Aquatic Invasive Species Early Detection and Monitoring Program for Lake Michigan: A Review of 2015

Report Number: 2017-016

Hayer, C-A., M. Petasek and B. Smith. 2017. Aquatic Invasive Species Early Detection and Monitoring Program for Lake Michigan: A Review of 2015. U.S. Fish and Wildlife Service, Green Bay Fish and Wildlife Conservation Office Report Number: 2017-016.

August 2017

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TABLE OF CONTENTS

Acronyms/Abbreviations ............................................................................ ii

Executive Summary ................................................................................... iii

Introduction ................................................................................................. 1

Hotspot sampling ........................................................................................ 4

Lower Green Bay ........................................................................................ 5

Milwaukee River Estuary ......................................................................... 14

Chicago Harbor ......................................................................................... 23

Calumet/Indiana Harbor ............................................................................ 30

Burns Harbor ............................................................................................. 37

Macroinvertebrate Sampling ..................................................................... 46

Environmental DNA (eDNA) ................................................................... 47

Ichthyoplankton Sampling ........................................................................ 55

Cooperative Science and Monitoring Initiative (CSMI) ........................... 56

Summary ................................................................................................... 61

Literature Cited ......................................................................................... 62

Appendix I Common and species names of fish captured during sampling in Lake Michigan by the GBFWCO early detection and monitoring program during 2015 ............................................................. 65

Appendix II Sampling gear specifications ................................................ 67

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Acronyms/Abbreviations

CPUE Catch Per Unit Effort

USFWS U.S. Fish and Wildlife Service

QAPP Quality Assurance Project Plan

AIS Aquatic Invasive Species

CAWS Chicago Area Waterway System

eDNA Environmental Deoxyribonucleic Acid

GBFWCO Green Bay Fish and Wildlife Conservation Office

CSMI Cooperative Science and Monitoring Initiative

SE Standard Error

SD Standard Deviation

PCR Polymerase Chain Reaction

WGL Whitney Genetics Lab

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Executive Summary

The Great Lakes region is home to a variety of unique wetland, near shore, and deep-water habitats that support a diverse array of plant, animal, and fish species. In addition to recreation and tourism, the Great Lakes provide a commercial and sport fishery valued at $7 billion a year (U.S. Fish and Wildlife Service 1994). The economic revenue of the Great Lakes has, and will continue to diminish due to the effects of Aquatic Invasive Species (AIS) (Great Lakes Commission 1999). The total cost of AIS is not only measured by the direct financial loss of consumers, industries, and governments, but also by indirect costs such as decreased productivity and reduced demand (Rosaen et al. 2012). It is estimated that AIS have reduced the current commercial and recreational fisheries in the Great Lakes by over 48% of their total value, effectively depriving the surrounding states from billions in annual income (Pimental 2005). Federal, state, tribal and nonprofit organizations also contribute to the total cost of AIS through management efforts that mitigate the effects of established species, as well as monitor for new ones.

The United States Fish and Wildlife Service’s Green Bay (USFWSGB) office has been tasked with creation and implementation of an Early Detection and Monitoring Program for Aquatic Invasive Species in Lake Michigan. Program development began in late 2012, with actual field work commencing in 2013. Monitoring activities involved collaboration with a variety of local, state, federal, and non-profit partners. Monitoring included both traditional gear efforts designed to assess community diversity and non-traditional efforts designed to specifically target individual species or ontogenies of fish species. The Program now consists of traditional gear sampling, ichthyoplankton sampling, macroinvertebrate sampling and environmental DNA (eDNA) testing. Sampling efforts in 2015 consisted of environmental DNA testing for Bigheaded carps (Bighead Carp and Silver Carp, collectively) and Eurasian Ruffe, sediment sampling for macroinvertebrates, bongo net and light trap sampling for larval fish, and traditional gear sampling for community fish species assessments. In addition to this work, the AIS program also participated in the Cooperative Science and Monitoring Initiative (CSMI): a project which assesses each of the Great Lakes on a rotating five year basis.

During the 2015 field season, USFWSGB collaborated with multiple state, federal and NGOs to conduct annual monitoring on Lake Michigan. Monitoring included the sampling of 19 primary sample sites in Wisconsin, Michigan, Illinois and Indiana. Traditional gears (i.e., boat electrofishing, fyke and mini-fyke nets, micromesh gillnets) were deployed in 5 priority hotspots accounting for 625 units of effort. Environmental DNA was collected from 6 tributaries (three times each) accounting for 2,400 total water samples; none of which resulted in any detections of bigheaded carp DNA. The ichthyoplankton communities of Green Bay were intensely sampled throughout the year accounting for 203 units of effort. There were also 410 units of effort (i.e.,

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gillnet, bong net) deployed at 8 near-shore (<18 m) sites on Lake Michigan as part of the CSMI effort. Additionally, the macroinvertebrate communities were assessed at all 5 priority hotspots as well as the eight CSMI sites using rock bags and sediment samplers. The combined units of monitoring effort in 2015 (i.e., eDNA, traditional gear, ichthyoplankton, macroinvertebrate, CSMI) amounted to 3,792 which captured a total of 37,810 fish from all life stages and represented 76 different species. While thirteen of these species were known to be existing or non-native fishes, no new AIS were detected in 2015; albeit, a single Grass Carp was found in each of two locations (i.e., Milwaukee Harbor, Burns Harbor).

Moving forward the AIS Program aims to broaden the scope of AIS sampling in Lake Michigan. Inclusion of larval fish sampling will increase the sensitivity of our hotspot sampling for invasive fish. A robust benthic macroinvertebrate sampling design will extend AIS sampling into the important near-shore benthic community. Given the level of effort used at locations likely to harbor new AIS, we find it unlikely that any new invasive fish species populations are being established in the near-shore zone of Lake Michigan at this time.

