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Conservation genetics of an undescribed species of Dionda (Teleostei: cyprinidae) in the Rio Grande drainage in Western Texas.

Cyprinids of the genus Dionda inhabit springs and spring-fed streams in New Mexico and Texas in the United States and in Mexico (Mayden et al., 1992; Schnohuth et al., 2012). Eight described species currently are recognized (Schnohuth et al., 2008, 2012): D. argentosa, D. diaboli, D. episcopa, D. flavipinnis, D. melanops, D. nigrotaeniata, D. serena, and D. texensis. Seven of these (all but D. melanops) inhabit central and western Texas, including spring-fed headwaters of the San Antonio, Colorado, Guadalupe, and Nueces river drainages, and portions of the lower Rio Grande drainage and its tributaries, including the Pecos and Devils rivers (Hubbs et al., 1991; Edwards et al., 2004; Schnohuth et al., 2008, 2012; Carson et al. 2010); D. melanops occurs in Mexico in various parts of the lower Rio Grande drainage (Schnohuth et al., 2008, 2012). Four additional, undescribed species of Dionda have been reported (Schnohuth et al., 2012): two from Mexico (Dionda species 1 from the Conchos and Nazas river drainages, and Dionda species 2 from Ojo de Agua de San Juan in the Mezquital River drainage and from El Vergel Spring, also in the Mezquital drainage), and two from the USA (Dionda species 3 from the San Saba and Concho rivers in the northern Colorado River drainage, and Dionda species 4 from the upper Pecos River drainage in New Mexico). These undescribed species apparently are being described by R. L. Mayden and colleagues (Schnohuth et al., 2008, 2012). Interested readers should be aware that the numbers used in Schnohuth et al. (2008) to annotate undescribed species differ somewhat from those used in Schnohuth et al. (2012).

Given their specific habitat requirements, species of Dionda are vulnerable to alteration of habitat (Garrett et al., 1992; Edwards et al. 2004). Of the seven species in Texas, only one (D. diaboli) is considered threatened (United States Fish and Wildlife Service, 1999); however, the remaining six species likely face challenges (Brune, 2002; Lopez-Fernandez and Winemiller, 2005; Texas Wildlife Action Plan, publications/), particularly during extended drought. Understanding the geographic distribution of Dionda in the United States and Mexico has been a central effort in the laboratory of R. L. Mayden (Schnohuth et al., 2008, 2012) and is critical to managing and conserving the biodiversity they represent. Recent fieldwork has led to discovery of new populations of Dionda in Texas, extending the known range of D. diaboli (Garrett et al., 2004) and D. argentosa (Carson et al., 2010).

Recent surveys in portions of Alamito Creek, a tributary of the Rio Grande in Big Bend Ranch State Park, Presidio County, Texas, uncovered a population of Dionda inhabiting small permanent pools. Dionda referred to as D. episcopa has been recorded in other parts of Alamito Creek ( Populations of Dionda in Big Bend State Park have been monitored sporadically by the Texas Parks and Wildlife Department for several years, but no biological assessment has been conducted. An analysis of sequences of the mitochondrial ND5 gene from a few individuals indicated that the fish in Alamito Creek were distinct from all other nominal Dionda in Texas. To investigate identity of the population in Alamito Creek, we acquired sequences of the mitochondrial cytochrome b (cytb) gene; genotypes at 34 nuclear-encoded microsatellites were used to evaluate the conservation-genetic status of the population. Morphometric and meristic characters were documented for future comparisons with other species of Dionda.

Materials and Methods--A total of 94 specimens was collected from Alamito Creek in Big Bend Ranch State Park, Presidio County, Texas. Sampling localities were between 29[degrees]42'36"N, 104[degrees]7'42"W and 29[degrees]42'23"N, 104[degrees]7'55"W. Specimens were preserved in 95% ethanol (for analyses of DNA) or fixed in 10% formalin, with subsequent transfer to 70% ethanol, for morphometric and meristic evaluation.

