Max Bangs, Marlis Douglas, & Michael Douglas Range-wide Assessment of Hybridization and Introgression in Bluehead Sucker (Catostomus discobolus) Richard T. Bryan and Wayne S. Starnes Max Bangs, Marlis Douglas, & Michael Douglas

Introduction Southwestern U.S. Harsh and fluctuating Local adaptation High endemism Fishes of the Southwestern United States represent a unique assemblage, locally adapted to dealing with the harsh and fluctuating environment. Sierra Club

Threats Over 140 Dams Irrigation Diversion Mining Invasive Species Climate Change Keith Yahl These unique fish now face numerous anthropogenic threats including introduction of non-native species, habitat alterations, and climate change driven drought. Zanden et al. 2004

Protection Potential impacts of these threats Hybridization Population structure Life history Genetic diversity Hybridization Can hamper population assessment Especially when introgressive In order to protect these species it is important to generate baseline data about population demographics and structure necessary to develop management plans. However, accurate assessments of species can be hampered if species identifications are ambiguous due to introgression, which is the backcrossing of hybrid individuals into one or both parental species.

Bluehead Sucker Range Snake River Basin Bonneville Basin WY CO NM AZ UT NV ID MT CA Colorado River Basin Bonneville Basin Snake River Basin Bluehead Sucker Range For example in the recent range wide assessment of BHS done by Hopken et al. several hybrids were found. The BHS is endemic to the Snake, Bonneville, and Colorado river basins and …

Bluehead Sucker Population decline Species of concern Hybridize Historical Contemporary w/ native species w/ invasive species … experienced drastic declines, now occurring in less than half of there historic range. This is mostly attributed to habitat degradation and introduction of sports fishes. For this reason it is one of three species of concern targeted for a basin-wide, multi-agency management and research focus. In addition, BHS have also been know to hybridize with both native and introduced congers and has been involved in several potential introgression events that have occurred in the past, both into and out of the species, as note in the red. [talk about figure] All of this can further complicate the assessment of this species. Also I should note in a basin-wide assessment of mtDNA haplotypes, BHS and MTS were not clearly distinct sharing several haplotypes. For this reason a basin-wide assessment was conducted across both species using 16 msat loci. Smith et al. (2013)

Bluehead Sucker Range wide assessment (Hopkens et al., 2013) 16 msats and two mtDNA genes (ATPase 6 & 8) Assigned several ESUs and MUs Noted and removed admixed samples In this assessment some samples were determined to be potentially hybrids based on either admixture between species in the msats, presence of a discordant mtDNA haplotype, or were identified as potential hybrids in the field. These samples were noted and removed from the analyses. For example, here is a structure plot using the msats for some of these potential hybrids. As you can see some of these samples come out as having admixed ancestry from several species, some of which do not even occur in the location were the sample was taken. This is the problem in using assessment test when multiple species are involved, the ambiguity in these markers can lead to the mis-assessment of the ancestry. Thus it is important to use unambiguous markers, to which species specific ancestry can be trace in order to demine the species involved in these admixtures.

Objectives Examine hybridization over the range of Bluehead Sucker Determine if introgression is occurring In order to resolve these potential hybrids we used three nuclear markers along with the two mitochondrial genes already analyzed. By doing so we attempt to examine range-wide patterns of hybridization in BHS and determine if introgression has occurred with any other species.

Collecting Sampling Fin Clip Ethanol Storage Samples for the range-wide assessment of BHS were collected from numerous sites for all three river basins, shown here by red dots. Total ~1,200 BHS, ~800 MTS (based on phenotype) Of these – only a small fraction were identified as hybrids or introgressed (~3-5%)

Collecting Sampling Fin Clip Ethanol Storage Of that the falling sites were identified as having potential hybrids. Fin clips or whole juveniles were collected and stored in ethanol and shipped to the lab.

Laboratory Methods DNA Extraction from Fin Clip PCR Amplify: Using a Qiagen or PureGene Kit PCR Amplify: mtDNA ATPase 6/8 genes nDNA S7 nDNA CK7 nDNA RB40 Direct Sequencing PCR Products ExoSAP cleanup ABI sequencer Once in the lab DNA was extracted and amplified for three nuclear DNA loci and two mtDNA genes. Amplification were then verified using gel electrophoresis and directly sequenced using Sanger sequencing.