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Introduction

The U.S. Fish and Wildlife Service (USFWS) is responsible for the prevention, early detection, rapid response, control, and management of invasive species (National Invasive Species Management Plan 2008). Nationally and internationally, invasive species have proliferated with the expansion of global trade, increased connectivity, degradation of native ecosystems and, more recently, climate change (Padilla and Williams 2004; Rahel and Olden 2008; Hulme 2009). Few places face more problems with aquatic invasive species (AIS) than the Laurentian Great Lakes (Mills et al. 1993; Ricciardi 2006). At least 184 non-native aquatic species are established in the Great Lakes and several have caused ecological alterations and economic harm (e.g., Round Goby, Zebra Mussel, Quagga Mussel, Spiny Water Flea; Vanderploeg et al. 2002). Prevention of further non-native introductions remains the best strategy for protecting the Great Lakes and is being actively pursued through regulation of ballast water, restrictions on the importation and sale of potentially harmful species, and public outreach and education. Concurrently, early detection and monitoring efforts are being developed to find any new aquatic invasive species (Vander Zanden et al. 2010). Lake Michigan is of particular concern due to connectivity not only to other Great Lakes but to the Mississippi River via the Chicago Area Waterway System (CAWS) where bigheaded carp (i.e., Bighead and Silver Carp) populations are expanding (Jerde et al. 2011).

In 2013, the USFWS established an AIS early detection and monitoring program for Lake Michigan at the Green Bay Fish and Wildlife Conservation Office (GBFWCO), hereafter referred to as the AIS Program. Monitoring for AIS in Lake Michigan has been challenging due to the size of the ecosystem and diversity of vectors by which new AIS can enter the lake. Early on, sites within the lake were prioritized for sampling based on their risk for harboring new AIS (Hayer et al. 2017). Vectors included frequency and volume of ballast water discharge, hydrologic connection to areas with high potential for AIS introduction, fishing activity (bait shops, anglers, boat ramp use, etc.), tourism, and other factors related to human population density. These vectors were used to construct heat maps identifying five areas in Lake Michigan that pose the highest risk for new AIS (Hayer et al. 2017).

In descending order, the areas of greatest concern, or “hotspots”, for introduction of new AIS in Lake Michigan are Calumet/Indiana Harbors, Chicago Harbor, Burns Harbor, Milwaukee River Estuary, and lower Green Bay. Since 2013, a goal of the AIS Program has been to intensively sample each hotspot annually to characterize their fish communities and use enough effort to detect new non-native species that would likely occur initially at low abundance. This goal was achieved for the first time during 2015. We estimate that by using ~100 units of effort, 90-96% of the fish community was represented in our samples (Figure 1-1). No new non-native fish were detected. In addition to fish, the AIS Program has expanded the scope of sampling to

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include benthic macroinvertebrates through a pilot study that will direct future efforts for this important and vulnerable ecological community.

The expansion bigheaded carp populations towards Lake Michigan has been particularly worrrisome for resource managers and policy makers (Cooke and Hill 2010; Jerde et al. 2013). To address these concerns, the AIS Program has conducted sampling of environmental DNA (eDNA) in habitats deemed most suitable for Asian carps; primarily large tributaries (Kocovsky et al. 2012; Murphy and Jackson 2013). This method is more sensitive and effective at detecting these species at low abundance than traditional fish sampling gears (Jerde et al. 2011). During 2015, the AIS Program sampled six large tributaries to Lake Michigan in Wisconsin and Michigan including the Fox River, Milwaukee River Estuary, St. Joseph River, Grand River, Kalamazoo River, and Muskegon River. Each river was sampled three times. There were no positive detections for the Silver or Bighead species indicating that bigheaded carp were not present in any sampled river. Future sampling of eDNA will be expanded to include the Galien, Black, and Macatawa rivers in Michigan.

To increase the scope and sensitivity of our early detection abilities, the AIS Program is developing an ichthyoplankton sampling program. During 2015, most ichthyoplankton sampling focused on Green Bay as part of a multifaceted cooperative project between the University of Wisconsin-Green Bay and USFWS. No new larval AIS were detected in Green Bay. In the future, ichthyoplankton sampling will be expanded into all five hotspots to complement current sampling efforts.

During 2015, the AIS Program had the opportunity to participate in the Cooperative Science and Monitoring Initiative (CSMI) coordinated by the U.S. Geological Survey, Environmental Protection Agency, and National Oceanic and Atmospheric Administration on a rotating five- year schedule for each of the Great Lakes. Participation involved sampling the near shore zone at eight locations lake-wide to study the influence of tributaries on the near shore fish communities of Lake Michigan. In addition to its main objective, CSMI had direct benefits for the AIS program. As river mouths are hypothesized to be suitable habitat for numerous non- native species, we took this as an opportunity to expand the spatial coverage of our sampling program. As a result, we found that river mouths were, in fact, preferred habitat for established non-native species including Rainbow Smelt and Alewife.

The remainder of this annual report will provide details for each major aspect of the AIS Programs’ activities including hotspot, macroinvertebrate, eDNA, ichthyoplankton, and CSMI sampling. Common and scientific names of all species collected are located in Appendix 1 and gear descriptions are listed in Appendix 2.

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Figure 1-1. Species accumulation curve (solid line) for six gears used to sample the near-shore zone of Lake Michigan at 13 locations during 2015 shown with 95% confidence intervals (dashed lines). Site data was added in a random order and subjected to 1,000 unique permutations. The extrapolated species richness (asymptote; mean ± SE) of the near-shore zone of Lake Michigan (dotted line) was estimated using bootstrapping techniques. We estimate that 96% of fish species present in the near-shore zone of Lake Michigan were collected using 858 units of effort from gillnets, paired fyke nets, electrofishing, and tandem set minnow traps.

