Reticulate evolution as a management challenge: Patterns of admixture with phylogenetic distance in endemic fishes of western North America

Abstract Admixture in natural populations is a long‐standing management challenge, with population genomic approaches offering means for adjudication. We now more clearly understand the permeability of species boundaries and the potential of admixture for promoting adaptive evolution. These issues particularly resonate in western North America, where tectonism and aridity have fragmented and reshuffled rivers over millennia, in turn promoting reticulation among endemic fishes, a situation compounded by anthropogenic habitat modifications and non‐native introductions. The melding of historic and contemporary admixture has both confused and stymied management. We underscore this situation with a case study that quantifies basin‐wide admixture among a group of native and introduced fishes by employing double‐digest restriction site‐associated DNA (ddRAD) sequencing. Our approach: (a) quantifies the admixed history of 343 suckers (10 species of Catostomidae) across the Colorado River Basin; (b) gauges admixture within the context of phylogenetic distance and “ecological specialization”; and (c) extrapolates potential drivers of introgression across hybrid crosses that involve endemic as well as invasive species. Our study extends across an entire freshwater basin and expands previous studies more limited in scope both geographically and taxonomically. Our results detected admixture involving all 10 species, with habitat alterations not only accelerating the breakdown of reproductive isolation, but also promoting introgression. Hybridization occurred across the genus despite phylogenetic distance, whereas introgression was only detected within subgenera, implicating phylogenetic distance and/or ecological specialization as drivers of reproductive isolation. Understanding the extent of admixture and reproductive isolation across multiple species serves to disentangle their reticulate evolutionary histories and provides a broadscale perspective for basin‐wide conservation and management.


| INTRODUC TI ON
Reticulated evolution is a product of several, often interacting phenomena, including horizontal gene transfer, polyploidization, and hybridization with introgression (Wendel & Doyle, 1998). All have been traditionally viewed as examples of "aberrant evolution," in that their occurrence was disruptive to the process of adaptation and speciation, with results translated as a network rather than a more traditional bifurcating tree. This supposition of aberrancy is best reflected in more legacy perspectives ([i.e., "… the grossest blunder in sexual preference which we can conceive of an animal making"; Fisher, 1930:130] and ["… the infection of one species with the genes from a second"; Du Rietz, 1930:376, 380, 386, 411]).
Rather than an evolutionary contradiction, hybridization, defined as the mixing of two species, offers instead an opportunity to grasp how evolution has been facilitated, in lieu of reproductive isolation (Good, Demboski, Nagorsen, & Sullivan, 2003). Hybridization, especially when coupled with introgression (i.e., the incorporation of alleles from one species into the gene pool of another), has long been thought to play a beneficial evolutionary role in both plants (Arnold, 1992) and animals (Dowling & Secor, 1997). It can promote evolution by (a) generating new genetic variation, (b) transferring adaptive traits, and (c) producing new lineages that exploit a novel niche within which neither parental taxa could succeed (Darras, Leniaud, & Aron, 2014;Edelman et al., 2019;Seehausen et al., 2014).
At the same time, it can have negative consequences, as with anthropogenic introductions, by either disrupting local adaptations or genetically swamping endemics, leading to the effective extinction of a species (Rhymer & Simberloff, 1996). These conflicting views have often complicated conservation and management (Allendorf, Leary, Spruell, & Wenburg, 2001), to include policies on how to adjudicate vonHoldt, Brzeski, Wilcove, & Rutledge, 2018).
Over the last 20+ years, genetic data have helped to inform biodiversity management, with both methodological and analytical approaches becoming more sophisticated. Genomics has been repeatedly advocated as a mechanism to better understand the complexities of conservation issues (Funk, McKay, Hohenlohe, & Allendorf, 2012), yet easy solutions are not apparent. For example, the appropriate application of genomic tools has become somewhat contentious (Benestan et al., 2016), with a common thread being the necessity for a practical, management-oriented approach .
In this regard, one issue of historic importance that would benefit from increased resolution is the occurrence and extent of admixture in natural populations (Allendorf et al., 2001). This practical problem fits easily into an evolutionary framework, particularly in relation to (a) quantifying the genetic erosion induced by invasive species (Lowe, Mulfeld, & Allendorf, 2015;Rhymer & Simberloff, 1996); (b) identifying cryptic species (Devitt, Wright, Cannatella, & Hillis, 2019); and (c) parsing admixture among endemics that stems from anthropogenic impacts (Abbott, Barton, & Good, 2016;Hamilton & Miller, 2016).
Over the past 20 years (Box 1), one focus of our team has been centered on desert fishes of the American Southwest, most recently by applying genomic methods to provide insights on species of conservation concern (Box 2). The arid southwest has been one of the most impacted environments, with demands for water driving both policy and socioeconomic agendas further exacerbated by climate-driven drought (Ficke, Myrick, & Hansen, 2007;Hinck, 2007). Here, we use the opportunity to illustrate the conservation challenges surrounding catostomids and how genomic tools can help clarify the manner by which hybridization and introgression have impacted three endemic species that face the combined threat of habitat alterations and introduced species.
Finescale Suckers (genus Catostomus) are known to hybridize, especially when invasive congeners have been introduced and/or habitats modified (Holden & Stalnaker, 1975. Introgression is also known to have occurred throughout the history of the genus as suggested by discordance between mitochondrial and morphological data (Smith, Stewart, & Carpenter, 2013;Unmack et al., 2014) and confirmed by genomic data (Bangs, Douglas, Mussmann, & Douglas, 2018), indicating a history of natural hybridization and introgression. Taken together, hybridization can occur between both endemics and introduced species and may have more far-reaching, albeit subtle effects such as potentially providing a bridge for introgression among native species that would not naturally hybridize , although the extent of this "hybrid bridge" has come under recent question (Mandeville, Parchman, McDonald, & Buerkle, 2015;Mandeville et al., 2017). These studies of hybridization, however, tend to focus on regional scales and with focus on two or three species at a time. Here, we expand on this work to examine an entire basin to include all species that are endemic or introduce in order to string together past work and elucidate larger patterns of hybridization and introgression.
The study area extends across the entire Upper Colorado River Basin adaptive management, ddRAD, hybridization, introduced species, introgression, reproductive isolation, species of concern C. P. clarkii)] and with two non-native species [White Sucker (WTS;

