Thirty Years of Hybridization Between Toads Along the Agua Fria River in Arizona

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5 AKAHASHI T IZUKI M AND 1 , ULLIVAN O. S EITH K 4 , CHWANER D. S ERRY T 3 , OOTEN W College of Sciences, University of Findlay, Findlay, Ohio 45840-3695 USA I. Evidence from Morphology and mtDNA 4 ESSICA J Department of Biology, Bucknell University, Lewisburg, Pennsylvania 17837 USA 5 1,2 , Department of Biology, Centre College, 600 West Walnut Street, Danville, Kentucky 40422 USA 3 School of Mathematical and Natural Sciences, Arizona State University, Phoenix, Arizona 85069 USA 1 ULLIVAN K. S RIAN B Thirty Years of Hybridization between Toads along the Agua Fria River in Arizona:

Thirty Years of Hybridization between Toads along the Agua Fria River in Arizona: I. Evidence from Morphology and mtDNA

1,2 3 4 1 5 BRIAN K. SULLIVAN, JESSICA WOOTEN, TERRY D. SCHWANER, KEITH O. SULLIVAN, AND MIZUKI TAKAHASHI

1School of Mathematical and Natural Sciences, Arizona State University, Phoenix, Arizona 85069 USA 3Department of Biology, Centre College, 600 West Walnut Street, Danville, Kentucky 40422 USA 4College of Sciences, University of Findlay, Findlay, Ohio 45840-3695 USA 5Department of Biology, Bucknell University, Lewisburg, Pennsylvania 17837 USA

ABSTRACT.—The Arizona Toad (Bufo [ = Anaxyrus] microscaphus) occupied the entire Agua Fria River drainage in central Arizona until relatively recently. By the 1980s, a close relative, Woodhouse’s Toad (Bufo woodhousii), colonized the lower reaches of the Agua Fria and replaced B. microscaphus at some sites. We tested the hypothesis that habitat disturbance drives replacement of B. microscaphus by B. woodhousii, via hybridization, by examining shifts in the distribution of these toads following the expansion of the Waddell Dam on the lower Agua Fria River in the early 1990s. As of 2010, the high elevation headwaters of the Agua Fria River were still occupied by B. microscaphus, the lower reaches near the conﬂuence with the Gila River were occupied by B. woodhousii, and along the middle reaches, hybridization between these two anurans occurred at the same three sites as documented in the early 1990s. Contrary to expectations, evidence of hybridization along middle reaches of the river is largely unchanged: B. microscaphus has not been replaced by B. woodhousii at any additional sites nor is there any evidence of introgression of woodhousii mtDNA into putatively ‘‘pure’’ microscaphus populations upstream of hybrid sites.