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Hotspot sampling

During summer and early fall of 2015, the AIS Program visited five locations identified by vector analyses to be the most vulnerable to invasion by new AIS. These locations were lower Green Bay, Milwaukee River Estuary, Chicago Harbor, Calumet/Indiana Harbors, and Burns Harbor. Potential species of concern included Monkey Goby Neogobius fluviatilis, Northern Snakehead Channa argus, Bighead Carp and Silver Carp to name a few (Rixon and Duggan 2005). Sampling was centered on harbors, river mouths, canals, and ship docking areas at each location (Hayer et al. 2017).

Five or six fish sampling gears with varying selectivities were used at each location to achieve a minimum of 100 units of total effort; a target identified by previous analyses as the amount of effort required to collect 95% of species present. We used micromesh gillnets, large-mesh gillnets, boat electrofishing, paired mini-fyke nets, paired fyke nets as well as minnow trap arrays at select locations. Sample locations for each gear were stratified by depth. Sampling required four crews; two sampled during the day with boat electrofishing and paired fyke nets while two crews conducted electrofishing and deployed gillnets at night. Each location took approximately one week to sample. Electrofishing represented the largest proportion of effort at all sites. Electrofishing was also the most effective sampling gear in near-shore habitats (<3 m). Minnow trap arrays were the least effective gear and only collected 5 species at each site.

We sampled 28-51 species at each location including established invasive species such as Round Goby, Alewife, Goldfish, Grass Carp, and White Perch. Grass Carp were collected in the Milwaukee River Estuary (N=1; diploid) and Burns Harbor (N=1; triploid). At each site, we achieved our objective by using >100 combined units of effort and we estimate that 90-95% of the species present at each site were represented in the data. No new non-native fish species were detected during hotspot sampling.

Details of the 2015 hotspot sampling are organized below by location and include sample area maps, gear-specific catch rates and diversity, size structure of the most abundant species, and species accumulation curves. Scientific names of collected species are found in Appendix 1 and gear descriptions are in Appendix 2.

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Lower Green Bay

Green Bay was sampled on several occasions from April 23rd to October 30th as part of the 2015 AIS early detection and monitoring program. Sampling included an 88 square kilometer area of southern Green Bay, stretching from Point Sable and Long Tail Point southward, and 11 kilometers of the Fox River (Figure 2-1).

One hundred seven electrofishing runs (≈ 10 min each) were conducted in diverse habitats including sand flats, lagoons, rip-rap break walls, weed beds, rocky structures and riverine habitat. Electrofishing yielded 11,445 fish from 44 species (Table 2-1). Catch per unit effort (CPUE) was 106.96 ± 18.26 (mean ± SE) fish per electrofishing run and was dominated by Gizzard Shad and juvenile Yellow Perch (Table 2-2). Twelve of the forty-four species were unique to this sampling method; electrofishing yielded specimens from all forty-four species captured in Green Bay in 2015. One Muskellunge captured using electrofishing was found to have a left vent clip (Appendix 2).

Fyke net and mini-fyke net sets were deployed in both targeted and randomly selected sites in the main bay areas of Green Bay. Total catch from 22 paired fyke nets was 2,676 fish from 25 species; CPUE was 121.64 ± 29.00 (mean ± SE) fish per paired net night and dominated by Yellow Perch and Gizzard Shad (Tables 2-1 and 2-2). Twenty-two mini-fyke nets yielded 3,689 fish with a CPUE of 167.68 ± 39.69 (mean ± SE) fish per paired net-night and consisted of 26 different species (Tables 2-1 and 2-2). Mini-fyke net samples were dominated by Yellow Perch, Round Goby, and Spottail Shiner.

Short-term (≤ 2 hours) benthic sets of large-mesh gillnets (N=26) and experimental micromesh gillnets (N=27) were performed throughout the study area. Large-mesh gillnets captured 697 fish averaging 26.81 ± 5.17 (mean ± SE) fish per set and included 9 species (Tables 2-1 and 2-2). Large-mesh gillnet catches were dominated by Freshwater Drum. Experimental micromesh gillnets captured an average of 83.52 ± 28.47 (mean ± SE) fish per set for a total of 2,255 fish comprised of 15 species (Tables 2-1 and 2-2). Micromesh gillnet catches were primarily comprised of Gizzard Shad, Yellow Perch, and White Bass.

Sixty-nine rock bags were deployed in sheltered shallow areas (< 4 m) throughout the study area in late August and early September. Rock bags were left to be colonized for one to two months then retrieved in late September and October. Forty-five rock bags were retrieved. On August 13th, 19 sediment samples were collected in areas of the bay that had soft substrates. Rock bag and sediment samples were dominated by Zebra and Quagga Mussels, Amphipods, and Chironomids.

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Light traps and bongo nets were used to sample larval fish and other ichthyoplankton for AIS in Green Bay as a collaborative effort between the GBFWCO and University of Wisconsin – Green Bay. A total of 35 bongo tows and 168 light traps were deployed in Green Bay and the Fox River from May 20th through August 13th, 2015. Bongo tows and light traps collected a total of 19,228 fish larvae and 49,626 eggs. Species captured include: Yellow Perch, Logperch, Common Carp, Freshwater Drum, Gizzard Shad, White Bass, Quillback, White Sucker, Round Goby, Burbot, Banded Killifish, Smallmouth Bass, Channel Catfish, and two other Cyprinid species only identifiable to genus – Pimephales spp. and Notropis spp.

Using 204 combined units of effort, we captured a total of 20,762 juvenile and adult fish from 44 species (Table 1). Known non-native species accounted for 7 of the 44 species present; no new non-native species were caught in the Green Bay sampling area. Size distributions of the ten most commonly encountered species are shown for all gears combined (Figure 2-2).