Analytical Methods Categories Pure Hybrid BHS x ? F1 or Bx Sequences were then manually edited and aligned to a database of pure samples of all species of Catostomus found in the Southwest. Samples were then classified as either pure for one species if they contained allele from only one species or hybrids if they contained alleles from multiple species. Hybrids were then classified by the species involved in the cross. Hybrids were also classified as either F1 if heterozygous for two species across all nuclear loci, or Bx if not heterozygous for the two species for all loci. Chaing et al. 2006

BHS x UTS Both native to Bonneville Basin 13 hybrids in Weber River 1 in Bear River UTS introduced to Colorado River Basin 1 hybrid in the Green River No introgression No asymmetry 42% BHS mtDNA The first hybrids I would like to talk about are BHS x UTS hybrids. Both species are native to the Bonneville Basin. In 2007 samples were collected from the Weber River and Bear River. Of the 30 samples in the Weber River 6 came back as F1 hybrids and 1 out of 4 in the Bear River came out as a F1 hybrid. Targeted sampling of hybrids were done on two sites in the Weber River in 2009 and 2011. All seven adults identified as hybrids were confirmed based on genotypes as F1 hybrids. I should also note that UTS has been introduced to the CRB and we detected one F1 hybrid in the Green River.

BHS x WTS WTS introduced to Colorado River Basin No introgression Upper Green River 1 in Little Sandy River 2 in Blacks Fork 2 in Ham’s Fork 1 in Muddy Creek (Sublette) Middle Green / Yampa 3 in Green River 34 in Yampa River 5 in Little Snake River 3 in Muddy Creek (Carbon) 6 in Navajo River No introgression WTS have been introduced in various parts of the CRB for over the last century, shown here by the orange polygons. Hybridization has previously been note in the literature in various parts of the CRB and here we found hybrids through out the range of introduction. In the Upper Green River we found one hybrid in LSR, 2 in BLF, 2 in HMF, and 1 in Muddy Creek in Sublette Co. In the middle Green River 3 hybrids were found below the confluence of the Yampa River, 34 were found in the Yampa river above the confluence of the Little Snake River, 5 were found in the Little Snake River, and 3 were found in the Muddy Creek in Carbon Co. I should note that 33 of the 34 hybrids for the Yampa River were found in on site near the town of Craig for which we analyzed over 200 samples. Six hybrids were also found in the Navajo River which is a tributary of the San Juan River. Hybrids have been noted in other tributaries of the San Juan to include the Animas River. All hybrids were F1, thus there appears to be no introgression. Also I should note that in all of these location BHS hybrids were much more rare than hybrids involving other species, which were mainly FMSxWTS hybrids.

BHS x WTS WTS introduced to Colorado River Basin No introgression Upper Green River 1 in Little Sandy River 2 in Blacks Fork 2 in Ham’s Fork 1 in Muddy Creek (Sublette) Middle Green / Yampa 3 in Green River 34 in Yampa River 5 in Little Snake River 3 in Muddy Creek (Carbon) 6 in Navajo River No introgression WTS have been introduced in various parts of the CRB for over the last century, shown here by the orange polygons. Hybridization has previously been note in the literature in various parts of the CRB and here we found hybrids through out the range of introduction. In the Upper Green River we found one hybrid in LSR, 2 in BLF, 2 in HMF, and 1 in Muddy Creek in Sublette Co. In the middle Green River 3 hybrids were found below the confluence of the Yampa River, 34 were found in the Yampa river above the confluence of the Little Snake River, 5 were found in the Little Snake River, and 3 were found in the Muddy Creek in Carbon Co. I should note that 33 of the 34 hybrids for the Yampa River were found in on site near the town of Craig for which we analyzed over 200 samples. Six hybrids were also found in the Navajo River which is a tributary of the San Juan River. Hybrids have been noted in other tributaries of the San Juan to include the Animas River. All hybrids were F1, thus there appears to be no introgression. Also I should note that in all of these location BHS hybrids were much more rare than hybrids involving other species, which were mainly FMSxWTS hybrids.

BHS x WTS WTS introduced to Colorado River Basin No introgression Upper Green River 1 in Little Sandy River 2 in Blacks Fork 2 in Ham’s Fork 1 in Muddy Creek (Sublette) Middle Green / Yampa 3 in Green River 34 in Yampa River 5 in Little Snake River 3 in Muddy Creek (Carbon) 6 in Navajo River No introgression WTS have been introduced in various parts of the CRB for over the last century, shown here by the orange polygons. Hybridization has previously been note in the literature in various parts of the CRB and here we found hybrids through out the range of introduction. In the Upper Green River we found one hybrid in LSR, 2 in BLF, 2 in HMF, and 1 in Muddy Creek in Sublette Co. In the middle Green River 3 hybrids were found below the confluence of the Yampa River, 34 were found in the Yampa river above the confluence of the Little Snake River, 5 were found in the Little Snake River, and 3 were found in the Muddy Creek in Carbon Co. I should note that 33 of the 34 hybrids for the Yampa River were found in on site near the town of Craig for which we analyzed over 200 samples. Six hybrids were also found in the Navajo River which is a tributary of the San Juan River. Hybrids have been noted in other tributaries of the San Juan to include the Animas River. All hybrids were F1, thus there appears to be no introgression. Also I should note that in all of these location BHS hybrids were much more rare than hybrids involving other species, which were mainly FMSxWTS hybrids.