Hybridization is a central issue in evolutionary biology, with Studies of hybridization have benefited immensely from relevance to the nature of species differences, the nature of recent advances in genetic techniques. For example, the level of speciation, and the nature of genetic variability and its role in introgression and directionality of hybridization in closely adaptation (Harrison, 1993; Howard, 1993; Roy et al., 2012). related species can be easily assessed using mtDNA (Avise Secondary contact leading to hybridization between close and Ellis, 1986; Chan and Levin 2005). For the toads, Bufo (= relatives was once viewed as a key step in the refinement of Anaxyrus) microscaphus and Bufo woodhousii, use of mtDNA species boundaries via the process of character displacement analysis allows for confident haplotype assignment (Gergus et (see review in Sullivan, 2009). Under this view, hybrid zones are al., 1999; Malmos et al., 2001). Morphological indicators of ephemeral, leading to introgressive fusion or reinforcement of hybridization between these toads in the southwestern United species-isolating barriers (e.g., Rhymer and Simberloff, 1996; States have been validated repeatedly using advertisement calls Krosby and Rohwer, 2010; Smith et al., 2013). However, (Sullivan, 1986), allozymes (Sullivan and Lamb, 1988), release investigators examining the proximate causes of hybridization calls (Sullivan, 1995), and mtDNA (Schwaner and Sullivan, have long recognized that hybridization can result from habitat 2009). disruption leading to reproductive interactions between other- In central Arizona, B. microscaphus and B. woodhousii wise allopatric taxa (e.g., Mebert, 2008; Vogel and Johnson, hybridize along intermediate reaches of the Agua Fria River 2008). north of Lake Pleasant (Fig. 1; Sullivan and Lamb, 1988; Malmos Some hybrid zones are spatially unstable, seemingly inde- et al., 2001). Based on specimens collected in this region during pendent of ecological gradients; some have argued (Barton and the 1940s and 1950s, and genetic analysis of populations in the Hewitt, 1989) that they may be determined by genetic 1980s, Sullivan and Lamb (1988) hypothesized that B. woodhousii interactions inherent to the taxa involved rather than the result recently (~1920s) gained access to the Agua Fria River, as a of some extrinsic, environmental circumstances (see reviews in result of construction of Waddell Dam (1927) and associated Trigo et al., 2008; Krosby and Rohwer, 2010; Smith et al., 2013). agricultural activity in northern Maricopa County (USDA, Hybrid zones should not necessarily be geographically stable 2007). In the 1980s, a hybrid zone between these toads was under this hypothesis because they are not determined by located 4 km upstream of the high lake level, and there was responses to specific ecological gradients: ‘‘they can move from genetic evidence of hybridization decades earlier along the place to place’’ (Barton and Hewitt, 1985, 1989). Consistent with lower reaches of the Agua Fria River (Sullivan and Lamb, 1988). this perspective, Roy et al. (2012) provided evidence that a During the 1990s, additional field observations (Malmos et al., hybrid zone between two anurans in Canada has shifted back 2001) revealed that B. woodhousii was syntopic with B. micro- and forth (‘‘oscillation’’) dramatically over the past 50 years, scaphus 10 km upstream of the high lake level, well above the independent of shifts in local biotic communities thought to 1984–86 contact zone described earlier (Sullivan, 1986). Analysis influence hybridization in these species historically. In another of mtDNA revealed that all F1 hybrids possessed mtDNA of B. example of hybrid zone shifts, Krosby and Rohwer (2010) woodhousii, presumably as a result of rare females of the argued that one species of warbler has replaced a second, expanding species mating with the more common males of independent of obvious environmental shifts, as a result of the resident species, B. microscaphus (Malmos et al., 2001). competitive advantages of one species over the other. Long-term Waddell Dam was expanded in the late 1980s, and after its studies of species interactions, including replacement of one by completion in 1994, the location of the 1984–86 contact zone was another, are critical to evaluating these hypotheses regarding the periodically flooded (BKS, pers. obs.) or exposed after the lake causes and consequences of hybridization. levels dropped dramatically. Under the hypothesis that hybridization between B. wood- 2Corresponding Author. E-mail: [email protected] housii and B. microscaphus is ephemeral, resulting from DOI: 10.1670/14-011 ecological disturbance and leading to the replacement of B. HYBRIDIZATION IN ARIZONA TOADS 151