C. commersonii) and Longnose Sucker (LNS; C. catostomus)].
Our study is based upon 20+ years of sampling and represents the first range-wide molecular evaluation of hybridization and introgression involving all members of a clade that occur in the basin, both native and introduced. As such, it provides a blueprint for management how to disentangle contemporary events from those historic.
Our results identify the breadth of invasive-endemic hybridizations and clarify the manner by which it is facilitated via habitat fragmentation, a second anthropogenic impact. We also bookmark the historic legacy of admixture among native species. Finally, we employ our results to define reproductive isolation among our study species as a component of phylogenetic distance, or potentially, as a stepping stone to ecological speciation.

| Sample acquisition
This range-wide study was made possible by collaborative sampling efforts conducted over 25+ years. Here, we attempt to disentangle historic versus contemporary signals of reticulate evolution in suckers of the Colorado River Basin. We employed reduced genomic approaches to analyze DNA extracted from fin clips and tissue plugs gathered across the basin during 1995-2015 (Douglas, Brunner, & Douglas, 2003;Hopken, Douglas, & Douglas, 2013). Additional samples were obtained from the Museum of Southwestern Biology (University of New Mexico). A total of 409 samples were used and included 343 samples captured in areas of known hybridization and 66 samples from outside of these areas that are known to be pure based on previous phylogenomic and population genetic work (Bangs et al., 2018 and served as reference to verify species identification. To properly assess hybridization, it is important to have a good reference database for the parental species. This study was made possible by our previous phylogenomic work (Bangs et al., 2018) that provided us with a solid reference database for parental species outside of the known hybridization areas. In addition to field-identified hybrids (N = 115), we included field-identified pure parental species (N = 228). This allowed us to test for genetic structure among natural populations that could be indicative of cryptic variation in comparison with our phylogenomic reference database. It also made it possible to verify potential cryptic hybridization that might not have been captured by simple field identifications.

Box 1 The shaping of professional trajectories
Two of us (MRD and PCB) were in the inaugural cohort of graduate students Louis mentored as a young, but visionary assistant professor at Laval University, in Quebec. Sadly, Patrick is no longer with us to make his own voice heard. I reflect on the opportunities Louis provided to both of us and how they positively affected our careers. I offer these thoughts as impetus for current graduate students and postdoctoral fellows to pursue their aspirations, but also to remind established professionals of their opportunity to promote younger colleagues. Thinking about my graduate years working with Louis made me to realize in retrospect how influential he was in shaping my professional growth as a scientist. I detail these in five vignettes below:

Facilitate and Provide Opportunities
Patrick and I were two Swiss graduate students with ideas and aspirations, but without the skills or environment to achieve them.
Although we received funding from the Swiss government to conduct conservation genetics studies on salmonids in Switzerland, we could not have completed them without the generous opportunity offered by Louis.
My PhD research on Coregonus, and Patrick's on Salvelinus, occurred in the Central Alpine lakes at the dawn of the microsatellite DNA era. We were fortunate to meet Louis Bernatchez, then a newly minted faculty at Laval, who kindly opened his laboratory to us. This was a tremendous opportunity, in that no one in Switzerland at that time employed this methodology. Coregonus in Swiss lakes has become a prime model for fish "species flocks," and this would not have occurred if Louis had not shared his knowledge of molecular techniques with two Swiss students and instilled in them his enthusiasm for biodiversity conservation.

Make it Work.
Louis' 1st laboratory was a single room (~20 m 2 ) where 10 of us literally worked elbow-to-elbow doing DNA extractions, allozymes, RFLP electrophoresis, and sequencing. A single bench in another laboratory was dedicated to PCR setup. Note: This was the early '90s-PCR was just being adopted as a standard method in fisheries genetics and automated sequencers were not yet available.
Although it was a bit crowded at times, the group made it work with a shared camaraderie and purpose in generating solid science.
This is a tribute to what enthusiasm and an entrepreneurial spirit can achieve.