BHS x FMS Both native WTS not need for hybrids No introgression 4 in Green River 2 in Yampa River 1 in White River WTS not need for hybrids No introgression No asymmetry 50% BHS mtDNA Both BHS and FMS are native to the CRB. Previous BHSxFMS hybrids have been noted in the literature and it was suggested that introduced WTS was acting as a “bridge” between the two native species. We confirmed BHSxFMS hybrids in the Yampa river below the confluence of the Little Snake River, 4 in the Green river between the confluence of the Yampa and White Rivers, and 1 in the White River. What is interesting about this is that some of these areas are were WTS are either rare or absent, especially in the White River were WTS have not been found. On top of this all hybrids were F1 between BHS and FMS, thus it appears that WTS are not needed to act as a bridge. Our data suggest that hybrids between BHSxFMS do occur naturally without the intermediary of an introduced species, but that these hybrids are very rare. Habitat alterations and/or severe population reductions may be a contributing factor, but would need to be further investigated if indeed BxF

BHS x MTS 1 sample in Little Sandy 6 samples in Blacks Fork Collected as BHS msats – BHS x MTS nDNA – MTS 6 samples in Blacks Fork msat – MTS nDNA – 4 MTS, 2 BHSxMTS Bx into MTS The last set of hybrids I would like to talk about are between BHS and MTS. As I noted earlier it has been suggested that admixture between BHS and MTS may have occurred – with presence of shared mitochondrial haplotypes an indication. We confirmed one potential hybrid in the LSR that was collected as a BHS, but msat data suggested a BHSxMTS hybrid. The nuclear loci however confirmed it as a pure MTS. This may be because we only use 3 species-diagnostic nuclear loci and thus if it is a high level backcross we may not be able to detect it with this limited number of markers. Six samples were as collect in the Blacks Fork as BHS, but identified as MTS in the msat analysis. The 3 nuclear loci suggested that 4 were pure MTS and two were BHSxMTS hybrids that had Bx into MTS. Both hybrids only had one BHS allele and five MTS alleles, thus this once again could just be evidence for historical introgression.

BHS x MTS 9 samples in Price River 2 samples in lower Collected as BHS mtDNA – MTS? nDNA – 1 BHS, 1 BHS x MTS Bx into BHS 7 samples in upper Collected as MTS msats – distinct pop. nDNA – 3 MTS, 4 BHS x MTS 1 F1, 3 Bx into MTS However in the Price River we found evidence for more contemporary hybrids. In the lower Price River two BHS were identified as potentially MTS based on mtDNA. The nuclear loci identified one as pure BHS and the other as a BHSxMTS hybrid that had Bx with BHS. This hybrid seemed to be more recent since it was heterozygous for two of the three loci. In the upper Price River seven samples of MTS were clustering as a distinct population based on msats. The nuclear loci identified 3 as pure MTS and 4 as BHSxMTS, of which one was a F1 and the other three Bx into MTS. This suggests a much more recent hybridization event in the Price River with introgression into MTS in the upper Price River and introgression into BHS in the lower Price River. Hmm, could it also be that the MTS in the upper Price is a distinct lineage, and we don’t have a representative sample for this?

Conclusions Bonneville Basin Colorado River Basin Decline in abundance of BHS (Webber et al., 2012) Hybridization with native UTS Threat of wasted reproductive effort Colorado River Basin Hybridizes with introduced WTS and UTS and native FMS and MTS Introgression only occurs with MTS How much historic verse contemporary? In the Bonneville Basin BHS has experienced recent drastic declines in both abundance and range. In this basin hybrids are found in all places were BHS are still abundant, and although there is no introgression and thus no threat of erosion of the genome, there is still a threat of lost of reproductive effort since hybrids appear to be common and also threat of competition for resources with the hybrids. Morphological identification appears to be accurate and should help in manage of the hybrids. In the CRB BHS hybridize with introduced WTS and UTS and native FMS and MTS. BHS hybrids appear to be less abundant than other hybrids and there seems to be no threat of introgression from both introduced species and FMS. Introgression does occur between BHS and MTS and appears to be not only historic but potentially also contemporary at least in the Price River.

Acknowledgements Collection agencies Matthew Hopken Steve Mussmann

Bluehead Sucker … experienced district declines, occurring in less than half of there historic range. For this reason they have been listed a species of concern in several states, and face anthropogenic threats include habitat alternation and hybridization with both introduced and native congeners. On top of this, BHS have been involved in several potential introgression events that have occurred in the past, both into and out of the species, as note in the red. [talk about figure] All of this can further complicate the assessment of this species. Smith et al. (2013)