FIG. 1. Collecting localities along the Agua Fria River from site #10 (uppermost) to site #2 (lowermost) and the single New River collecting locality (site #1). Map modified from USDA (2007); components of the watershed shown in blue, major roadways in grey. 152 B. K. SULLIVAN ET AL. microscaphus by B. woodhousii, we predicted that having gained BlackCanyonCity(#7). These two sites are approximately 30 access to the system, B. woodhousii would continue moving km north of Waddell Dam. Along the Agua Fria River 10–30 upstream along the Agua Fria River, especially subsequent to km north of Black Canyon City, we sampled sites (#8–10) 40– the expansion of Waddell Dam. Specifically, we expected to find 60 km north of Waddell Dam that represent pure populations the three areas of hybridization along the Agua Fria River (10 of B. microscaphus to determine whether introgression of B. km upstream of the new high lake level), near Black Canyon woodhousii mtDNA has occurred, similar to that documented City (identified in the 1990s) to have shifted to predominately B. by Schwaner and Sullivan (2009) for hybrid zones along woodhousii by 2010. Also, we expected to find some indication of tributaries of the Virgin River in Utah. introgression of B. woodhousii mtDNA further upstream of these Hybrid Indices.—We collected toads by hand along the Agua areas of hybridization and loss of evidence of B. microscaphus Fria River and its immediate tributaries (primarily New River, along the lower reaches of the Agua Fria River. Herein, we use Big Bug Creek) in the spring breeding period (February hybrid indices and mtDNA to examine the extent of hybridiza- through July) during 1992–97 and 2009–10. Ten toads from tion between these two toads at 10 sites along the Agua Fria each hybrid site each decade were scored for morphological River, updating surveys at sites from the 1980s (Sullivan, 1986) characters, euthanized by overdose of MS 222, and tissues with those previously unpublished from the 1990s and 2000s. A harvested for genetic analysis (see below), prior to fixation in future contribution will examine evidence of introgression 10% formalin, and transfer to 70% ethanol for permanent based on microsatellites. storage. Toads collected between 1984 and 2010 are now housed in the Arizona State University Vertebrate Collection MATERIALS AND METHODS (BKS); for the majority of toads (> 90%) observed in the 1990s and 2000s, toads were not euthanized. Tissue for protein and Field Methods.—The Agua Fria River watershed (Fig. 1) was DNA analysis came from two toe clips taken with sterile sampled over three disjunct time periods: 1984–87 (= ‘‘1980s’’); instruments, both placed in sterile Eppendorf tubes, filled 1992–97 (= ‘‘1990s’’); and 2009–10 (= ‘‘2000s’’). Results of the with 99% ethanol, and later stored at -758Cinanultracold earlier surveys were published in part in Sullivan (1986), Sullivan freezer. and Lamb (1988), and more recently, in Malmos et al. (2001). We recorded scores for morphological hybrid indices at the However, the bulk of the data presented herein are unpublished; time of capture, and individuals were subsequently released at previous data are referenced only to establish baselines for the capture site. Specimens collected during the present study subsequent comparisons. were scored for morphological characters indicative of hybrid- A total of 655 individual adult (> 50 mm snout–vent length) ization using a hybrid index (Sullivan 1986, 1995). In brief, we toads were sampled over the course of our surveys, including assessed each toad for four qualitative characters: degree of 121during the 1980s, 365 during the 1990s, and 176 during ventral spotting; presence of a dorsal stripe; presence of cranial 2009–10. Hybrid indices (HI; see below) were obtained for all crests; and presence of a pale bar between eyelids (for more 655 individuals, whereas 251 were subjected to genetic analysis details, see Stebbins, 2003, and Schwaner and Sullivan, 2009). (mtDNA designation). The majority (80%) of individuals We obtained a value for each of the four separate characters, subjected to genetic analysis were obtained from the primary respectively, of 0, 1, 2, or 3: 0 = typical B. microscaphus,3= areas of hybridization and only small numbers (20%) from the typical B. woodhousii, and 1–2 are degrees of intermediacy presumed ‘‘pure’’ populations (sites at the lower or higher between the two species (B. microscaphus-like = 1, B. woodhousii- reaches). Because of limitations in sampling for genetic analysis like = 2). Scores for these four characters were summed to and the absence of breeding activity attributable to extreme produce a single (cumulative) hybrid index score (HI = 0–12) flooding in some years, only selected sites were subjected to for each individual (scores of 0–3 = B. microscaphus, scores of 4– analysis across all time periods (6 of 10). 8 represent hybrids, and 9–12 represent B. woodhousii). Because We surveyed a total of 10 sites along the Agua Fria River prior studies have documented some ontogenetic variation in over a straight-line distance of 75 km, beginning near Phoenix > in the south (confluence of the Agua Fria and New Rivers), these characters, we used only adult individuals ( 50 mm below Lake Pleasant (formed by Waddell Dam in 1927; USDA, snout–vent length) for all morphological analyses (Sullivan, 2007), to the headwaters of the Agua Fria River near Prescott 1986). Valley. Three sites were along the lower reaches of the Agua mtDNA Analysis.—Whole genomic DNA was extracted from Fria River (Figs. 1, 2): from 1–2 km (site = #3) and 10–12 km toe clips with a DNeasy Kit (Qiagen, Valencia, CA). The 16S south (#2) of the new, expanded Waddell Dam; and at one site rRNA portion of the mitochondrial genome was amplified using 0 0 0 along the primary tributary of the Agua Fria River, New River, LGL286 (5 -AGATAGAAACCCACCTGGAT-3 ) and LGL381 (5 - 0 15 km east of Waddell Dam (#1). Based on prior work (Sullivan ACCCCGCCTGTTTACCAAAAACAT-3 ) primers (Bickham et and Lamb, 1988), these sites were expected to represent pure al., 1996) in a 25-uL reaction for 35 cycles at 948C for 1 min, 558C populations of B. woodhousii, except for rare evidence of for 1 min, and 728C for 2 min. Restriction digests using Tsp509 ‘‘hybridization long past’’ in the form of mtDNA of B. (New England Biolabs, Beverly, MA) with a recognition site of 0 0 microscaphus. Upstream from these sites along the Agua Fria 5 ...AATT...3 were then conducted on the 16S rRNA PCR River, we sampled above (north) Lake Pleasant, 8 km north of products using the manufacturer’s specifications. Digestion the dam (#4), 24 km north of Waddell Dam and immediately fragments were separated on 4% agarose gels stained with south of Black Canyon City, the area of known hybridization in ethidium bromide, and results observed and photographed the 1990s (#5). In the vicinity of Black Canyon City, we under ultra-violet light. All fragments were compared to a 100- surveyed two additional sites, established as areas of hybrid- bp DNA ladder from New England Biolabs (Beverly, MA). The ization in prior studies (1990s; Malmos et al., 2001), along resulting fragments allow for the differentiation between B. BlackCanyonCreek,justnorthofitsconfluencewiththeAgua microscaphus and B. woodhousii (Lamb et al., 2000; Malmos et al., Fria River (#6) and along the Agua Fria River, just northeast of 2001). HYBRIDIZATION IN ARIZONA TOADS 153