Do it Right
Even though the early laboratory was small, it worked because Louis established an efficient workflow, subsequently adopted in my own laboratories: Workspace was assigned to tasks that employed standardized protocols. Metadata were recorded on standardized forms, rather than individual notebooks, to ensure consistency across long-term projects.
Louis provided guidance when needed and was an invested, but hands-off advisor. He achieved productivity by making resources available and challenging us to give our best. We prospered in such an environment; it requires independence and self-motivation and is successful when mentees set high standards for themselves. I adopted this approach in my own mentoring, but also realized that such a "free spirited" environment works for some but not all.

Think Big-and Outside the Box
Louis always thinks "big" and did not let convention limit the goals he set for his team. Over his career, Louis not only had a huge impact on Conservation Genetics, but also helped shape the emerging field of Molecular Ecology. In this sense, we not only received a great start by being his students, but also prospered beyond our graduate years by tracking the slipstream of his ideas and innovations. Louis inspired us to think outside the box and not be confined by circumstance or dogma. We were pushed to "think outside the box" and pursue novel ideas, but also to generate solid data and always consider alternative hypotheses when interpreting results.

To Go Boldly
Embracing new opportunities is a key aspect of my professional trajectory. Each change demonstrated that taking reasonable professional chances will benefit in the long run. The hard part for me was to convince myself to take that next big step. In this sense, I moved among major institutions (two each for PhD and postdocs and three for faculty positions). I am currently an endowed professor at the University of Arkansas. For sure, each transition was challenging, but each provided amazing opportunities. This all began in the laboratory of Louis Bernatchez. His welcoming and entrepreneurial spirit guided me professionally. From feedback by my own former students now in established careers, I also realize that it is not the "big things" that guide them throughout their careers, but rather those small actions that resonate most with young students and inspire them to go boldly.
reference species, region, and hybrid type were randomly distributed across several libraries and lanes so as to reduce the potential bias in library preparation or lane effects. Sequencing was performed at the University of Wisconsin Biotechnology Center (Madison).

| Filtering and alignment
Illumina reads were filtered and aligned using pyR AD v.3.0.5 (Eaton & Ree, 2013) following the parameters determined in our previous ddRAD work in this system (Bangs et al., 2018). This included: removal of restriction site sequences and barcodes, and clustering at a threshold of 80% based the uncorrected sequence variation in catostomid fishes (Bangs et al., 2017;Chen & Mayden, 2012).
In addition, loci were removed if they displayed: (a) <5 reads per individual, (b) >10 heterozygous sites within a consensus, (c) >2 haplotypes for an individual, (d) >75% heterozygosity for a site among individuals, and (e) <50% of individuals at a given locus.
Filter 1 reduces the chance for false homozygosity, filters 2-4 remove paralogs, and filter 5 decreases the amount of missing data.
This filtering process has worked well in our previous work (Bangs et al., 2018 for phylogenetic, population genetic, and hybrid analyses.

| Clustering algorithms
All analyses employed unlinked SNPs generated by pyRAD, which samples one SNP at random from each RAD locus. Average pairwise genetic distances were calculated between all species using the complete sequence alignment for all 14,007 loci (Bangs et al., 2018). Distances were calculated using the default F84 model in DNADiSt, as implemented in phylip (Felsenstein, 1993).

| Hybrid detection
For hybrid analyses, we used unlinked SNPs with additional filtering to include only fixed differences between the two parental species and the removal of loci that contained <80% individuals.
Confirmation of pure parental species for determining fixed SNPs was based on q = 1.0 for a single cluster (species) in the Bayesian clustering analysis above. Only fixed differences between species were used to ensure accurate interspecific heterozygosity. Both hybrid analyses require the designation of parental populations, with only two parental species per test.