DNA was extracted from muscle tissue, using a Chelex-resin-extraction protocol (Estoup et al., 1996). Initially, polymerase-chain-reaction (PCR) primers L12328 (5'-AACTCTTGGTGCAAMTCCAAG-3'; Miya et al., 2006) and DS-H (5'-AAAAATTTGTTGATTTCTCGGA-3'; Carson et al. 2010) were used to sequence a 585 base-pair (bp) fragment of the ND5 gene from three individuals; these were compared to ND5 sequences in Carson et al. (2010) of six species of Dionda (D. argentosa, D. diaboli, D. flavipinnis, D. serena, D. texensis, and Dionda species 4) known from Texas and New Mexico. The change, relative to nomenclature used in Carson et al. (2010), from D. episcopa sampled in the upper Pecos River to D. species 4, D. nigrotaeniata to D. flavipinnis (Dionda in the Guadalupe River drainage), and D. serena (Dionda in the upper Nueces River) to D. texensis reflects nomenclatorial changes suggested by Schonhuth et al. (2012). Laboratory methods were as in Carson et al. (2010). Phylogenetic analyses (not shown) revealed that fish from Alamito Creek possibly represented a new species of Dionda. Fifteen additional specimens were then sequenced for the 585 bp ND5 fragment and all 18 specimens were sequenced for the complete mitochondrial cytb gene (1,141 bp). PCR primers Glu-F and Thr-R (5'-GAAGAACCACCGTTGTTATTCAA-3' and 5'ACCTCCRATCTYCGGATTACA-3', respectively; Zardoya and Doadrio, 1998) were used to amplify cytb sequences, using amplification protocols described in Carson et al. (2010). PCR products from each individual were band-cut from 2% agarose gels and purified using the QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, California). Sequencing was carried out by the Interdisciplinary Center for Biotechnology Research at the University of Florida (, using the forward primer, Glu-F. Sequences were analyzed using Sequencher version 3.0 (Gene Codes, http://www.genecodes. com/) and truncated to 969 orthologous bp for comparison across species. Haplotypes were identified using Mega version 4.0.2 (Kumar et al., 1994).

Cytochrome b sequences of Dionda from Alamito Creek were compared to available sequences of described and undescribed species of Dionda from Texas, New Mexico, and Mexico (Mayden et al., 2007; Schonhuth et al., 2008, 2012). Sequences (GenBank accession numbers) examined were: D. argentosa (EU082498.1, EU082499.1), D. diaboli (DQ324085.1, DQ324086.1, EU082493.1, EU082494.1), D. episcopa (DQ324077.1, EU082490.1), D. flavipinnis (EU082501.1, EU082502.1), D. melanops (EU082495.1, EU082496.1, EU082497.1), D. serena (DQ324080.1), D. texensis (EU082504.1, EU082505.1), Dionda species 1 (DQ324084.1, EU082492.1), Dionda species 2 (DQ324081.1, DQ324082.1, DQ324083.1, EU082491.1), Dionda species 3 (EU082503.1), and Dionda species 4 (DQ324078.1). Sequences for Dionda species 4, D. flavipinnis, and D. texensis are in GenBank under the names D. episcopa, D. nigrotaeniata, and D. serena, respectively; the change to Dionda species 4 (Dionda in the upper Pecos River), D. flavipinnis (Dionda in the Guadalupe River drainage), and D. texensis (Dionda in the upper Nueces River) reflects nomenclatorial changes suggested by Schonhuth et al. (2012). Sequences (GenBank accession numbers) from five species of Campostoma (DQ324062.1, DQ324063.1, DQ324064.1, DQ324065.1, EU082476.1, EU082477.1) and Nocomis leptocephalus (EU082468.1) were included as outgroup taxa. Sequences were aligned by eye, using Textwrangler version 2.3 ( and McClade version 4.05 (Maddison and Maddison, 1997).

Maximum-parsimony analysis of the cytb dataset employed heuristic searches in PAUP* version 4.0b10 (Swofford, 2002), using TBR branch swapping with the MULTREES option and 1,000 random-addition sequence replicates. Bootstrapping with 1,000 pseudoreplicates (random-addition sequence and TBR branch swapping) was used to evaluate nodal support. Maximum-likelihood analysis and non-parametric bootstrapping (1,000 pseudoreplicates) were conducted using Garli version 0.951 (Zwickl, 2006), using a GTR model of nucleotide substitution with all parameters set to default.

Genotypes at 38 nuclear-encoded microsatellites were acquired from 40 individuals. PCR primers and reaction conditions for each microsatellite are given in Renshaw et al. (2009). Four microsatellites (Dep2, Dep44, Dep57, and Dep102) were discarded due to inconsistency of scoring. The remaining 34 microsatellites were genotyped using fluorescently labeled DNA following Renshaw et al. (2009) and an ABI PRISM 377 DNA Sequencer (Applied Biosystems, Carlsbad, California). Alleles were sized using the 400 HD Rox size-standard (Applied Biosystems, Carlsbad, California). Chromatograms were analyzed in Genescan (version 3.1.2, Applied Biosystems, Carlsbad, California); alleles were scored using Genotyper (version 2.5, Applied Biosystems, Carlsbad, California).