Using data from all traditional fish sampling gears, we calculated a species accumulation curve with associated 95% confidence intervals using the package vegan in R version 3.1.2. The order in which data was added to the analysis was randomized and 100 unique permutations were performed. The combined species accumulation curve for all gears indicates that our sampling most likely encountered the majority of species present in the study area (Figure 2-3). Extrapolated species richness in the Green Bay study area was estimated to be 45.9 ± 1.2 species (mean ± SE) using bootstrapping techniques, indicating that we likely collected 95.8% of the species present in the Green Bay study area. To capture the total predicted number of species present, we estimate that 220 total units of effort would be required.

Figure 2-1. Map of the Green Bay sampling area with locations of all sampling gears used during April 23rd through October 30th, 2015. 7

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Table 2-1. Total catch of 44 species collected using five gears in the Green Bay sampling area during April 23rd to October 30, 2015. Light trap deployments did not count towards total effort.

Gear type

Electro- Large-mesh Micromesh Paired Paired mini- Grand

Species

fishing

Gillnet

Gillnet fyke net

fyke net

Total

Alewife 40 1 163 123 5 332

Banded Killifish 10 - - - - 10

Bigmouth Buffalo 7 - - - - 7

Black Bullhead 1 - - - - 1

Black Crappie 15 - - 218 115 348

Bluegill 24 - - 2 75 101

Bluntnose Minnow 4 - - - 1 5

Bowfin 5 - - 4 - 9

Brown Bullhead 2 - - 2 2 6

Burbot 109 - - - 1 110

Channel Catfish 15 43 1 1 1 61

Chinook Salmon 1 - - - - 1

Common Carp 293 - - 6 1 300

Common Shiner 9 - 1 - 2 12

Emerald Shiner 837 - 15 1 17 870

Fathead Minnow 8 - - - - 8

Flathead Catfish 22 1 - - - 23

Freshwater Drum 269 568 44 27 2 910

Gizzard Shad 6247 22 1095 486 32 7882

Golden Shiner 2 - - - - 2

Green Sunfish 5 - - 1 1 7

Lake Whitefish 20 - - - - 20

Largemouth Bass 25 - - - 7 32

Logperch 35 - - - 2 37

Longnose Gar 5 - - - - 5

Mottled Sculpin 1 - - - - 1

Muskellunge 5 - - - - 5

Northern Pike 10 - - 2 - 12

Pumpkinseed 1 - - 6 12 19

Quillback 1 1 - 3 - 5

Rock Bass 52 - - 10 5 67

Round Goby 39 - 4 67 994 1104

Shortnose Gar 9 - - 1 - 10

Smallmouth Bass 222 - - 2 2 226

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Smallmouth Buffalo 3 - - - - 3

Spotfin Shiner 1 - 2 - - 3

Spottail Shiner 26 - 80 26 589 721

Trout-perch 6 - 5 17 16 44

Walleye 201 43 15 12 1 272

White Bass 73 17 257 122 14 483

White Perch 230 - 15 101 14 360

White Sucker 11 1 1 5 2 20

Yellow Bullhead 1 - - - - 1

Yellow Perch 2543 - 557 1431 1776 6307

Grand Total

11445

697

2255

2676

3689

20762

* Table continued from previous page

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Table 2-2. Catch per unit (mean ± SE) for 44 fish species sampled with five gears in the Green Bay study sampling area during April 23rd to October 30th 2015.

Gear type

Electro- Large-mesh Micromesh Paired Paired mini- fishing Gillnet Gillnet fyke net fyke net

Species (fish/run) (fish/net) (fish/net) (fish/net night) (fish/net night)