Box 2 Admixture in Southwestern Fishes
One of the major challenges in conservation today is how to deal with the complexities of hybridization and reticulate evolution (Allendorf, Hohenlohe, & Luikart, 2010).
Historically, hybridization has been viewed as a negative, and thus, management has focused on removal of hybrids.
However, recent genomic work has highlighted the importance of reticulate evolution in the emergence of biological diversity and adaptation of species leading to management perspectives that accept it as a key process (Hamilton & Miller, 2016 (Bangs et al., 2018), and (c) delineate species of conservation concern (Bangs, . These studies, and others, actively promote ongoing management by defining historic introgression and contemporary hybridization in native fishes, as well as developing a genetic database that can be used to accurately parse contemporary introgression among species. Suckers (family Catostomidae) readily hybridize, as do many cypriniform fishes, and particularly the genus Catostomus, a situation exacerbated anthropogenically by introducing invasive congeners and the extensively modifying riverine habitat (Bangs, Douglas, Thompson, & Douglas, 2017;Holden & Stalnaker, 1975).
This phenomenon has also been hypothesized to include more subtle effects, such as providing a "hybrid bridge" for introgression among species that would not do so naturally . Hybridization without the influence of introduced congeners has been observed in native sympatric fishes (Hubbs, Hubbs, & Johnson, 1943;Nelson, 1968) and seemingly occurs between genera within families (Buth, Haglund, & Minckley, 1992;Dowling et al., 2016;McAda & Wydoski, 1980;Tranah & May, 2006). However, the manner by which these genera should be taxonomically categorized is the subject of debate (Bangs et al., 2018).
We developed a hybrid index (Gompert & Buerkle, 2009) for each cross by implementing the R-package iNtRogReSS (Gompert & Buerkle, 2010). This involved a test of hybridization between the following species: (a) Flannelmouth × White (FMS × WTS), Flannelmouth × Sonora (FMS × SOS). The same package (above) was used to create a triangle plot depicting hybrid index by interspecific heterozygosity for each admixture test and (occasionally) by location as well.
We then utilized NewhybRiDS (Anderson & Thompson, 2002) to test the probability of assignment to a hybrid class, including first-filial (F1), second-filial (F2), and first-and second-generation backcross (Bx). Additional crossings, while of interest, would fail to assign individuals to any of the designed hybrid or parental categories.
Only first-and second-generation backcrosses were so designated, given the potential for ancestral crosses to be spuriously assigned to later-generation backcross categories (i.e., third and fourth). If this occurred, individuals would then be erroneously designated as more contemporaneous.

Box 3 The ecological theater of Western North America
The geomorphic history of western North America (synopsized from Minckley, Hendrickson, & Bond, 1986;Spencer, Smith, & Dowling, 2008) has catalyzed the evolution of its resident aquatic fauna. The tectonics of the region have alternately fractured and coalesced drainages, consequently reshuffling the distributions of aquatic species over time. For example, the Basin and Range physiographic province spanned much of Western North America during Miocene ( Figure 1a) and was replete with small-bodied species that were subsequently coalesced by vicariant tectonism into reproductively isolated refugia. Antecedent streams (i.e., those previously formed) on the adjacent Colorado Plateau (Spencer et al., 2008, Figure 1) deeply incised the Plateau as it uplifted, eroding headwater canyons and subsequently isolating not only aquatic but terrestrial fauna as well (Douglas et al., 2016). During this process, other streams drained internally to form several closed Plateau lakes that eventually emptied into the newly formed Colorado River as it transitioned across the Basin and Range, circa 5 mya. This allowed a rather depauperate assemblage of lacustrine-evolving, larger-bodied species (Uyeno & Miller, 1965) to disperse downstream into diverse habitats replete with new ecological niches.
Quaternary glaciation in western North America was limited to high elevation peaks, in contrast to its overwhelming presence in Eastern North America as the Laurentide Ice Sheet. Nevertheless, climate processes still resonated in the west, with broad impacts on resident biodiversity (Douglas, Douglas, Schuett, & Porras, 2006 In contrast to vicariant events, the Quaternary also provoked frequent admixture as glacial periods were supplanted by warmer interglacials (nine of which were recorded during the last 0.8 mya; Douglas, Douglas, Schuett, & Porras, 2009). Drainage reorganization (i.e., stream captures, diversions, beheadings) occurred frequently during these more pluvial periods, and not only extended fish distributions into adjacent but previously isolated basins, but also promoted admixture that subsequently confounded taxonomy (Dowling et al., 2016;Smith, Hall, Koehn, & Innes, 1983). Of late, this situation has been exacerbated in Western North America by ongoing admixture between endemic and introduced fishes, with management alternatives limited due to the weak resolution provided by legacy approaches. Again, interglacials were global in their occurrence and served to promote gene flow and secondary contact on a broadscale (Douglas & Brunner, 2002;Douglas, Brunner, & Bernachez, 1999;Ericson et al., 2019;Kohli, Fedorov, Waltari, & Cook, 2015;Licona-Vera, Ornelas, Wethington, & Bryan, 2018).

| Bayesian clustering
The most likely number of genetic clusters (gene pools) was k = 10, corresponding to the 10 species in our study ( Figure S1).
All 66 reference samples from outside the known hybridization area were assigned to a single cluster ( Figure S2). All 115 field identify hybrids had mixed assignments as did 19 fieldidentified pure specimens. These included ( One sample, collected in the Navajo River, was assigned to three species: Bluehead Sucker (q = 0.50), White Sucker (q = 0.37), and Flannelmouth Sucker (q = 0.13). Since this sample has assignment to more than two species, it could not be used for calculating a hybrid index or for assignment to a hybrid category in NewHybrids. All other samples were assigned to two clusters at most and thus were utilized for hybrid analyses. However, the high interspecific heterozygosity value for this sample indicates that it was a first-generation cross of a Bluehead Sucker and with a White Sucker × Flannelmouth Sucker hybrid. (Figure 2c).
Average pairwise genetic distances between species, as computed in phylip, are presented in Table 2. Introgression was detected F I G U R E 2 (a) Network depicting crosses among study species. Solid lines = those recorded in this study; dashed = previous studies (Clarkson & Minckley, 1988). Red lines = introgression; black lines = hybridization without introgression. Species abbreviations as in Appendix A and are colored by subgenus or species (shades of green and red = Catostomus; blue = Pantosteus; orange = Xyrauchen; purple = Longnose Sucker). (b) Species relationships, as depicted in Bangs et al. (2018). (c) Bayesian clustering plots by region for those populations with admixed ancestry (343 samples). These plots do not include the 66 samples used as reference, which can be found in Figure S2 only between species separated by genetic distances < 2%. On the other hand, hybridization without introgression was only recorded in species pairs exhibiting genetic distances between 2.2% and 2.9%.