FIG. 2. Sampling sites along the Agua Fria River, near Black Canyon City; site #7 (upper photo) and site #5 (lower photo) during the spring breeding season, 2010 (note the evidence of extensive ﬂooding from previous months). These represent the primary areas of hybridization during the 1990s and 2000s.

Statistical Analysis.—Statistical methodology follows Hollander periods (1990s, 2000s); a Kruskal-Wallis test was used to assess and Wolfe (1973) and Zar (1999); SPSS (version 16.0, SPSS for shifts in HI across sampling periods for sites grouped by Windows, Rel. 16.0.2., 15 November 2007, Chicago, SPSS Inc.) was taxonomic status. Mean HIs are provided for heuristic purposes used for all calculations. Because our data were nonparametric by (when comparing results across survey periods). deﬁnition (qualitative hybrid indices and frequencies), we used a

Wilcoxon signed-ranks, matched pairs test to evaluate HI scores RESULTS by site over the three survey periods (1980s, 1990s, 2000s). We used a Mann-Whitney U-test to examine the species-speciﬁc Morphology.—The sites representing pure B. woodhousii (#1, #2, proportions of mtDNA of each species by site for the latter survey #3), hybrids (#4, #5, #6, #7) and B. microscaphus (#8, #9, #10) 154 B. K. SULLIVAN ET AL.

TABLE 1. Sampling information and mtDNA results by selected sites, comparing proportion of mtDNA of Bufo woodhousii (BW) to Bufo microscaphus (BM) from two sites on the lower reaches (#1, #2), the three hybrid sites on the middle reaches surrounding Black Canyon City (#5– 7), and upper reaches of the Agua Fria River (#9). 1990s samples represent toe clips collected in 1993–95; 2000s samples were collected in 2009–10. * data from Sullivan and Lamb (1988).