Alewife 0.37 ± 0.23 0.04 ± 0.04 6.04 ± 1.25 5.59 ± 1.96 0.23 ± 0.09

Banded Killifish 0.09 ± 0.04 - - - -

Bigmouth Buffalo 0.07 ± 0.04 - - - -

Black Bullhead 0.01 ± 0.01 - - - -

Black Crappie 0.14 ± 0.08 - - 9.91 ± 2.93 5.23 ± 2.75

Bluegill 0.22 ± 0.09 - - 0.09 ± 0.06 3.41 ± 1.26

Bluntnose Minnow 0.04 ± 0.03 - - - 0.05 ± 0.04

Bowfin 0.05 ± 0.02 - - 0.18 ± 0.08 -

Brown Bullhead 0.02 ± 0.01 - - 0.09 ± 0.09 0.09 ± 0.06

Burbot 1.02 ± 0.20 - - - 0.05 ± 0.04

Channel Catfish 0.14 ± 0.06 1.65 ± 0.74 0.04 ± 0.04 0.05 ± 0.04 0.05 ± 0.04

Chinook Salmon 0.01 ± 0.01 - - - -

Common Carp 2.74 ± 0.34 - - 0.27 ± 0.15 -

Common Shiner 0.08 ± 0.04 - 0.04 ± 0.04 - 0.09 ± 0.09

Emerald Shiner 7.82 ± 2.34 - 0.56 ± 0.24 0.05 ± 0.04 0.77 ± 0.62

Fathead Minnow 0.07 ± 0.04 - - - -

Flathead Catfish 0.21 ± 0.05 0.04 ± 0.04 - - -

Freshwater Drum 2.51 ± 0.32 21.85 ± 3.92 1.63 ± 0.24 1.23 ± 0.38 0.09 ± 0.06

Gizzard Shad 58.38 ± 16.11 0.85 ± 0.41 40.56 ± 10.04 22.09 ± 11.46 1.45 ± 0.57

Golden Shiner 0.01 ± 0.01 - - - -

Green Sunfish 0.05 ± 0.02 - - 0.05 ± 0.04 0.05 ± 0.04

Lake Whitefish 0.19 ± 0.14 - - - -

Largemouth Bass 0.23 ± 0.06 - - - 0.32 ± 0.16

Logperch 0.33 ± 0.10 - - - 0.09 ± 0.09

Longnose Gar 0.05 ± 0.02 - - - -

Mottled Sculpin 0.01 ± 0.01 - - - -

Muskellunge 0.05 ± 0.03 - - - -

Northern Pike 0.09 ± 0.04 - - 0.09 ± 0.06 -

Pumpkinseed 0.01 ± 0.01 - - 0.27 ± 0.22 0.55 ± 0.45

Quillback 0.01 ± 0.01 0.04 ± 0.04 - 0.14 ± 0.07 -

Rock Bass 0.49 ± 0.11 - - 0.45 ± 0.17 0.23 ± 0.11

Round Goby 0.36 ± 0.11 - 0.15 ± 0.07 3.05 ± 0.92 45.18 ± 10.68

Shortnose Gar 0.08 ± 0.05 - - 0.05 ± 0.04 -

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Smallmouth Bass 2.07 ± 0.35 - - 0.09 ± 0.09 0.09 ± 0.06

Smallmouth Buffalo 0.03 ± 0.02 - - - -

Spotfin Shiner 0.01 ± 0.01 - 0.07 ± 0.07 - -

Spottail Shiner 0.24 ± 0.08 - 2.96 ± 1.28 1.18 ± 0.73 26.77 ± 12.74

Trout-perch 0.02 ± 0.01 - 0.19 ± 0.07 0.77 ± 0.71 0.73 ± 0.67

Walleye 1.88 ± 0.28 1.65 ± 0.45 0.56 ± 0.19 0.55 ± 1.92 0.05 ± 0.04

White Bass 0.68 ± 0.22 0.65 ± 0.28 9.52 ± 6.53 5.55 ± 1.92 0.64 ± 0.34

White Perch 2.15 ± 0.85 - 0.56 ± 0.21 4.59 ± 0.96 0.64 ± 0.28

White Sucker 0.10 ± 0.04 0.04 ± 0.04 0.04 ± 0.04 0.23 ± 0.16 0.09 ± 0.09

Yellow Bullhead 0.01 ± 0.01 - - - -

Yellow Perch 23.77 ± 4.64 - 20.63 ± 13.34 65.05 ± 25.17 80.73 ± 29.37

Grand Total

106.96 ± 18.26

26.81 ± 5.17

83.52 ± 28.47

121.64 ± 29.00

167.68 ± 39.69

* Table continued from previous page

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Figure 2-2. Histograms of length-frequency for the ten most abundant species collected across all gear types at Green Bay during April 23rd – October 30th, 2015. Note that axes are different across species.

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Figure 2-3. Species accumulation curve (solid line) for five gears used to sample the near-shore zone of Lake Michigan in Green Bay during 2015 shown with 95% confidence intervals (dashed lines). Site data was added in a random order and subjected to 100 unique permutations. The extrapolated species richness (Asymptote; mean ± SE) at Green Bay (dotted line) was estimated using bootstrapping techniques. We estimate that 95.9% of fish species present at Green Bay were collected during traditional gear sampling.

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Milwaukee River Estuary The Milwaukee River Estuary was sampled August 17th - 30th, September 30th, and October 28th as part of the 2015 AIS early detection and monitoring program. Sampling encompassed approximately 8 km along the Milwaukee Harbor shoreline and several km of the Milwaukee River (Figure 2-4).

Forty-seven electrofishing runs (≈ 10 min each) were completed around sand flats, rip-rap break walls, weed beds and harbors. Electrofishing yielded 1,425 fish from 34 species (Table 2-3). Catch per unit effort (CPUE) was 30.32 ± 4.86 (mean ± SE) fish per run and was dominated by Alewife, White Sucker, and Gizzard Shad (Table 2-4). Twenty-one of the 37 species were unique to this sampling method. Three Lake Trout captured by electrofishing were found to have fin clips (Appendix 2).

Fyke nets and mini-fyke nets were deployed in the harbors and extensive sand flats. The total catch from 16 paired fyke nets was 409 fish from 11 species; CPUE was 25.56 ± 4.96 (mean ± SE) fish per paired net night and dominated by Rock Bass, Alewife, and Round Goby (Tables 2-3 and 2-4). Sixteen mini-fyke nets yielded 880 fish with a CPUE of 55.00 ± 14.79 (mean ± SE) fish/paired net-night. Catch was dominated by Round Goby and Rock Bass (Tables 2-3 and 2-4). Black Bullheads were only captured with this gear.

Large-mesh gillnets (N=7) captured 2.71 ± 0.63 (mean ± SE) per net set for a total of 20 fish comprised of five species (Tables 2-3 and 2-4). The only Lake Sturgeon and Lake Whitefish caught during the survey were captured in

Dalton Hendricks (USFWS) with a Brown Trout captured using electrofishing.

large-mesh gillnets. Micromesh experimental gillnets (N=10) captured 2.70 ± 1.60 (mean ± SE) fish per net set for a total of 27 fish comprised of 2 species (Tables 2-3 and 2-4). All fish captured with micromesh gillnets were either Alewife or Round Goby. Large-mesh gillnets captured 16 fin clipped fish: 15 Lake Trout and 1 Lake Sturgeon (Appendix 2).

Twenty rock bags were deployed August 17th-20th in shallow sandy areas and rip-rap break walls (< 4 m) throughout the harbor portion of the study area. Several rock bags were retrieved on September 30th and the rest of the bags were retrieved on October 28th. Only seven rock bag samples were recovered. The rest of the bags were lost due to human tampering and large storm events. Rock bag samples were dominated by Zebra and Quagga Mussels, Amphipods, and Chironomids. Sixteen light traps were also deployed; one light trap caught two juvenile Round Goby.