| Hybridization with invasive species
The two introduced species differ in their distributions, with White

| Hybridization between endemic species
Hybrids between the two widespread species, Flannelmouth and Bluehead sucker, were found in two areas: The Yampa River, and throughout the Middle Green River region, to include the White River and the mainstem Green River between above its confluence with the White River (Figures 1b and 2c). All seven individuals reflected hybrid indices of ~0.50 with high interspecific heterozygosity ( Figure 4d) and were categorized as F1 by NewhybRiDS. These assignments were consistent with q-scores that approximated 0.50 (Figure 2c).
Hybrids involving the widespread, but rare Razorback Sucker were only found in the mainstem San Juan River near its confluence with the Colorado River. These included one F1-hybrid with Bluehead Sucker (Figure 4e) and one F1-hybrid with Flannelmouth Sucker (Figure 4r). The F1-classification was consistent across all three analyses. Introgressed hybrids between Razorback and Flannelmouth sucker were found in the southwestern area, the Virgin River and Grand Canyon . All are seemingly high-level backcrosses to Flannelmouth Sucker, given that NewhybRiDS failed to assign them to any hybrid category. In addition, contained q-scores and hybrid indexes > 0.75 for Flannelmouth Sucker (Figures 2c and 3r).
Other hybrids involved the two endemic species from the Lower Colorado River basin: A Bluehead × Desert sucker hybrid and a Flannelmouth × Sonora sucker hybrid were found in Grand Canyon, and one Bluehead × Desert sucker hybrid in the Virgin River. These assignments were consistent across both Bayesian clustering ( Figure 2c) and hybrid index (Figure 4p,q), but their low interspecific  Bayesian clustering (Figure 2c).

| D ISCUSS I ON
A more formal exploration of hybridization, and of reticulated evolution in general, has been promoted by contemporary advancements in sequencing technology, with more expansive datasets developed as a consequence (Eaton & Ree, 2013;Kane et al., 2009). Given this, a much less cumbersome view of introgressive hybridization has emerged, one that promotes instead the maintenance of semipermeable species boundaries, the consequences of which have impacted evolutionary thought (Nosil, Funk, & Ortiz-Barrientos, 2009;Harrison, 2012;Michel et al., 2010). For example, we now understand that introgression can occur without subsequent dismantling of species boundaries (Fontaine et al., 2015), and likewise, with a rather precise transmission of adaptive traits Nadeau et al., 2012). This has reshaped both our view of speciation, as well as the manner by which reproductive isolation can evolve in the face of contemporary and historic hybridization (Edmands, 2002). It also broadens our concept of how admixture can facilitate adaptation. For example, a gene region that controls color pattern expression in Heliconius butterflies has been identified as part of a chromosomal inversion that is transferred intact during admixture, allowing for color patterns to be switched (Edelman et al., 2019). How these insights affect conservation and management of wild species is still evolving, especially when the perceived negative impacts of invasive hybridization are superimposed onto a complex system with a long history of reticulate evolution among native species. Here, we build on our previous work to demonstrate how genomic tools can not only resolve this complexity, but also promote new perspective that can facilitate the adaptive management of species that are of conservation concern.
Catostomid fishes are a good system to gauge the manner by which reproductive isolation, or lack thereof, has evolved for several reasons. They display (a) an historic tendency to hybridize (Buth et al., 1992;Dowling et al., 2016;Hubbs et al., 1943;Nelson, 1968 all of these questions at once but instead build on the recent literature that has already quantified historic hybridization and reticulate evolution (Bangs et al., 2018), explored species delimitation , and explored the extent of hybridization and introgression on a local scale (Mandeville et al., 2015;Mandeville et al., 2017), by examining hybridization on a basin-wide scale and then evaluating results from these recent genomic studies in a comparative framework to better understand and disentangle the history of hybridization in the system.
Our case study documents all possible patterns of hybridization that have occurred across an array of hybrid crosses involving 10 species in the Colorado River Basin (Figures 1b and 2). Our results highlight a level of reproductive isolation that increases with phylogenetic distance, as well as a recognition of the variability in the outcomes of hybridization, as displayed across an entire basin.
These data provide insights into the evolution of reproductive isolation, a consequence that can not only inform conservation, but also predict potential patterns of admixture as rivers inevitably dwindle due to drought and anthropogenic water use (Cayan et al., 2010).