Exact probability tests as implemented in Genepop version 4.0.10 (Raymond and Rousset, 1995) were used to test genotypes for conformance to Hardy-Weinberg expectations and for genotypic disequilibrium. Sequential Bonferroni correction (Rice, 1989) was applied for multiple tests of the same hypothesis. Each microsatellite was evaluated for amplification errors, null alleles, or both, using Microchecker (van Oosterhout et al., 2004). F-stat version (Goudet, 1995) was used to obtain number of alleles, gene diversity (expected heterozygosity), and the inbreeding coefficient [F.sub.IS] (f of Weir and Cockerham, 1984); SPSS software (International Business Machines, Armonk, New York) was used to compute 95% confidence intervals (CI) around means for number of alleles and gene diversity.

The Bayesian coalescent approach in Msvar version 4.1b (Beaumont, 1999; Storz and Beaumont, 2002) was used to estimate parameters [N.sub.0] and [N.sub.1] (effective number of chromosomes at sampling and at the beginning of an expansion-decline phase, respectively), [t.sub.a] (number of generations since effective change in size began), and [mu] (average rate of mutation across all microsatellites). Run parameters are available from AHH. Effective number of breeders ([N.sub.b]) was estimated using the linkage-disequilibrium method in LdNe (Waples and Do, 2008). Alleles were excluded using the 2% threshold recommended by Waples and Do (2010); 95% CIs were estimated using the jackknife method. Finally, average, long-term effective size of population ([N.sub.eLT]) was estimated using the maximum-likelihood approach in Migrate version 3.0.3 (Beerli and Felsenstein, 1999, 2001). A short run was used to provide an initial estimate of theta ([THETA]) for longer runs, which used 10 short chains (10,000 sampled gene trees) and four long chains (5,000,000 sampled gene trees). Average, long-term effective size was then estimated as [THETA] = 4[N.sub.e][mu], where [mu] was generated using Msvar version 4.1b (Beaumont, 1999; Storz and Beaumont, 2002).

A total of 15 morphometric characters was taken from 12 specimens; six of these were cleared and double-stained after Taylor and van Dyke (1985) for counts of fin-rays and vertebrae. Measurements and counts followed Hubbs and Lagler (1958). Males and females were identified by presence or absence, respectively, of snout tubercles and rows of tubercles along the anterior-most pectoral fin-rays. Voucher specimens were deposited at the Texas Cooperative Wildlife Collection (TCWC 14782.01-17). Compiled morphological data and a photograph of male and female specimens are at < doc/> under morphological data and Dionda from Alamito Creek.

RESULTS--No variation in cytb (969 bp) or ND5 (564 bp) sequences was observed among the 18 individuals sequenced. GenBank accession numbers are JQ412818 (cytb) and JQ412817 (ND5). Topologies resulting from maximum-parsimony and maximum-likelihood analyses of cytb sequences recovered a monophyletic group comprising Dionda from Alamito Creek and Dionda species 1 (sensu Schonhuth et al., 2008) from the Rio Conchos and Rio Nazas basins of Mexico, with 100 and 98% bootstrap support, respectively. The mtDNA haplotype from Alamito Creek differed from those of Dionda species 1 by 2-7 substitutions, and from all other nominal species of Dionda by 64-143 substitutions. Both topologies are at <> under phylogenetic topologies. Topologies may be of interest as GenBank accession numbers for all described and undescribed species of Dionda suggested by Schonhuth et al. (2012) are provided.

Of 34 microsatellites assayed, 25 were monomorphic. One microsatellite, Dep3, deviated significantly from Hardy-Weinberg expectations before, but not after, sequential Bonferroni correction. There was no evidence of amplification errors or null alleles at any microsatellite. Five of 45 tests of genotypic equilibrium were significant before Bonferroni correction; none was significant after correction. Average number of alleles and average gene diversity among the nine polymorphic microsatellites were 2.7 and 0.437, respectively (Table 1); considering all 34 microsatellites, averages were 1.4 and 0.116, respectively.