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Using 96 combined units of effort (i.e., fyke nets, gill nets, boat electrofishing), a total of 2,761 juvenile and adult fish from 37 species were captured (Table 2-3). Non-native species accounted for 8 of the 37 species present; no new non-native species were caught in the Milwaukee River Estuary. A single male Grass Carp (895-mm TL, 10.5 kg), an invasive species of concern, was captured in the Milwaukee River using electrofishing (location: 43.05687, -087.89639). This fish was further evaluated by USFWS personnel at the La Crosse Fish Health Center and determined to be diploid. Natal origins of the grass carp are being investigated by USGS personnel from the Columbia Environmental Research Center and Great Lakes Science Center using hard-part microchemistry; however, preliminary findings suggest that this fish is likely not hatchery-derived. Size distribution of the ten most commonly encountered species are shown for all gears combined (Figure 2-5).

Using data from all traditional fish sampling gears, we calculated a species accumulation curve with associated 95% confidence intervals using the package vegan in R version 3.1.2. The order in which data was added to the analysis was randomized and 100 unique permutations were performed. The combined species accumulation curve for all gears indicates that our sampling most likely encountered the majority of species present in the study area (Figure 2-6). Extrapolated species richness in the Milwaukee River Estuary was estimated to be 41.10 ± 1.90 species (mean ± SE) using bootstrapping techniques, indicating that we likely collected 90.0% of the species present in the Milwaukee River Estuary. To capture the total predicted number of species present, we estimate that 157 total units of effort would be required.

Figure 2-4. Map of the Milwaukee River estuary sampling area with locations of all sampling gears used during August 17th through October 30th, 2015.

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Table 2-3. Total catch of 37 species collected using five gears in the Milwaukee River Estuary sampling area during August 17th through October 30th, 2015.

Gear type

Electro- Large-mesh Micromesh Paired Paired mini- Grand

Species fishing Gillnet Gillnet fyke net fyke net Total

Alewife 401 1 12 81 5 500

Black Bullhead - - - - 2 2

Bluegill 37 - - 21 17 75

Bluntnose Minnow 15 - - - - 15

Brown Bullhead 3 - - 7 - 10

Brown Trout 76 - - - - 76

Chinook Salmon 2 - - - - 2

Coho Salmon 7 - - - - 7

Common Carp 24 - - - - 24

Emerald Shiner 17 - - - - 17

Fathead Minnow 2 - - - - 2

Freshwater Drum 2 - - - - 2

Gizzard Shad 224 - - - - 224

Golden Redhorse 5 - - - - 5

Golden Shiner 33 - - - - 33

Grass Carp 1 - - - - 1

Greater Redhorse 1 - - - - 1

Green Sunfish 6 - - 1 1 8

Lake Sturgeon - 1 - - - 1

Lake Trout 3 16 - - - 19

Lake Whitefish - 1 - - - 1

Largemouth Bass 23 - - 5 9 37

Longnose Dace 1 - - - - 1

Longnose Sucker 1 - - - - 1

Northern Pike 7 - - - - 7

Pumpkinseed 7 - - 3 2 12

Quillback 1 - - - - 1

Rainbow Trout 29 - - - - 29

River Redhorse 7 - - - - 7

Rock Bass 128 - - 152 132 412

Round Goby 32 - 15 77 668 792

Silver Redhorse 7 - - - - 7

Smallmouth Bass 25 - - 2 8 35

Spottail Shiner 6 - - - 28 34

Walleye 2 - - - - 2

18

White Sucker 283 1 - 52 8 344

Yellow Perch 7 - - 8 - 15

Grand Total

1425

20

27

409

880

2761

* Table continued from previous page

19

Table 2-4. Catch per unit effort (mean ± SE) for 37 fish species sampled with five gears in the Milwaukee River Estuary study sampling area during August 17th through October 30th, 2015.

Gear type Electro- Large-mesh Micromesh Paired Paired mini- fishing Gillnet Gillnet fyke net fyke net

Species (fish/run) (fish/net) (fish/net) (fish/net night) (fish/net night)