| Reproductive isolation as a component of phylogenetic distance
Reproductive isolation is expected to increase with phylogenetic divergence, especially if phenotypic differences promote ecological specialization among taxa (Coyne & Orr, 2004). Ecological divergence, an important driver of reproductive isolation (Funk, Nosil, & Etges, 2006), has been suggested as such in Catostomus despite repeated occurrences of hybridization and introgression (Mandeville et al., 2015). Here, we find that while hybridization transects all phylogenetic levels within the genus, barriers to introgression increase with phylogenetic distance, particularly between those subgenera that display different life histories and habitat preferences.
The phylogeny of Catostomus includes two subgenera (Catostomus and Pantosteus as described Smith et al., 2013) with Longnose Sucker as sister to the two subgenera ( Figure 2b). These subgenera were described morphologically (Smith et al., 2013) and confirmed with mitochondrial (Unmack et al., 2014) and genomic (ddRAD) data (Bangs et al., 2018) and represent two ecologically specialized types (i.e., mainstem river versus mountain stream specialist). Crosses between subgenera (i.e., Flannelmouth × Bluehead sucker, White × Bluehead sucker, Razorback × Bluehead sucker; currently within a different genus, but with nuclear (Bangs et al., 2018) and mitochondrial (Chen & Mayden, 2012) et al., 2014). Lake suckers also show evidence of multiple hybridization events throughout their evolutionary history, often associated with droughts, and may have an evolutionary benefit of parasite avoidance (Smith et al., 2018). Thus, for simplicity sake, we included Razorback Sucker in the subgenus Catostomus given the overall genetic similarity (Table 2).
This pattern of introgression within subgenera, and a lack thereof between them, remains consistent even when expanded This pattern may also be ecologically driven. For example, species within the subgenus Pantosteus prefer cooler, higher elevation habitats as compared to those within the subgenus Catostomus . In addition, Pantosteus also demonstrates a series of specialized morphological adaptions that facilitate the scraping of diatoms and biofilm from the substrates of high-velocity streams (Smith, 1966). Thus, ecological specializations may also promote reduced introgression, in that the fitness of hybrids is depressed in either parental environment. As noted above, the Razorback Sucker is ecologically specialized for lake or large bodies of water; however, they can readily introgress with Flannelmouth Sucker which goes against the idea of ecological specialization playing a major role in levels of isolation. Still, introgression may have an evolutionary benefit for in Razorback Sucker as mechanism to reduce parasite risk (Smith et al., 2018).
Regardless of the reason, the pattern of reduce introgression across subgenera is pronounced in this study and has previously been suggested in population-level studies of three species (Mandeville et al., 2015) that focused on quantifying levels of introgression at the local scale. Our study documents that this trend is maintained at a broad geographic scale and across a wider breath of species.

| Invasive species hybridize with native speciesbut without a hybrid bridge
A non-native species, White Sucker, has been introduced throughout the Upper Green, Yampa, and San Juan rivers Sublette, Hatch, & Sublette, 1990) and now hybridizes with both Flannelmouth and Bluehead sucker Holden & Stalnaker, 1975;Quist, Bower, Hubert, Parchman, & McDonald, 2009 where White Sucker is absent, or at best uncommon. All were found in the Green River above its confluence with the White River, as well as White and Yampa rivers themselves (Figure 1a).
This area on the Green River is impacted by Flaming Gorge Dam (Figure 1b), which altered downstream habitat, reshuffling the distribution and abundance of native suckers, and consequently disrupted reproductive isolation of endemic species in the Middle Green, White, and Yampa rivers. These habitat modifications also promoted the distribution of White Sucker and its hybrids (Holden & Stalnaker, 1975).
If an introduced species (i.e., White Sucker) does in fact serve as a hybrid bridge, one would also expect hybrids to be found with DNA from all three species. Yet, only one such individual was found (i.e., Navajo River in the San Juan River drainage), despite the presence Flannelmouth × White sucker and Bluehead × White sucker in other areas ( Figure 2c). Thus, three-way crosses are not only extremely rare, but also restricted to particular geographic regions where introgression between Flannelmouth and White sucker is more common, and where Bluehead Sucker is abundant. This is also reflected in a recent study confirming the presence of admixed individuals between the three species in Muddy Creek (Yampa River), but not the Big Sandy, even though Flannelmouth × White sucker and Bluehead × White sucker are present in both (Mandeville et al., 2015). Three-way hybrids were found to be 50% ancestral to Bluehead Sucker, and may thus represent a first-generation cross between Bluehead Sucker and Flannelmouth × White sucker hybrids. This also fits well with our previous argument indicating a lack of introgression across subgenera.
In either case, introgression was not detected for any Bluehead Sucker hybrids, except within the subgenus Pantosteus.
Thus, admixture with Flannelmouth Sucker, White Sucker, and Flannelmouth × White sucker hybrids is not a threat to the genetic integrity of Bluehead Sucker and does not to contribute to a hybrid swarm (per McDonald et al., 2008). However, it does represent a loss of reproductive effort, and management should therefore be aimed at this aspect.
In comparison with White Sucker, the impact of Longnose  Table 2).
In addition, other studies in the Big Sandy River found but a few Bluehead × Longnose sucker hybrids, all of which were presumably F1s (Mandeville et al., 2015).

