Identifying genetic variation in the western , aberti, of the Black River Basin using mitochondrial and nuclear markers

Summary: There is strong evidence that the western fanshell, is a complex consisting of an undetermined number of distinct genetic lineages. However, little is known about the genetic composition of populations of C. aberti an in particular, populations in the Black River System, which contains several different shell morphotypes. Previous work (Serb 2006) has suggested that a population of C. aberti in the Black River is more closely related to the federally endangered C. stegaria. We proposal to determined how many genetically distinct populations of C. aberti exist within the Black River Basin, and what the taxonomic affinities of those populations are.

Project Leader: Dr. Jeanne M. Serb, Assistant Professor Department of Ecology, Evolution and Organismal Biology, 253 Bessey Hall, Iowa State University, Ames, IA 50010 [email protected] voice: (515) 294-7479 fax: (515) 294-1337

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Project Partner: Dr. Kevin J. Roe, Adjunct Assistant Professor Department of Natural Resource Ecology and Management, 229 Science II, Iowa State University, Ames, IA 50010 [email protected] voice: (515) 294-8332 fax: (515) 294-2995

Total amount requested: $9,131

Amount and source of matching funds or inkind services: $4565 from 0.5 month of PI salary and 36.5% ISU indirect cost savings.

1 NEED: Freshwater mussels are one of the most imperiled taxonomic groups in North America. Nearly one third of recognized species are threatened, endangered, or presumed extinct (Williams et al. 1993). One such imperiled freshwater mussel species is the western fanshell, Cyprogenia aberti, endemic to the Arkansas, Ouachita, White, and St. Francis river systems in Arkansas, Kansas, Oklahoma, and Missouri. Like many freshwater mussels in North America, current data suggest that the range of C. aberti is diminishing (Obermeyer 2000). Although C. aberti is still relatively common at a few locations in rivers in the Ouachita and White systems, a large portion of its former range in these two basins has been reduced, primarily because of habitat loss due to large impoundments (Obermeyer, pers. observ.). In the Arkansas River system, populations are only found sporadically in three streams (Verdigris, Spring, and Fall rivers), and these populations are geographically isolated by a great distance from the remaining range of the species (Obermeyer et al. 1997). In the St. Francis River system in Missouri, populations are extremely rare and are restricted to a small portion of the upper St. Francis River (Ahlstedt and Jenkinson 1991). Populations in Oklahoma are thought to be extirpated (Mather 1990). Cyprogenia aberti is currently listed as a species of special concern by the U.S. Fish and Wildlife, and is listed in the state of Kansas as endangered.

Restoration programs based on propagation of have increased in recent years. Understanding how genetic variation in partitioned across a species range is a critical first step to initiating a successful captive breeding program. If care is not taken to first identify genetic partitioning among populations the augmentation program can have negative effects. Mixing of breeding stock from genetically distinct populations has the potential to result in outbreeding depression and loss of co-adaptive gene complexes, while failure to augment a population with a suitable number of genetically diverse individuals can result in a genetic bottleneck and the loss of valuable natural variation (Avise 1989).

Recent genetic analysis of certain species has revealed that a great amount of unrecognized genetic diversity can exist among and within mussel populations that were previously assumed to be a homogeneous, single species. For example, the genus Cyprogenia, which includes the western fanshell, C. aberti and the fanshell, C. stegaria. Several recent studies suggest that populations classified as C. aberti in Kansas, Missouri, and Arkansas may actually consist of at least 3 and possibly more species (Serb 2006, Eckert 2003; see also Figures 1 and 2).

Fig. 1) Fig. 2)

Figures: 1) Strict consensus of 32 trees from the maximum parsimony analysis (Serb 2006). 2) Conglutinate color variation identified among populations of C. aberti (Eckert 2003).

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Of particular concern is to determine the species boundaries between C. aberti and C. stegaria. Molecular and morphological data suggest that the current taxonomic designations of these taxa are not biologically accurate. Because C. stegaria is listed as a federally endangered species, understanding how C. aberti and C. stegaria populations are related to one another will be important for the legal status of some C. aberti populations which may have genetic affinity to C. stegaria. Genetic data indicate that C. aberti of the Black River (at RM 72.8, AR) is more genetically similar to the fanshell, C. stegaria, than it is to other C. aberti populations (Serb & Kuhadja 2004, Serb 2006; see red box in Figure 1). For conservation management of these species, there is a need to further test these relationships.

Funding and Conservation Priorities: Our proposed research will provide biological information and scientific evaluation to fill data gaps identified by the Arkansas Wildlife Action Plan. These data will aid in accurately assessing the biological diversity contained within C. aberti, so that biologically appropriate management plans can be development and implemented. The regional focus of this project will be in the Mississippi River Alluvial Plain Ecoregion (73). Ultimately, the project will fulfill the need to conduct final stages of genetic research to determine the existence of cryptic species of freshwater mussel within the species currently defined as Cyprogenia aberti.

Goals and Objectives: By 2008 we will have determined how many genetically distinct populations of Cyprogenia aberti exist within the Black River Basin, and what the taxonomic affinities of those populations are. Armed with these results we will be able to make recommendations with regards to the management and conservation of Cyprogenia aberti within the Black River Basin as well as throughout the state of Arkansas.

