a monophyletic pair, as did I and N from the Colorado Fig. 2. Phylogenetic relationships among 18 haplotypes River Delta. mtDNA haplotypes (A to R) from desert pupfish and haplotypes from two other western pupfishes, Cypri- Bootstrap analysis of the ND2 data alone pro- nodon nevadensis and C. radiosus. Cyprinodonvariegatus duced a topology (180 steps; CI = 0.90) iden- was the designated outgroup. Numbers above each tical to that for the combined ND2/D-loop data node indicate bootstrapsupport (> 50%) in a maxi- except that it did not support the clade contain- mum the parsimony bootstrap analysis (outside pa- ing haplotypes B and F; instead these haplo- rentheses are resultsfor combined data; ND2/D-loop types clustered as separate parts of the large po- inside parentheses are results for ND2 alone). Num- lytomy in Figure 2 that includes the remaining bers below each interior node are number of syna- for the ND2 and the Salton Sea/Colorado River Delta haplotypes. pomorphies gene (top) D-loop for most internal nodes of (bottom); for each, number of transitionsis to the left Bootstrap support of the slash mark and number of transversionsis to the tree were greater with the ND2 data alone the right. Only the total number is given for autapo- than with the combined ND2/D-loop data (Fig. morphic changes. 2).

Notes on nuptial colorationin males.-Males from haplotypes in the ingroup differed from the Rio Sonoyta/Quitobaquito and those from the outgroup (C. variegatus) haplotype by a shared remainder of the historic range of the desert base substitution. Only 13 sites (12 ND2, 1 D- pupfish complex are diagnostically different in loop) were informative regarding relationships male breeding coloration. Miller (1943:4, 11) among haplotypes of the desert pupfish com- reported that breeding males of C. macularius plex. The heuristic search for the shortest tree from the Salton Sea and from a location near with the combined ND2/D-loop data produced our sites 8 and 9 in Sonora had "bright yellow two equally parsimonious topologies (230 steps; to brilliant yellow orange" on the caudal pe- CI = 0.89) after collapsing all nodes with no duncle and caudal fin. R. R. Miller's (pers. synapomorphies. The strict consensus tree was comm.) fieldnotes from April 1950 on males identical to the 50% majority-rule consensus from a locality on the Rio San Pedro, a tributary tree from the bootstrap analysis (Fig. 2). These of the Gila River in northern Sonora and south- trees, and similar trees produced by analysis of eastern Arizona, mention "yellow to yellow or- ND2 separately, supported monophyly of the ange" on the caudal fin and caudal peduncle. desert pupfish complex and the previously re- Thus, the now-extirpated eastern populations ported (Echelle and Dowling, 1992; Duvernell apparently had the bright yellows seen in the and Turner, 1998) close relationship between extant populations in the Salton Sea and the haplotypes of the complex and those of C. ra- Colorado River Delta. In contrast, males from diosus (Fig. 2). D-loop data also supported Quitobaquito Springs and the associated pond monophyly for the desert pupfish complex do not exhibit bright yellow in the caudal re- (bootstrap support = 99%). gion (Liu, 1969; Loiselle, 1982; M. R. Douglas, Haplotypes of the desert pupfish complex pers. comm.; pers. obs.), although Miller and formed two monophyletic clades, one compris- Fuiman (1987:599) reported "yellow to olive-

This content downloaded from 128.123.44.23 on Tue, 22 Jul 2014 12:05:11 PM All use subject to JSTOR Terms and Conditions 360 COPEIA, 2000, NO. 2 yellow" in the caudal fin and "just encroaching Ranking C. eremusand C. macularius as species onto the posterior third of caudal peduncle." under the phylogenetic species concept avoids Recent observations by one of us (COM) and ambiguity regarding their ontological status by P. Unmack indicate that males from the Rio (Cracraft, 1983, 1989). Treating them as subspe- Sonoyta population do not exhibit bright yellow cies would connote the possibility that, like colors. This is supported by R. R. Miller's field- many recognized subspecies, they are arbitrarily notes for 14 April 1950: "color of males [from defined parts of a taxon rather than discrete en- a Rio Sonoyta locality was] essentially same as at tities existing independently of human con- Quitobaquito [Springs].... No yellow on body, cepts. but some males with yellowish caudal [fin] and Recent speciation probably explains the lack caudal [fin] base." of