| Introgression increases with habitat alteration
Introgression between native Flannelmouth Sucker and introduced White Sucker can, however, be construed as a threat to the genetic integrity of Flannelmouth Sucker, already listed as a "species of concern" throughout its range. Yet, this threat varies by region. Some (i.e., Upper Green River and Blacks Fork regions) reflect greater levels of introgression than do others (Figure 4h and 4j). In the Yampa River region, F2 and Bx hybrids were detected, but solely from Muddy Creek, a drainage where suckers were previously impacted by extensive introgression (Mandeville et al., 2015;. Despite the presence of several F1 hybrids, no evidence for introgression was found in the mainstem Yampa and Little Snake rivers, a result consistent with that of . The Big Sandy River contained only one Bx and several F1 hybrids (Figure 4i), again juxtaposing with the limited introgression found in this region (Mandeville et al., 2015).
The extent of introgression between species documented in our analyses can be attributed to habitat alterations. All sites with obvious introgression are found in Wyoming, an area of the Upper Colorado River basin characterized by anthropogenic impacts.
These include (a) dumping of industrial pollutants and raw effluent in the 1940s (Bosley, 1960); (b) development of Flaming Gore and Fontenelle dams in the early 1960s; (c) extensive rotenone treatment to remove "trash" fish in 1962; and (d) introduction of numerous invasive fishes . Collectively, these actions reduced native fish densities, particularly suckers, as well as greatly modified the habitat of the region (Quartarone, 1995;Wiley, 2008).
The probability is thus elevated that habitats in this region have been homogenized, reproductive behaviors impacted, and hybridization promoted, such that hybrid survival is facilitated. This is especially apparent in the Upper Green River, where the brunt of these impacts occurred, with Bluehead and Razorback sucker now rare or absent (Wiley, 2008). These regions also manifest the greatest levels of introgression between Flannelmouth and White sucker ( Figure 4j).

| Contemporary hybrids between native species
Along with Bluehead × Flannelmouth hybrids (mentioned above), several other contemporary hybrids were detected between native species, to include Bluehead × Mountain sucker as well as hybrids with Razorback Sucker.
Bluehead and Mountain sucker share a long history of introgression in the Colorado and Bonneville river basins and also hybridize in the Little Sandy River (Mandeville et al., 2015). However, our range-wide assessment found introgressive hybridization between these species in Blacks Fork ( Figure 4n) and Price River (Figure 4o), as well as two F1 hybrids in the Price River. These data emphasize how contemporaneous the hybridization between these species has been and, in turn, reflects not only habitat alterations in the Upper Green River (WY) but also the introduction of Mountain Sucker from the Bonneville Basin into the Price River .
Razorback Sucker was historically distributed throughout the entire Colorado River Basin, but has experienced drastic declines Minckley, 1983), leading to its listing as an endangered species (US Fish and Wildlife Service, 1991). Declines are attributed to habitat alteration, to include development of dams that not only disrupt recruitment but increase opportunities for hybridization with Flannelmouth Sucker (Buth, Murphy, & Ulmer, 1987).

Several hybrids involving Razorback Sucker in the Grand Canyon and
Virgin River were high-level backcrosses with Flannelmouth Sucker, as would be expected from an initial hybridization followed by several generations of backcrossing (Figures 2c and 4r).
Similar results were found in a four-year mark-recapture study of Flannelmouth Sucker in Grand Canyon , where the hybrid population was estimated to be ~ 30 . Over the four-year study, 41 morphologically diagnosed Flannelmouth × Razorback hybrids were not only captured but subsequently recaptured 60 times. Twelve of these were evaluated using molecular markers (T.E. Dowling, pers. comm.), and eight were determined to be of hybrid origin with Flannelmouth Sucker (but none designated as F1).
In addition, two F1 Razorback hybrids were also found in the San Juan River (Figure 1b). One was with Flannelmouth Sucker, which has been known to hybridize, and the second was an F1 cross with Bluehead Sucker sampled from the mainstem San Juan River that, to our knowledge, has not been previously documented. Finding a couple of these hybrids may not represent much of a loss of natural recruitment and reproductive output, both of which have been drastically reduced in Razorback Sucker (Minckley, 1995). However, these hybrids may be important to note given that stocking programs to rehabilitate Razorback Sucker were initiated in 1991, with several populations subsequently augmented to include the San Juan River (Dowling, Minckley, & Marsh, 1996;Dowling et al., 2014;Minckley, 1995). While two hybrids might not represent much of a threat to this program, it does underscore that hybridization is occurring. Importantly, these documented instances may not represent the true level of hybridization and introgression in this area, since both were random samples sent to us for analysis and do not represent a true population-level assessment of suckers in this area.