Bayesian coalescent analysis (Table 2) revealed a negative, posterior distribution of [log.sub.10] (r) value of -3.295, consistent with a three-orders-of-magnitude decline in effective size of the population in Alamito Creek; the modal estimate ofcurrent effective size ([N.sub.0]) of the population was 21.8. Given possible generation times of 1 and 3 years, estimated time since decline was 13-38,550 years ago (mode, 538-1,614 years ago). Estimated effective number of breeders in the population in Alamito Creek was 28 (CI = 7.9-infinity); estimate of average, long-term effective size ofpopulation was 660 (CI = 585-755).

Discussion--Cytochrome b sequences of Dionda in Alamito Creek are essentially the same as those from Dionda species 1 (sensu Schonhuth et al., 2008), a species known to date only from the Rio Conchos and Rio Nazas basins of Mexico and in the USA from Cibolo Creek, a small tributary of the Rio Grande. We have not had the opportunity to examine other specimens of Dionda species 1, but the cytb data support the hypothesis that the Dionda in Alamito Creek is conspecific with Dionda species 1. Additional surveys of spring habitats in the vicinity of Big Bend Ranch State Park and Big Bend National Park may lead to discovery of additional populations of Dionda species 1 in Texas or even undiscovered populations of other described, undescribed, or both, species of Dionda.

Genetic diversity in Dionda from Alamito Creek is extremely low. Only one mtDNA haplotype (1,533 bp) was recovered among 18 individuals assayed. In contrast, average ([+ or -]SE) number of ND5 haplotypes (585 bp) and diversity of haplotypes across 10 populations representing five of the described species in waters of the USA was 4.6 [+ or -] 1.3 and 0.446 [+ or -] 0.096, respectively (Carson et al., 2010). In addition, 25 of 34 microsatellites assayed were monomorphic in the population in Alamito Creek. Including the monomorphic microsatellites, average number of alleles and gene diversity were 1.4 and 0.116, respectively. In comparison, average number of alleles and gene diversity, based on 28-34 microsatellites, in 10 populations representing five described species of Dionda in waters in the USA were 6.2 [+ or -] 1.0 and 0.463 [+ or -] 0.159, respectively (A. H. Hanna et al., in litt.).

Bayesian estimates of Ne (current effective size of population) were 0.4-525.8, with highest probability of modal [N.sub.e] of 21.8. This modal value was similar to the effective number of breeders ([N.sub.b] = 28) obtained from the linkage-disequilibrium approach in LdNe. The latter ([N.sub.b]) provides information about the effective number of breeding adults that produced the sampled cohort(s) (Waples and Do, 2010); however, relating [N.sub.b] to [N.sub.e] is problematic for iteroparous species because of the potential for overlap between sets of parents producing offspring in successive years (Waples, 2010). Nonetheless, both [N.sub.e] and [N.sub.b] are estimates on a recent time scale (Beaumont, 2003) and their near identity for Dionda in Alamito Creek is striking. Finally, the Msvar-derived [N.sub.0]:[N.sub.1] ratio indicated a three-orders-of-magnitude decline in effective size of the population in Alamito Creek, occurring 13-38,550 years ago, with modal estimates of 538-1,614 years ago. A decline in effective size of population also was indicated by the estimate of average, long-term effective size of population ([N.sub.eLT] = 660), where [N.sub.eLT] represents a harmonic mean of [N.sub.e] over ca. 4[N.sub.e] generations (Hare et al., 2011).

The minimum effective size of population needed to ensure long-term genetic integrity remains a matter of debate. In theory, the equilibrium between loss of adaptive genetic variance, stemming from genetic drift, and its replacement, by mutation, necessitates an effective size of a few hundred to a few thousand individuals (Schultz and Lynch, 1997; Lynch and Lande, 1998). An effective size of population <50, as in Dionda in Alamito Creek, indicates high vulnerability to inbreeding depression (Rieman and Allendorf, 2001) and risk of extinction due to fixation of deleterious alleles and loss of adaptive genetic variance (Franklin, 1980; Anderson, 2005). The low estimates of [N.sub.e] (22) and [N.sub.b] (28) for Dionda in Alamito Creek, together with low levels of genetic diversity, clearly indicate the population is compromised genetically.