fixed allelic differences for eight polymorphic proteins (Turner, 1983) and the low RFLP DIscuSSION estimate of overall mtDNA sequence-divergence (0.34%; Echelle and Dowling, 1992) between C. Reciprocal monophyly between the mtDNA macularius and C. eremus.The level of mtDNA haplotypes of the Rio Sonoyta/Quitobaquito divergence is similar to that (0.16-0.64%, x = populations and those of the Salton Sea/Colo- 0.42) within a monophyletic group of pupfish rado River Delta suggests long, mutually exclu- haplotypes in which those of C. nevadensis are sive evolutionary histories (Neigel and Avise, paraphyletic (Echelle and Dowling, 1992; Du- 1986) for the two groups, a hypothesis that is vernell and Turner, 1998) with respect to the consistent with geological history. Isolation of Devils Hole Pupfish, C. diabolis, a morphologi- the ancestral Rio Sonoyta/Quitobaquito drain- cally divergent species, which, as Miller (1981) age from the Colorado River Delta probably oc- suggested, may have evolved within the last curred sometime in the last 100,000 years when 20,000 years. The species flock of five pupfishes lava beds resulting from eruptions of the Sierra in Lake Chichancanab on Mexico's Yucatan Pinacate Volcanic Field diverted the flow of the Peninsula provides another example of rapid Rio Sonoyta southward to the Gulf of California morphological divergence with negligible diver- and away from its previously westward course to- gence in allele frequencies for protein-encoding ward the Colorado River Delta (Ives, 1964; Don- genes (Humphries, 1984) and a lack of recip- nelly, 1974; Turner, 1983). More recent separa- rocal mtDNA monophyly (Strecker et al., 1996). tion of the Rio Sonoyta and Quitobaquito In this example, speciation and striking mor- Springs populations would explain the lack of a phological divergence (Humphries and Miller, significant difference in haplotype frequencies 1981) might have occurred in as little as 8000 between our samples from these areas. years (Strecker et al., 1996). Geological history and the mtDNA genealogy The relatively low level of morphological di- indicate that the desert pupfish complex com- vergence between C. macularius and C. eremus prises two separate entities that have been di- despite levels of molecular divergence similar verging for perhaps 100,000 years. They are di- to, or higher than, that in the other two pupfish agnosable on the basis of two independent char- examples just described probably reflects mode acters, mtDNA and male breeding colors, and of speciation. Vicariant speciation involving we believe they should be treated as separate large populations is not necessarily associated species, the Quitobaquito pupfish, C. eremus with divergence in morphology or other out- Miller and Fuiman, in Rio Sonoyta and the Qui- ward expressions of adaptational differences tobaquito Springs area, and the desert pupfish (Smith and Todd, 1984; Dimmick et al., 1999). C. macularius in the remainder of the range of Rapid morphological change in C. diabolis, an the desert pupfish complex. The two groups example of speciation by peripheral isolation, clearly qualify as species under both the evolu- probably reflects the unique, cavernous habitat tionary species concept (Wiley, 1978; Frost and in Devils Hole and the consistently small size of Hillis, 1990) and the various forms (Mayden the resident population (Miller, 1950). Rapid and Wood, 1995) of the phylogenetic species morphological evolution in the Lake Chichan- concept. Analyses of morphometrics, meristics, canab species flock, which represents intrala- and protein electrophoretic variation show no custrine (possibly sympatric) speciation, has fixed or otherwise marked differences between been attributed to ecological divergence driven the two groups (Miller, 1943; Turner, 1983; Mill- by competition (Humphries, 1984). er and Fuiman, 1987). At the same time, those Recent gene flow probably explains the lack studies reveal no patterns of geographic varia- of significant genetic divergence among pupfish tion that are discordant with the grouping populations within the Salton Sea area and in based on mtDNA and male breeding colors. the Colorado River Delta. Dunham and Minck-