| Historic hybridization between Lower and Upper Basin species
An interesting result in our analyses was an echo of historic hybridization between species currently allopatric. Sonoran and Desert sucker, found below Grand Canyon in the Lower Colorado River Basin (Figure 1a) Douglas et al., 2003).
Introgression between these sister species was also found in the Virgin River, an area in close proximity to Grand Canyon (Figure 1a).

| Adding to the growing body of genomic work and its implications on management
We hope that this work along with the growing body of conservation genomic literature on Catostomus can lay out a blueprint on how to disentangle the complexity of hybridization and introgression.
Hybridization in the genus has been suggested to occur throughout their history leading to historic introgression events that resulted in discords between mitochondrial and morphological phylogenies that hinder species delimitation and studies of contemporary hybridization. On top of this, contemporary hybridization can occur between both endemic species as well as with introduced species making it harder to decipher anthropogenic and natural processes.
In order to resolve these issues, there is a need to (1) examine historic introgression in a phylogenomic framework to resolve discords in previous mitochondrial and morphological phylogenies, then (2) use this framework to examine species delimitation, (3) examine contemporary hybridization in the absence of introduce species to understand natural hybridization and introgression processes, and then (4) examine the correlation with anthropogenic impacts, to include introduced species and habitat change, on rates and patterns of hybridization and introgression.
Points 1 and 2 have been addressed in recent genomic studies. Bangs et al. (2018). used ddRAD to examine the phylogeny of the genus and showed that historic introgression had occurred and in turn explains discordance between morphological and mitochondrial phylogenies. This allowed   Here, we show that hybridization occurs between endemic species in the Upper Colorado River Basin, even in the absence of introduced species and that introgression is limited with increasing phylogenetic distance, either due to ecological specialization or genetic incompatibilities. While our study could not quantify the exact levels of introgression in each population, it does corroborate the results of previous population-level genomic studies (Mandeville et al., 2015(Mandeville et al., , 2017) that suggested introgression is rare and might be limited to certain crosses, populations or areas with increased habitat modification. This pattern is maintained by our analyses at a larger geographic scale (basin-wide) and a broader taxonomic spectrum to include all 10 species that occur in the basin.
Due to minimal rates of introgression found in most locations and the rarity of hybrids with ancestry of more than two species across the basin, as well as in a focused population study in the upper reaches of the Upper Colorado River Basin (Mandeville et al., 2017), the capacity for White Sucker to serve as a "hybrid bridge" between native species is negligible, and the implication that multiple species will potentially collapse into a "mutt sucker" (per McDonald et al., 2008) is improbable. The concern of a complete collapse of reproductive isolation to the point of a multispecies hybrid swarm is unlikely. Management efforts should therefore not focus on the removal of hybrids, an arduous endeavor at best with marginal effects, but instead be directed toward habitat restoration, since hybridization and introgression appear to be promoted by habitat disturbance.
These studies demonstrate how multiple conservation genomic studies can work in tandem to provide synergistic insights into complex and challenging systems. Future work is still needed on understanding why these patterns of introgression have occurred, with particular interest on quantifying what factors of habitat disturbance lead to increased introgression, the fitness impacts of different hybrid genotypes, the uniformity of introgression, or lack thereof, across genomic clines (i.e., super invasive alleles), and detecting loci that might increase genetic incompatibles, all of which can play an important role in conservation decisions (i.e., see Arnold, 2016).

| CON CLUS IONS
While hybridization is increasingly recognized as a common evolutionary phenomenon among fishes, our case study of catostomid fishes from the Colorado River basin suggests introgression seemingly decreases with phylogenetic distance and may be driven by ecological specializations that separate subgenera. Furthermore, introgression between a native and introduced species has increased concomitant with habitat disturbance (also suggested by Mandeville et al., 2015). However, the capacity of an introduced species to serve as a "hybrid bridge" between native species, as suggested for White Sucker (per McDonald et al., 2008), is negligible at a larger scale, particularly given the extreme influence of habitat alterations in promoting breakdown of reproductive isolation among native species (per Middle Green, Yampa, and White rivers). Based on our analyses, the implication that multiple species will potentially collapse into a "mutt sucker"  is improbable, due to minimal rates of introgression found in most locations coupled with the increased level of reproductive isolation concomitant with phylogenetic divergence.
The presence of historic admixture between native species also provides an example of how species boundaries can be maintained, even in the presence of anthropogenically induced introgression.
This study examines hybridization and introgression across an entire freshwater basin, to include all native or introduced catostomids in the system. Understanding the existing patterns of hybridization and reproductive isolation across this diverse range of species provides a baseline necessary to disentangle the long history of hybridization among fishes in western North America. These data, in turn, will promote region-wide adaptive management and conservation.

ACK N OWLED G EM ENTS
Numerous agencies contributed field expertise, specimens, technical assistance, collecting permits, funding, or comments. Rio Grande and