Identifying Origins and Pathways of Spread of Zebra Mussels using Genetics and Genomics

Michael McCartney and Sophie MallezMN AIS Research Center (MAISRC)University of Minnesota, St. Paul

Upper Midwest Invasive Species ConferenceLaCrosse WI

October 17th, 2016

North American invasion

• Several introductions to the Great Lakes in ship ballast water

• Appeared in Lake St Clair (1988: arrow)

• Through navigable waters (Great Lakes and Mississippi Basins, Hudson and Susquehanna Rivers)—they reached Louisiana to the south, Quebec and New York to the east, Oklahoma and Minnesota to the west in 5 years!

2011: Brown and Stepien

Spread to inland lakes: much slower, less predictable

• In Minnesota…

• Introduced in 1989, in Lake Superior

• Upper Mississippi and St Croix Rivers: early 1990’s

• First natural inland lake infested in 2003: Ossawinnamakee

• A long lag, 10 years longer than other US states. Now invasion rate of inland lakes is very high

Data sources:• US Geological Survey (NAS:

2014)• MnDNR (2015)

Minnesota’s rate of new inland invasions is now among the highest in the US

From Mallez and McCartney (in review)

We have the time, the will, and the resources to slow spread and prevent infestation of many prized water bodies!

• Prevention works, but must be targeted by

• Studying transport pathways (boats, docks, lifts…)—both on the map and to understand the vectors

• Using genetics and genomics to directly pinpoint invasion sources and determine routes

Hypotheses for inland spread in MN

• Longer lag• Prevention success

• Slow dispersal from Miss/St Croix Rivers, into inland “hub” lakes

• Post-2008 increase• Secondary spread

from inland hub(s) (e.g. Mille Lacs Lake)

DNA markersVariable (ie. polymorphic) genes or DNA fragments whose

chromosomal location is known

Distinguishes individualsAllows their assignment to population-of-origin

Invasion genetics and genomics

DNA markers to “track” invasion paths, both past and present

Genetic markers for zebra mussel

• Microsatellite markers• Repeated motifs – GTTAGTCCAGAGAG….AGAGAGTTCGATCT• Polymorphic – numerous alleles

• Genotyping of 9 microsatellite markers• Obtained from the literature • Optimized for this study

Waterbodies sampled— 2014-2016

• Sampling throughout Minnesota (and nearby regions)• 59 sites - 35 water bodies – 1747 individuals

Waterbodies sampled— 2014-2016

• Sampling throughout Minnesota (and nearby regions)• 59 sites - 35 water bodies – 1747 individuals

Detroit Lakes area (9 lakes)

Analyzing the invasion in Minnesota

• 1747 individuals genotyped at 9 microsatellite markers• 35 lakes, 3 river systems• Several lakes (e.g. Gull, Mille Lacs, Minnetonka, Prior,

Pelican, Superior); Mississippi & St. Croix Rivers at multiple sites

Each brand of analysis targets a different invasion process:

- Analysis of genetic diversity = Number of individuals introduced, severity and duration of bottleneck when lakes were colonized

- Analysis of genetic structure/clustering = Genetic differences between lakes set up by unique “histories”

- Analysis of invasion models = Origins and routes of introduction. Contrast of detailed scenarios to describe invasion history. Most lessons for

management are generated here.

Analysis of genetic diversity

*Mille Lacs Lake Prior Lake 8 lakes in Alexandria area

Moderate founder effects due to bottlenecks

Broad pattern: large numbers of mussels and/or larvae cause infestations

Analysis of genetic structure/clustering

Some well-defined genetic clusters distinguish important lake infestations

K = 2

K = 3

K = 4

K = 5

K = 6

K = 7

K = 8

Mille Lacs Prior Alexandria area Brainerd area

Analysis of invasion models

• Comparisons of scenarios of invasion• Approximate Bayesian Computation• Selection of the most likely scenario of invasion based on probabilities

Focus on distinguishable lakes

“Super-spreader” lakes Clustered invasion

Mille Lacs LakePrior Lake

Alexandria-area Lakes

Mille Lacs Lake – A source for spread to other inland lakes infested later (post-2005)?

Analysis of invasion models – “Super-spreader” lakes

Independent invasion scenario

Mille Lacs Lake

Successive invasion scenario

Mille Lacs Lake

X

Gull Lake

Mille Lacs LakeMille Lacs Lake

Gull Lake

Mille Lacs Lake – A source for spread to other inland lakes infested later (post-2005)?

Analysis of invasion models – “Super-spreader” lakes

The answer ‘’No’’ was strongly preferred for 10 of 11 lakes (Prior is ambiguous)(Posterior Probabilities from 0.86 to 0.99)

Independent invasion scenarioSuccessive invasion scenario

X

Posterior P = 0.89

Mille Lacs Lake

Gull Lake

Mille Lacs LakeMille Lacs Lake

Gull Lake

Mille Lacs Lake did not infest Carlos, LeHomme Dieu, Darling, Gull, Pelican (Otter Tail), Minnetonka, Xmas, Pike (St. Louis), Bass, Sand (Itasca)

Where are we going with this?