This content downloaded from 128.123.44.23 on Tue, 22 Jul 2014 12:05:11 PM All use subject to JSTOR Terms and Conditions ECHELLE ET AL.-DESERT PUPFISH COMPLEX 361 ley (1998) reported homogeneous allele fre- tions on the delta (Hendrickson and Varela, quencies for four polymorphic allozyme loci in 1989). their samples of C. macularius from four sites in The dynamics of populations in the Salton the Salton Sea area. Samples from two captive Sea/Colorado River Delta area include periods stocks, including the DNFH stock we examined, of "boom and bust" (Dunham and Minckley, gave indirect evidence of divergence among 1998:12), with population expansion and dis- populations on the delta (Dunham and Minck- persal alternating with population declines, iso- ley, 1998), but this might reflect genetic drift in lation, and local extinctions. This effect is par- captivity. The DNFH stock began with 280 ticularly pronounced for fishes in the arid re- founders captured in 1983 from our Santa Clara gions of western North America (Minckley et Slough site (Dunham and Minckley, 1998; their al., 1986). For C. macularius, decline, isolation, "Canal Sanchez Toboada"). In our survey, this and extinction of populations have been greatly stock had only the common haplotype of C. exaggerated by anthropogenic environmental In macularius, whereas all samples from the wild alterations. such instances, populations had two to five haplotypes. This suggests found- should be managed to simulate historic patterns of with er effect or subsequent genetic drift during the dispersal (Meffe and Vrijenhoek, 1988), when conditions are 15 years of captivity. Correspondingly, the pop- periodic intermixing and, transfers into areas of ulation also showed reduced allozyme variation favorable, extirpation. Dunham and also recommend- relative to other samples of the species (Dun- Minckley (1998) ed of local habitats to the ham and Minckley, 1998; 1 polymorphic locus manipulation provide unstable conditions that would favor vs 3 to 4). In contrast with our results for the harsh, over the less tolerant nonnative Salton Sea area and the allozyme homogeneity pupfish usually reported by Dunham and Minckley (1998), species. The of Turner (1983) found significant heterogeneity significant among-region component variation in C. macularius that the Sal- for four of eight polymorphic allozyme loci in suggests from the area. Dunham and ton Sea and the Colorado River Delta regions samples Minckley should be as units. (1998:12) that the managed separate Although suggested present homoge- the same mtDNA was common reflect "a recent dramatic increase in haplotype neity may both the distribution of abundance and flow." throughout regions, pupfish gene less common indicates a lack of A but rather haplotypes significant, small, among-region wholesale Under natural condi- of mtDNA in intermixing. component genetic diversity C. tions in the brief of surface-water macularius occurs between the Salton Sea area past, periods connection between Salton Sea and the delta and the Colorado River Delta. Similarly, Turner were separated by decades or longer with no (1983) and Dunham and (1998) re- Minckley connection 1937). A conservative man- between wild (Sykes, ported allozyme divergence pop- would avoid ulations in the Salton Sea area and agement approach intermixing captive pupfish between the two regions beyond what stocks derived from the delta, although this occurs when the reflect drift in the stocks. present human-regulated hy- might genetic captive drology fails under the forces of nature. Within The low level of detected sequence divergence these regions, neither the available genetic data for mtDNA reflects the na- probably dynamic nor the morphological analysis by Miller and ture of the extant regional hydrology. Although Fuiman (1987) provide a basis for partitioning are of populations separated by large expanses populations into separate conservation units. A there has been historic desert, ample opportu- similar conclusion might be reached for C. ere- nity for gene flow. For example, two of the more mus based on mtDNA variation alone, but the isolated the Salton Sea and Sala- areas, Laguna potentially long history of isolation between are in sinks in re- da, topographic that, even these two populations and the evidence of some corded history, have experienced repeated cy- degree of morphological divergence (Miller cles of flooding and desiccation as the river me- and Fuiman, 1987) indicate a need for conser- andered across the deltaic plain (Carpelan, vative management with no artificial intermix- 1961). The last flow of the river into the Salton ing of the populations in Quitobaquito Springs Sink occurred from 1905 to 1907 when the Col- and Rio Sonoyta. orado River breached an irrigation-canal system and created the present Salton Sea (Carpelan, ACKNOWLEDGMENTS 1961). Laguna Salada last received flow from the river during high flows in 1983 and 1984 Collections from Mexico were made under that apparently were associated with dispersal permit number 221196-213-03 from the Secre- and increased abundance of pupfish popula- tario de Medio Ambiente Recursos Naturales y