Factors affecting decline in effective size of the population of Dionda in Alamito Creek undoubtedly include deterioration of habitat and water quality. Alamito Creek is an intermittent stream in the Chihuahuan Desert that contains segments of healthy riparian habitat and perennial pools that historically have supported populations of endemic fishes, amphibians, and aquatic invertebrates. This region of Texas has the highest percentage of species of vertebrates of conservation concern, and Alamito Creek alone contains three other species of fish (Campostoma ornatum Mexican stoneroller, Notropis chihuahua Chihuahua shiner; Cyprinodon eximus Conchos pupfish) that are listed as threatened by the state of Texas. Persistent drought and withdrawal of groundwater have damaged many existing spring-associated communities in this region (Garrett and Edwards, 2001), and the current, exceptional drought in much of Texas raises an even greater risk of deterioration of habitat and water quality. In addition to >10 km of Alamito Creek that occurs on Big Bend Ranch State Park, an additional 5.5-km segment upstream of the park is protected by the Trans Pecos Water and Land Trust. This upstream segment has been recognized by the state of Texas as meeting criteria of an ecologically unique river and stream segment, and a coordinated plan for a holistic approach to watershed conservation is underway.

The paucity of genetic variation and low estimates of [N.sub.e] and [N.sub.b] for the population of Dionda in Alamito Creek clearly suggest a need for continued monitoring and perhaps assignment as an officially recognized population of conservation concern. Further, there is a need for additional surveys of tributaries and streams in the Rio Grande drainage to assess whether there are additional populations of Dionda species 1 in the basin. It also would be important for Dionda species 1 to be described as a means to assist in its conservation and protection.

We thank M. Lockwood, M. Bean, S. Shelton, D. Wilson, P. Bean, and R. Edwards for assistance in sampling, and C. Santamaria for assistance with translation of the abstract into Spanish. Research was supported by State Wildlife Grant 199634 of the Texas Parks and Wildlife Department and projects H-6703 and TEX09452 of Texas AgriLife Research. This paper is the third in the series of conservation genetics of cyprinid fishes (genus Dionda) in southwestern North America, contribution 207 of the Center for Biosystematics and Biodiversity, and contribution 1,426 of the Texas Cooperative Wildlife Collection. Views presented do not necessarily reflect those of the Texas Parks and Wildlife Department.

Submitted 19 October 2011. Accepted 9 November 2012.

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Associate Editor was Robert J. Edwards.

A. H. Hanna, K. W. Conway, E. W. Carson, G. P. Garrett, and J. R. Gold *

Center for Biosystematics and Biodiversity, Texas A&M University, College Station, TX 77843-2258 (AHH, KWC, EWC, JRG) Inland Fisheries Division, Heart of the Hills Fisheries Science Center, Texas Parks and Wildlife Department, 5103 Junction Highway, Mountain Home, TX 78058 (GPG)

* Correspondent:
TABLE 1--Summary statistics for nine polymorphic
microsat-ellites in 40 individuals of Dionda sampled from Alamito
Creek, Presidio County, Texas: [F.sub.IS] is an inbreeding
coefficient; P is the probability that [F.sub.IS] = 0.

                   Number       Gene
Microsatellite   of alleles   diversity   [F.sub.IS]       P

Dep3                 5          0.592       -0.141     0.007 (a)
Dep7                 2          0.444       -0.127       0.489
Dep9                 2          0.491       0.136        0.513
Dep20                3          0.535       0.114        0.764
Dep38                2          0.096       -0.040       1.000
Dep40                2          0.468       -0.232       0.181
Dep91                3          0.344       -0.165       0.088
Dep93                3          0.488       0.026        0.487
Dep100               2          0.475       -0.161       0.495

(a) Non-significant following Bonferroni correction.

TABLE 2-Summary statistics for posterior distributions of
parameters [mu], [N.sub.0], [N.sub.1], and [t.sub.a].
The parameter r is the ratio [N.sub.0]/[N.sub.1].
Estimates of [t.sub.a] are given for generation times
of 1 and 3 years.

Parameter      Mode               0.025 quartile     0.975 quartile

[mu]           2.2 x [10.sup.4]   2.6 x [10.sup.5]   1.9 x [10.sup.3]

[N.sub.0]      21.8               0.4                525.8

[N.sub.1]      43,052.7           3,270.4            617,731.9

[log.sub.10]   -3.295             -3.916             -3.062

[t.sub.a]      538-1,614          13-39              12,850-38,550
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Author:Hanna, A.H.; Conway, K.W.; Carson, E.W.; Garrett, G.P.; Gold, J.R.
Publication:Southwestern Naturalist
Article Type:Report
Geographic Code:1USA
Date:Mar 1, 2013
Previous Article:Population metrics and use of saltcedar (Tamarix) habitats by common side-blotched lizards (Uta stansburiana).
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