Site Sample N BWmtDNA BMmtDNA % BWmtDNA 1* 1980s 11 9 2 82 1 1990s 14 13 1 93 2000s 18 18 0 100 2* 1980s 12 9 3 75 2 1990s 9 9 0 100 2000s 10 10 0 100 5 1990s 36 4 32 11 FIG. 4. Hybrid index scores for individuals from Black Canyon 2000s 26 9 17 35 Creek, just north of its confluence with the Agua Fria River, Black 6 1990s 8 4 4 50 Canyon City (site #6), in the 1990s (solid bars; 1990–94; N = 34) and in 2000s 24 11 13 46 the 2000s (open bars; 2009–10; N = 33); scores from 0–3 are associated 7 1990s 33 4 29 12 with Bufo microscaphus and 9–12 with Bufo woodhousii. Contrary to 2000s 26 2 24 8 predictions, there was not a shift toward scores indicative of B. 9 1990s 5 0 5 0 woodhousii over the past 20 yr (Kruskal Wallis, H = 3.37, P = 0.067). 2000s 22 0 22 0 cantly, than in the 2000s (X = 4.45, N = 33), counter to the were grouped for ease of comparison of HI across time periods expectation of an increase (higher) HI indicative of replace- (Fig. 1, Table 1). The putatively pure populations of B. woodhousii ment by B. woodhousii. did not vary across sampling periods with respect to HI (for #1, Overall, the HIs were indistinguishable across all 10 sites H = 0.56, P = 0.76, N = 96; for #2, H = 0.58, P = 0.45, N = 41). between the 1990s and the 2000s sampling periods, whether The putatively pure populations of B. microscaphus similarly did using individual HI scores for all sites (Kruskal-Wallis ANOVA, not vary across sampling periods (for #9, H = 0.79, P = 0.37, N = H = 2.1, P > 0.25) or signed ranks for median HI scores for each 55). Hence, morphologically, there was no change in the site summed (i.e., a pairwise, site by site evaluation of medians; putatively pure populations along the lower or upper reaches Wilcoxon: Z = 0.21, P = 0.60). For selected well-studied sites, of the Agua Fria River across these 20–30 years of surveys. there was no trend across sampling periods (Table 2). Thus, As expected, there was more variation in HIs among the there was no detectable shift in HI scores toward B. woodhousii, hybrid sites. For greater resolution of potential shifts in HI, the falsifying the species replacement hypothesis presented by three sites near Black Canyon City were analyzed separately Sullivan and Lamb (1988). for patterns of morphological change. For site #7 (Fig. 3), the mtDNA Variation.—The proportion of mtDNA of B. wood- most thoroughly surveyed site with respect to morphology housii at each of the primary hybridization sites near Black (Malmos et al., 2001), there was no significant variation across Canyon City (sites #5–7) was not significantly higher (Wilcox- all time periods (H = 0.05, P = 0.83, N = 269); mean HI was ~2, on: Z = 0.71, P = 0.46) in the latter sampling period (2009–10) typical of B. microscaphus, during both the 1990s and the 2000s than from the early 1990s (Table 2). However, at the lower survey periods. Similarly, site #5 (Fig. 4) did not differ in HI reaches, evidence of hybridization diminished over the past 30 scores across the two latter surveys (H = 3.37, P = 0.067, N = years, with no mtDNA evidence of B. microscaphus at site #1 67). Mean HI in the 1990s (X = 6.32, N = 34) was actually and site #2 in the more recent surveys. Thus, the hybrid sites at higher (more ‘‘woodhousii-like’’ earlier), although nonsignifi- themiddlereachesoftheAguaFriaRiverarelargely

TABLE 2. Mean hybrid index scores by selected sites, comparing scores from two sites on the lower reaches (#1, #2), the three hybrid sites on the middle reaches surrounding Black Canyon City (#5–7), and upper reaches of the Agua Fria River (#10). Scores from 0–3 are associated with Bufo microscaphus and 9–12 with Bufo woodhousii.