This content downloaded from 128.123.44.23 on Tue, 22 Jul 2014 12:05:11 PM All use subject to JSTOR Terms and Conditions 362 COPEIA, 2000, NO. 2

Pesca. We thank R. Miller for sharing his field- - , ANDA. F. ECHELLE.1993. Allozyme perspec- notes, S. Hoofer for generous help with se- tive on mitochondrial DNA variation and evolution B. and G. Knowles for of the Death Valley pupfishes (Cyprinodontidae: Cy- quencing, Bagley help 1993:275-287. with fieldwork in Mexico, T. for first prinodon). Copeia Dowling AND 1. 1998. the for A. F. - , Evolutionary relationships suggesting ND2 gene analysis, of in the eximius Echelle for in the field and for pupfishes Cyprinodon complex help reviewing (Atherinomorpha: Cyprinodontiformes). Ibid. the manuscript, T. Tibbets and P. Barrett for as- 1998:852-865. sistance with collections in Ari- permits and/or EXCOFFIER, L., P. E. SMOUSE, AND J. M. QUATTRO. zona and California, S. Keeney for extensive 1992. Analysis of molecular variance inferred from hospitality and assistance during fieldwork in metric distances among DNA haplotypes: applica- the Salton Sea area, P. Marsh, W. Minckley, and tion to human mitochondrial DNA restriction data. P. Unmack for valuable information, A. Loran- Genetics 131:479-491. FAN, E., D. B. LEVIN,B. W. GLICKMAN,AND D. M. Lo- ger for administrative assistance, and M. E. GAN. in the use of SSCP and M. R. for discus- 1993. Limitations analysis. Douglas Douglas helpful Mut. Res. 288:85-92. sions and for a providing way-station during FELSENSTEIN, J. 1985. Confidence limits on phyloge- was NAFTA Bor- fieldtrips. Funding provided by nies: an approach using the bootstrap. Evolution derlands Funds (contract 1448-00002-95-0840) 39:783-791. to Imperial National Wildlife Refuge and Ari- FROST,D., AND D. M. HILLIS. 1990. Species concepts zona Fishery Resources Office, Parker Unit, Re- and practice: herpetological applications. Herpe- gion II, U.S. Fish and Wildlife Service; the funds tologica 46:87-104. AND A. VARELA-R. 1989. Conser- were administered by the Oklahoma Coopera- HENDRICKSON, D. A., vation status of desert macula- tive Fish and Wildlife Research Unit, a cooper- pupfish, Cyprinodon ative of the U.S. rius, in Mexico and Arizona. Copeia 1989:478-483. program Geological Survey (Bi- M. 1984. Genetics of in Resources the Oklahoma De- HUMPHRIES,J. speciation pup- ological Division), fishes from Chichancanab,Mexico, 129- of Wildlife Oklahoma Laguna p. partment Conservation, 139. In: Evolution of fish species flocks. A. A. State University, and the Wildlife Management Echelle and I. Kornfield (eds.). Univ. of Maine Institute. Press, Orono. --, ANDR. R. MILLER.1981. A remarkable spe- LITERATURECITED cies flock of pupfishes, genus Cyprinodon,from Yu- catan, Mexico. Copeia 1981:52-64. CARPELAN,L. H. 1961. History of the Salton Sea, p. IVES,R. L. 1964. The Pinacate region, Sonora, Mex- 9-15. In: The ecology of the Salton Sea, California, ico. Occ. Pap. Calif. Acad. Sci. 47:1-43. in relation to the sportfishery.B. W. Walker (ed.). KOCHER,T. D., W. K. THOMAS,A. MEYER,S. V. 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vation genetics in the management of desert fishes. SYKES,G. 1937. The Colorado Delta. Carnegie Inst. Conserv. Biol. 2:157-169. Wash. Publ. 460:1-193. MEYER,A., T. D. KOCHER,P. BASASIBWAKI,AND A. C. THOMPSON, J. D., D. G. HIGGINS, AND T. J. GIBSON. WILSON. 1990. Monophyletic origin of Lake Vic- 1994. CLUSTAL W: improving the sensitivity of toria fishes suggested by mitochondrial DNA se- progressive sequence alignment through sequence quences. Nature 347:550-553. weighting, position-specific gap penalties and MILLER, R. R. 1943. The status of Cyprinodonmacular- weight matrix choice. Nucleic Acid Res. 22:4673- ius and Cyprinodonnevadensis, two desert fishes of 4680. western North America. Occ. Pap. Mus. Zool., Univ. TURNER,B. J. 1974. Genetic divergence of Death Val- Mich. 473:1-25. ley pupfish species: biochemical versus morpholog- 1. 1950. Speciation in fishes of the genera Cy- ical evidence. Evolution 37:690-700. 1. 