Expanded sampling of water bodies, both in MN and throughout the Upper Mississippi and Great Lakes Basins

• To examine, more comprehensively the causes of clustered invasions

• To obtain several more recently-infested lakes to increase confidence in results for “hubs”

• Mille Lacs was heavily infested only ≥ 2009

• Minnetonka ≥ 2010

Where are we going with this?

• New lakes sampled in 2016—for Mille Lacs analyses• Alexandria— was this large cluster “founded” from Mille Lacs?• Brainerd Lakes: geographically proximate to Mille Lacs• Many new infestations 2009 or later

Alexandria Lakes Region Brainerd Lakes Region

Mille Lacs Lake

AIS-infestedZM-infestedZMSampled

Where are we going with this?

• New lakes sampled in 2016—for Mille Lacs analyses

Pelican Rapids/Detroit Lakes Region

Cass/Winnie Region

• Lakes (e.g. Cass) that are “next stop” destinations for boaters

Mille Lacs Lake – A source for spread to other inland lakes infested later (post-2005)?

Analysis of invasion models – “Super-spreader” lakes

The answer ‘’No’’ was strongly preferred for 26 of 27lakes tested so far (Prior Lake is ambiguous)

Independent invasion scenarioSuccessive invasion scenario

X

Posterior P = 0.89

Mille Lacs Lake

Gull Lake

Mille Lacs LakeMille Lacs Lake

Gull Lake

Mille Lacs Lake – A source for spread to other inland lakes infested later (post-2005)?

Analysis of invasion models – “Super-spreader” lakes

Independent invasion scenario

X

Posterior P = 0.89

Mille Lacs Lake

Gull Lake

Mille Lacs Lake did not infest Alexandria Region Lakes: Brophy, Cowdrey, Darling, Carlos,Irene, Miltona, Ida, Victoria, Geneva, LeHomme Dieu, Mary, Maple; Brainerd Region Lakes: Gilbert, Gull, Round, Rice, North Long, Cross, Lower Hay, Ossie, Pelican (Crow Wing); also

Minnetonka, Christmas, Pike (St. Louis), Bass, or Sand (Itasca)

The Zebra Mussel Genome Project

• We sequenced the zebra mussel genome using Illumina “short read” technology

• 100s of millions of fragments, each 50-300 base pairs

• Piled up and “stitched together” using bioinformatics

• We used this draft genome to help genotype Single Nucleotide Polymorphism (SNP) markers

• 5,000 to 200,000 markers per mussel!

• Genotyped using Sequence Based Genotyping (SBG)—

new technology at UMGC

These thousands of variable SNP markers providehigher resolution for studying sources and pathways of spread

Genomic Markers for Studying Spread

24 mussels from Minnetonka and 24 mussels from Gull Lake (2 putative “super spreader” lakes)

• Genotyped with 9 microsatellite markers (left panel)

• Genotyped with > 5,500 genomic (SNP) markers (right panel)

• Analyzed the same way (Principal Component Analysis) to examine

genetic differences between lakes

Lake Minnetonka

Gull Lake

Co

ord

. 2

Coord. 1

Higher genetic resolution = more complex scenario testing = more targeted management

Lake Michigan

Upper Miss

Mille Lacs

Minnetonka

Tim

e

Scenario I: Successive invasions inland

Management: target boat traffic between inland lakes

Scenario II: Chronic reintroductions

Management: increase surveillance of chronic sources (e.g, Mississippi boat ramps, or sources out of state)

Mille Lacs

Minnetonka

Tim

e

Lake Michigan

Analyses of “super

spreader” lakes (Mille Lacs,

Prior) have so far indicated

a surprising lack of

contribution to spread

• We continue to analyze lakes

infested post-2009 for better

confidence

Management implications?

Lakes infested post-2009

• Watercraft inspection of boats departing Mille Lacs may be working

• Invasions are not following patterns of transport of trailered boats (veligers in residual water, mostly)

Mille Lacs

Upper Miss

Lake Michigan

Mille Lacs

Lakes infested post-2009

• Time is now to consider “other pathways” (docks, lifts, transport of resident boats from marinas on St. Croix/UMR)

• How to better monitor and intercept these less frequent but high risk events?

© www.nps.gov© www.marinedocklift.com

Upper Miss

Lake Michigan

Management implications?

Thanks to…

UMN: • Grace Van Susteren, Sarah Peterson, Maxwell Kleinhaus , Melody Truong

for collecting help and lab support• UM Genomics Center: Kenneth Beckman, Daryl Gohl, Shea Anderson,

Aaron Becker for molecular biology; John Garbe for informatics

NPS: Byron Karns, Michelle Prosser for field support on St. Croix & Mississippi

MnDNR: Dan Swanson, Rich Rezanka, Keegan Lund for field support and

advice on collecting MN waters

USGS: Mary-Anne Evans for collections in western Lake Erie

Clear Water Fund, ENRTF for funding