Methods: Methods to identify genetically distinct populations will include DNA sequencing of both mitochondrial genes (CO1 and ND1) as well as either a nuclear gene (ITS-1) or microsatellite marker developed for C. stegaria (pers. comm. J. Jones). DNA sequences will be analyzed for the presence of distinct haplotypes within Black River and compared phylogenetically to C. aberti populations throughout its range. The proposal builds on the existence of a large molecular data set generated by one of us (Serb 2006). In addition, we are coordinating specimen collection activities with Dr. John Harris, Environmental Division of the Arkansas Highway Department, and Dr. Chris Barnhart of Missouri State University

Measurable outcomes: One of the products of the project will be a report detailing the results and their management implications for Cyprogenia aberti. In addition, taxonomic descriptions of any new species or subspecies of Cyprogenia aberti will be published in the appropriate refereed journal. . Budget: Budget for this project will include 0.5 month salary and benefits (26.4%) for one Principal Investigator, hourly wage for an student researcher (plus benefits at 4.6%), and funds to cover lab supplies. The estimated total cost is $9,131. We are requesting 50% of the total budget from the State Wildlife Grants ($4565.50).

3 Proposed Budget FY2007 Proposed ISU Project contributed Total Salaries and Wages Principal Investigator 3,111 3,111 Student hourly help 640 640 Subtotal 3,751 0 3,751

Benefits Principal Investigator (26.4%) 821 0 821 Student hourly help (4.6%) 29 0 29 Subtotal 850 0 850

Supplies 3,700 3,700 Services 0 Other Direct Costs 0

Total Direct Costs 8,301 0 8,301 Indirect Costsa 0 830 830 Total Project Costs 8,301 830 9,131 a Indirect costs are contributed by Iowa State University, per Cooperative Agreement 1434-HQ- 97_RU-01560.

The University's full provisional rate is 47% of total direct costs less equipment and tuition.

4 INVESTIGATORS:

Dr. Jeanne Serb has been working with freshwater bivalve genetics and molecular systematics for over nine years. She has worked and published on Cyprogenia aberti since 2000. Her work has been funded by multiple state and federal agencies including U.S. Fish and Wildlife Service, Arkansas Game and Fish, Ouachita National Forest, Alabama Department of Conservation and Natural Resources, Kansas Department of Wildlife and Parks, and National Science Foundation. Currently, she has a laboratory in the Department of Ecology, Evolution and Organismal Biology at Iowa State University. The lab has all of the necessary equipment to complete the genetic analyses. DNA sequencing and genotyping will be conducted at Iowa State University DNA Sequencing and Synthesis Facility

Dr. Kevin Roe has over 13 years of experience working with freshwater bivalve genetics, molecular systematics, and . He has worked with and has been funded by both state and federal agencies including U.S. Fish and Wildlife Service, U.S. Forest Service, Alabama Department of Conservation and Natural Resources, and National Science Foundation. Currently, he is in the Department of Natural Resource Ecology and Management at Iowa State University where he teaches Conservation Genetics and conducts molecular phylogenetic research on unionid species.

Literature Cited Ahlstedt, S.A. and J.J. Jenkinson. 1991. Distribution and abundance of capax and other freshwater mussels in the St. Francis river system, Arkansas and Missouri, USA. Walkerana 5: 225-261. Avise, J.C. 1989. A role for molecular genetics in the recognition and conservation of endangered species. Trends in Ecology and Evolution 4: 279-281. Eckert, N.L. 2003. Reproductive biology and host requirement differences among isolated populations of Cyprogenia aberti (Conrad 1850). Unpublished M.S. Thesis, Southwest Missouri State University. 87 pp. Mather, C.M. 1990. status survey of the western fanshell and Neosho mucket in Oklahoma. Report to the Oklahoma Department of Wildlife Conservation. Oklahoma City, OK. Pp 22. Obermeyer, B.K. 2000. Recovery plan for four freshwater mussels in southeast Kansas: Neosho mucket (Lampsilis rafinesqueana), Ouachita kidneyshell (Ptychobranchus occidentalis), rabbitsfoot (Quadrula cylindrica cylindrica), and western fanshell (Cyprogenia aberti). Kansas Department of Wildlife & Parks, Pratt, KS. 51 pp. Serb, J.M. and B.K. Obermeyer 2001. Genetic systematics of the western fanshell (Cyprogenia aberti) in Arkansas, Kansas, and Missouri. Report to Kansas Department of Wildlife and Parks. 10 pp. Serb, J.M. and B.K. Kuhajda. 2004. Identifying cryptic species within the western fanshell, Cyprogenia aberti, of Arkansas using DNA sequencing techniques. Report to Ouachita National Forest. 17 pp. Serb, J.M. 2006. Discovery of genetically distinct sympatric lineages in the freshwater mussel Cyprogenia aberti (: ). Journal of Molluscan Studies 72: 425-434. Williams, J.D., M.L. Warren, Jr., K.S. Cummings, J.L. Harris, and R. J. Neves. 1993. of the freshwater mussels of the United States and Canada. Fisheries 18:6-22.

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