Alewife 8.53 ± 3.76 0.14 ± 0.13 1.20 ± 0.81 5.06 ± 2.45 0.31 ± 0.19

Black Bullhead - - - - 0.13 ± 0.08

Bluegill 0.79 ± 0.68 - - 1.31 ± 0.82 1.06 ± 0.85

Bluntnose Minnow 0.32 ± 0.32 - - - -

Brown Bullhead 0.06 ± 0.04 - - 0.44 ± 0.31 -

Brown Trout 1.62 ± 0.36 - - - -

Chinook Salmon 0.04 ± 0.03 - - - -

Coho Salmon 0.15 ± 0.11 - - - -

Common Carp 0.51 ± 0.13 - - - -

Emerald Shiner 0.36 ± 0.34 - - - -

Fathead Minnow 0.04 ± 0.04 - - - -

Freshwater Drum 0.04 ± 0.03 - - - -

Gizzard Shad 4.74 ± 1.21 - - - -

Golden Redhorse 0.11 ± 0.04 - - - -

Golden Shiner 0.70 ± 0.46 - - - -

Grass Carp 0.02 ± 0.02 - - - -

Greater Redhorse 0.02 ± 0.02 - - - -

Green Sunfish 0.13 ± 0.06 - - 0.06 ± 0.06 0.06 ± 0.06

Lake Sturgeon - 0.14 ± 0.13 - - -

Lake Trout 0.06 ± 0.04 2.29 ± 0.66 - - -

Lake Whitefish - 0.14 ± 0.13 - - -

Largemouth Bass 0.49 ± 0.18 - - 0.31 ± 0.25 0.56 ± 0.34

Longnose Dace 0.02 ± 0.02 - - - -

Longnose Sucker 0.02 ± 0.02 - - - -

Northern Pike 0.15 ± 0.07 - - - -

Pumpkinseed 0.15 ± 0.07 - - 0.19 ± 0.10 0.13 ± 0.08

Quillback 0.02 ± 0.02 - - - -

Rainbow Trout 0.60 ±0.19 - - - -

River Redhorse 0.13 ± 0.06 - - - -

Rock Bass 2.72 ± 1.21 - - 9.50 ± 3.81 8.25 ± 3.18

Round Goby 0.68 ± 0.31 - 1.50 ± 0.82 4.81 ± 1.79 41.75 ± 14.95

Silver Redhorse 0.15 ± 0.09 - - - -

Smallmouth Bass 0.53 ± 0.18 - - 0.13 ± 0.12 0.50 ± 0.18

Spottail Shiner 0.13 ± 0.07 - - - 1.75 ± 1.69

20

Walleye 0.04 ± 0.03 - - - -

White Sucker 6.15 ± 1.99 0.14 ± 0.13 - 3.25 ± 1.08 0.50 ± 0.27

Yellow Perch 0.15 ± 0.09 - - 0.50 ± 0.29 -

Grand Total 30.32 ± 4.86 2.71 ± 0.63 2.70 ± 1.60 25.56 ± 4.96 55.00 ± 14.79

* Table continued from previous page

21

Figure 2-5. Histograms of length-frequency for the ten most abundant species collected across all gear types at Milwaukee River Estuary during August 17th through October 30th, 2015. Note that axes are different across species.

22

Figure 2-6. Species accumulation curve (solid line) for five gears used to sample the near-shore zone of Lake Michigan in the Milwaukee River Estuary during 2015 shown with 95% confidence intervals (dashed lines). Site data was added in a random order and subjected to 100 unique permutations. The extrapolated species richness (Asymptote; mean ± SE) in the Milwaukee River estuary (dotted line) was estimated using bootstrapping techniques. We estimate that 90.0% of fish species present at Burns Harbor were collected during traditional gear sampling.

23

Chicago Harbor The Chicago Harbor study area was sampled during September 21st – 23rd as part of the 2015 AIS early detection and monitoring program. Sampling encompassed a 10.5 km stretch of shoreline along the waterfront of downtown Chicago from the First Street Harbor to North Avenue Beach (Figure 2-7).

Forty-one electrofishing runs (≈ 10 min each) were completed around sand flats and rip-rap break walls. Electrofishing yielded 514 fish from 20 species (Table 2-5). Catch per unit effort (CPUE) was 12.50 ± 2.09 fish/run (mean ± SE) and was dominated by Smallmouth Bass, Gizzard Shad, and Common Carp (Table 2-6). Nine of the 28 species were unique to this sampling method. Nine fin-clipped Rainbow Trout were also captured (Appendix 1).

Fyke nets and mini-fyke nets were deployed in shallow areas throughout the study area. The total catch from 12 paired fyke nets was 196 fish from 12 species; CPUE was 16.30 ± 3.69 fish/paired net night (mean ± SE) and Rock Bass comprised 75% of the catch (Tables 2-5 and 2- 6). Three species were unique to this gear; Black Bullhead, Bluegill, and Yellow Bullhead. Twelve mini-fyke nets yielded 473 fish from eight species with a CPUE of 39.40 ± 28.65 fish/paired net-night (mean ± SE); Spottail Shiner comprised 73% of the catch (Tables 2-5 and 2- 6). The only Longnose Sucker of the survey was captured with this gear.

Thirteen short-term sets of large-mesh gillnets and an equivalent number of micromesh gillnets were performed throughout the study area. The nets were concentrated between the First Street Harbor and the North break wall of Chicago Harbor. Large-mesh gillnets captured 43 fish at 3.30 ± 1.02 fish/set (mean ± SE) and included five species (Tables 2-5 and 2-6). The only Channel Catfish (N=4) of the survey were captured with this gear. Freshwater Drum (40%) and Gizzard Shad (44%) were the most common species captured using large-mesh gillnets. Experimental micromesh gillnets captured 57 fish with a CPUE of 4.40 ± 1.51 fish/set (mean ± SE) and included 5 species (Tables 2-5 and 2-6). The only Chinook Salmon encountered during this survey (N=2) were collected using this gear. Nearly the entire catch was comprised of Alewife (19%), Spottail Shiners (42%), and Yellow Perch (30%).

Eighteen minnow trap arrays collected 469 fish comprised of five species with a CPUE of 26.10 ± 5.20 fish/array (mean ± SE; tables 5-6). Round Goby (64%) and Spottail Shiner (29%) were the most common species collected. Five Lake Chub, an infrequently encountered native species, were also collected.

Twenty rock bags were deployed on September 22nd along the outer break wall of the First Street Harbor at the Southern end of the study area. Rock bags were deployed for approximately one

24

month, with an attempt to retrieve them on October 22nd. No rock bag samples were recovered due to burial by sediment during high wave events.

Using 109 combined units of effort, a total of 1,752 fish from 28 species were captured (Table 2- 5). Five light traps were set; although, failed to capture any fish. Non-native species accounted for 8 of the 28 species present; no new non-native species were caught in the Chicago Harbor sampling area. Size distribution of the ten most commonly encountered species are shown for all gears combined (Figure 2-8).

Using data from all traditional fish sampling gears, we calculated a species accumulation curve with associated 95% confidence intervals using the package vegan in R version 3.1.2. The order in which data was added to the analysis was randomized and 100 unique permutations were performed. The combined species accumulation curve for all gears indicates that our sampling most likely encountered the majority of species present in the study area (Figure 2-9). Extrapolated species richness in the Chicago Harbor study area was estimated to be 31.40 ± 1.90 species (mean ± SE) using bootstrapping techniques, indicating that we likely collected 89.2% of the species present. To capture the total predicted number of species present, we estimate that 209 total units of effort would be required.