Site Sample N Hybrid index (X) 1 1980s 10 11.0 1990s 30 10.9 2000s 20 11.5 2 1980s 10 11.2 1990s 10 11.1 2000s 12 10.9 5 1990s 10 3.8 2000s 30 2.9 6 1990s 33 6.2 FIG. 3. Hybrid index scores for individuals from the Agua Fria River, 2000s 34 4.2 Black Canyon City (site #7), in the 1990s (solid bars; 1990–92; N = 60) 7 1990s 50 1.6 and in the 2000s (open bars; 2009–10; N = 57); scores from 0–3 are 2000s 60 1.7 associated with Bufo microscaphus and 9–12 with Bufo woodhousii. 10 1990s 18 1.5 Contrary to predictions, there was not a shift toward scores indicative 2000s 15 2.2 of B. woodhousii over the past 20 yr (Kruskal Wallis, H = 0.05, P = 0.83). HYBRIDIZATION IN ARIZONA TOADS 155 unchanged morphologically over the past two decades, hybrid zone between two bufonids in western Canada (Bufo whereas sites in the lower reaches suggest that evidence of americanus and Bufo hemiophrys); they found that shifts occur past hybridization continues to diminish (reduction in mtDNA independent of any disturbance or natural ecotones in the local of B. microscaphus) and is virtually absent by 2009–10. biotic communities. Consistent placement of hybrid zones with particular environmental features strengthens hypotheses im- plicating an important role for ecological factors in determining DISCUSSION hybridization (Walters et al., 2008; Engler et al., 2013). Long- In contrast to the hypothesis of Sullivan and Lamb (1988), we term studies are especially valuable in providing information on found no morphological evidence of species replacement: B. temporal and spatial stability of hybrids zones in relation to woodhousii has not replaced B. microscaphus at sites along the human induced habitat alterations (see reviews in Allendorf et Agua Fria River north of Lake Pleasant. Similarly, the al., 2001; Krosby and Rohwer, 2010; Roy et al., 2012). proportion of toads with mtDNA indicative of B. woodhousii, Our results suggest that the hybrid zone between B. micro- possessed by all hybrids observed at hybrid sites analyzed in scaphus and B. woodhousii is stable, with little shift in proportions the 1990s, has diminished, albeit nonsignificantly, at all three of parentals and hybrids at sites over the past three decades. hybrid sites near Black Canyon City (Table 1), contrary to the Nonetheless, it does appear that B. woodhousii has entered some predictions of Malmos et al. (2001). Additionally, in contrast to systems only in the vicinity of anthropogenic disturbance, along the findings of Schwaner and Sullivan (2009), who documented the lower reaches of the Agua Fria River near Lake Pleasant or introgression of B. woodhousii mtDNA into otherwise pure B. in the vicinity of Black Canyon City with widespread microscaphus populations upstream from hybrid zones along the anthropogenic impacts (e.g., ponds for livestock, damming of Virgin River, we found no indication of introgression in higher side channels for flooding agricultural fields, etc.; reviewed in elevation populations of B. microscaphus along the upper Agua USDA, 2007). However, after gaining entry to a riparian Fria River. corridor, B. woodhousii does not appear to move upstream The expansion of Waddell Dam on the Agua Fria River in unless further disturbance occurs upstream to yield lotic 1994 resulted in repeated flooding of the hybrid site reported on habitats. by Sullivan (1986) and Sullivan and Lamb (1988). During the 1990s, we rarely observed toads breeding at the historic site, Acknowledgments.—We thank D. Anderson, M. Demlong, E. either because it was under water, attributable to high lake Gergus, J. Hall, T. Harris, C. Heidemann, M. Kwiatkowski, K. levels, or relatively barren when lake levels dropped rapidly. Malmos, L. Schwaner, and E. Sullivan for assistance with some That site was not used for breeding by toads during the 2009–10 field observations; R. Bowker was especially helpful over all sampling period. The absence of lotic habitat attributable to the three decades of study. All work was conducted under permits impoundment expansion explains the absence of B. microscaphus and protocols provided by the Arizona Game and Fish at the historic hybrid site; perhaps the vicinity of large, Department scientific collecting permits issued to BKS and introduced fish predators (dozens of introduced fish occur in IACUC-approved protocol of Arizona State University also Lake Pleasant) accounts for the absence of B. woodhousii. issued to BKS. However, the hybrid zones established upstream at Black Canyon City, where B. woodhousii was first observed in 1992, have remained consistent over the past 20 years. Further, there LITERATURE CITED is no evidence of introgression into pure populations of B. ALLENDORF, F. W., R. F. LEARY,P.SPRUELL, AND J. K. WENBURG. 2001. The microscaphus upstream from Black Canyon City. Although we problems with hybrids: setting conservation guidelines. 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