1983. Genic variation and differentiation of prinodon and Empetrichthysinhabiting the Death Val- ley region. Evolution 4:155-162. remnant natural populations of the desert pupfish, 1 1979. Freshwater fishes. Red data book. Vol. C. macularius. Ibid. 37:690-700. 4. Pisces. Rev. Ed. IUCN, Morges, Switzerland. . 1984. Evolutionary genetics of artificial re- •- the 1. 1981. Coevolution of deserts and pupfishes fugium populations of an endangered species, desert 1984:364-369. (genus Cyprinodon)in the American Southwest, p. pupfish. Copeia 39-94. In: Fishes in North American deserts. R. J. UNITEDSTATES DEPARTMENT OF THE INTERIOR.1986. and threatened wildlife and de- Naiman and D. L. Soltz (eds.). John Wiley and Endangered plants; Sons, New York. termination of endangered status and critical habitat for the desert Fed. 51:10842- - I, AND L. A. FUIMAN. 1987. Description and pupfish. Reg. 10850. conservation status of Cyprinodonmacularius eremus, E. 0. 1978. The a new subspecies of pupfish from Organ Pipe Cac- WILEY, evolutionary species concept reconsidered. Zool. 27:17-26. tus National Monument, Arizona. Copeia 1987: Syst. 593-609. MINCKLEY,W. L. 1973. Fishes of Arizona. Arizona De- (AAE, RAVDB, TPM, MJH) DEPARTMENTOF ZO- partment Fish Game, Phoenix. OLOGY AND COOPERATIVE FISH AND WILDLIFE - , D. A. HENDRICKSON,AND C. E. BOND. 1986. RESEARCH UNIT, OKLAHOMA STATE UNIVERSI- of North American freshwater Geography western TY, STILLWATER, OKLAHOMA 74078; AND fishes: and to intraconti- description relationships (COM) ARIZONA FISHERYRESOURCES OFFICE, nental tectonism, 519-614. In: of p. Zoogeography U.S. FISH AND WILDLIFESERVICE, PARKER ARI- North American freshwater fishes. C. H. Hocutt ZONA 85344. E-mail: (AAE) echelle@okstate. and E. O. Wiley (eds.). John Wiley and Sons, New York. edu. Send reprint requests to AAE. Submit- ted: 1999. 7 1999. MOYLE,P. B. 1976. Inland fishes of California. Univ. 26 April Accepted: Sept. Section editor: D. McEachran. of Calif. Press, Berkeley. J. NEIGEL,J. E., AND J. C. AVISE. 1986. Phylogeneticre- of mitochondrial DNA under various lationships APPENDIX demographic models of speciation, p. 515-534. In: Evolutonary processes and theory. E. Nevo and S. SAMPLELOCATIONS FOR 11 NATURAL Karlin (eds.). Academic Press, New York. POPULATIONS OF THE DESERT PUPFISH COMPLEX ORITA, M., T. SEKIYA,AND K. HAYASHI.1989. Y. SUZUKI, and sensitive detection of mutations Rapid point Locality numbers correspond with those in and DNA polymorphisms using the Polymerase 1 and Table 1. Chain Reaction. Genomics 5:874-879. Figure RAYMOND, M., AND F. ROUSSET.1995. An exact test Salton Sea area.-(1) drain, 100 m for population differentiation. Evolution 49:1280- Irrigation 1283. from shore of Salton Sea near boundary be- 3 km RICE,W. R. 1989. Analyzing tables of statistical tests. tween Riverside and Imperial Counties, Ibid. 43:223-225. north of Desert Shores, Imperial County, Cali- SCHOENHERR,A. A. 1988. A review of the life history fornia; (2) San Felipe Creek at Highway 86 and status of the desert pupfish, Cyprinodonmacu- bridge, 18 km southeast of Salton City, Imperial larius. Bull. So. Calif. Acad. Sci. 87:104-134. County, California; (3) shoreline pool of Salton SMITH,G. R., AND T. N. TODD. 1984. Evolution of Sea near Trifolium 20A drain, about 15 km species flocks of fishes in north temperate lakes, p. northwest of Westmorland, Imperial County, 45-68. In: Evolution of fish species flocks. A. A. California. Echelle and I. Kornfield (eds.). Univ. of Maine Press, Orono. Colorado River A near Cerro STRECKER,U., C. G. MEYER,C. STURMBAUER,AND H. Delta.-(4) slough Prieto and 0.8 km north of a WILKINS.1996. Genetic divergence and speciation geothermal power a area at east in an extremely young species flock in Mexico plant, Baja California; (5) seep formed by the genus Cyprinodon(Cyprinodontidae, edge of Laguna Salada, 20.6 km south of High- Teleostei). Mol. Phyl. Evol. 6:143-149. way 2, Baja California; (6) Santa Clara Slough

This content downloaded from 128.123.44.23 on Tue, 22 Jul 2014 12:05:11 PM All use subject to JSTOR Terms and Conditions 364 COPEIA, 2000, NO. 2 at terminus of Wellton-Mohawk canal, Sonora, Rio Sonoyta/Quitobaquito area.-(10) Quitobaqui- Mexico; (7) a canal at Flor del Desierto, Sonora to Springs/Pond, Organ Pipe Cactus National on Highway 003 (Highway 40 on some maps; Monument, Pima County, Arizona; (11) Rio (8) a spring at El Doctor, Sonora; (9) a spring Sonoyta 11.5 km west, 1.5 km south Sonoyta, about 150 m northwest of locality 8. Sonora.

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