Figure 2-7. Map of the Chicago Harbor sampling area with locations of all sampling gears used during September 21 - 23, 2015.

25

26

Table 2-5. Total catch of 28 species collected using six gears in the Chicago Harbor sampling area during September 21 - 23, 2015.

Gear type

Electro- Large-mesh Micromesh Fyke Mini- Minnow Grand Species fishing gillnet gillnet net fyke net trap Total

Alewife 10 - 11 21 - - 42

Black Bullhead - - - 1 - - 1

Bluegill - - - 2 - - 2

Brown Trout 2 - - - - - 2

Brown Bullhead - 2 - 2 - - 4

Channel Catfish - 4 - - - - 4

Chinook Salmon - - 2 - - - 2

Common Carp 60 - - - - - 60

Freshwater Drum 5 17 - - - - 22

Gizzard Shad 229 19 - - - - 248

Goldfish 1 - - - - - 1

Lake Chub 8 - - - - 5 13

Largemouth Bass 3 - - - 2 - 5

Longnose Dace 2 - - - - - 2

Longnose Sucker - - - - 1 - 1

Pumpkinseed 1 - - - - - 1

Rainbow Trout 10 - - - - - 10

Rock Bass 16 - - 147 60 22 245

Round Goby 20 - 3 12 37 298 370 Shorthead Redhorse 17 - - - - - 17

Silver Redhorse 2 - - - - - 2

Smallmouth Bass 99 - - 1 4 - 104

Spottail Shiner 11 - 24 2 345 136 518

Walleye 4 - - - - - 4

White Perch 3 - - 1 - - 4

White Sucker 11 - - 4 1 - 16

Yellow Bullhead - - - 1 - - 1

Yellow Perch - 1 17 2 23 8 51

Grand Total

514

43

57

196

473

469

1752

27

Table 2-6. Catch per unit effort (mean ± SE) for 28 fish species sampled with six gears in the Chicago Harbor study sampling area during September 21 - 23, 2015.

Gear type

Species

Electro-

fishing (fish/run)

Large-mesh

gillnet (fish/set)

Micromesh

gillnet (fish/set)

Paired

fyke net (fish/net night)

Paired mini-

fyke net (fish/net night)

Minnow

trap (fish/array)

Alewife 0.24 ± 0.10 - 0.85 ± 0.34 1.75 ± 0.61 - -

Black Bullhead - - - 0.08 ± 0.08 - -

Bluegill - - - 0.17 ± 0.16 - -

Brown Trout 0.05 ± 0.05 - - - - -

Brown Bullhead - 0.15 ± 0.10 - 0.17 ± 0.11 - -

Channel Catfish - 0.31 ± 0.17 - - - -

Chinook Salmon - - 0.15 ± 0.10 - - -

Common Carp 1.46 ± 0.44 - - - - -

Freshwater Drum 0.12 ± 0.06 1.31 ± 0.30 - - - -

Gizzard Shad 5.59 ± 1.33 1.46 ± 1.12 - - - -

Goldfish 0.02 ± 0.02 - - - - -

Lake Chub 0.20 ± 0.13 - - - - 0.28 ± 0.22

Largemouth Bass 0.07 ± 0.05 - - - 0.17 ± 0.11 -

Longnose Dace 0.05 ± 0.05 - - - - -

Longnose Sucker - - - - 0.08 ± 0.08 -

Pumpkinseed 0.02 ± 0.02 - - - - -

Rainbow Trout 0.24 ± 0.09 - - - - -

Rock Bass 0.39 ± 0.12 - - 12.25 ± 3.65 5.00 ± 1.11 1.22 ± 0.86

Round Goby 0.49 ± 0.15 - 0.23 ± 0.17 1.00 ± 0.37 3.08 ± 1.06 16.56 ± 4.59

Shorthead Redhorse 0.41 ± 0.17 - - - - -

Silver Redhorse 0.05 ± 0.03 - - - - -

Smallmouth Bass 2.41 ± 0.66 - - 0.08 ± 0.08 0.33 ± 0.18 -

Spottail Shiner 0.27 ± 0.15 - 1.85 ± 0.53 0.17 ± 0.11 28.75 ± 27.53 7.56 ± 3.16

Walleye 0.10 ± 0.06 - - - - -

White Perch 0.07 ± 0.04 - - 0.08 ± 0.08 - -

White Sucker 0.27 ± 0.11 - - 0.33 ± 0.18 0.08 ± 0.08 -

Yellow Bullhead - - - 0.08 ± 0.08 - - Yellow Perch - 0.08 ± 0.07 1.31 ± 1.15 0.17 ± 0.11 1.92 ± 1.42 0.44 ± 0.18

28

Figure 2-8. Histograms of length-frequency for the ten most abundant species collected across all gear types at Chicago Harbor during September 21 - 23, 2015. Note that axes are different across species.

29

Figure 2-9. Species accumulation curve (solid line) for six gears used to sample the near-shore zone of Lake Michigan at Chicago Harbor during 2015 shown with 95% confidence intervals (dashed lines). Site data was added in a random order and subjected to 100 unique permutations. The extrapolated species richness (Asymptote; mean ± SE) at Chicago Harbor (dotted line) was estimated using bootstrapping techniques. We estimate that 89.2% of fish species present at Chicago Harbor were collected during traditional gear sampling.

30

Calumet/Indiana Harbors

The Port of Chicago - Calumet Harbor, hereafter referred to as Calumet Harbor, was sampled during September 14th – 17th as part of the 2015 AIS early detection and monitoring program. Sampling encompassed a 10.7 km reach along the shoreline from Calumet Harbor to Indiana Harbor (Figure 2-10).

Fifty-two electrofishing runs (≈ 10 min each) were completed around sand flats, rip-rap break walls, and harbors. Electrofishing yielded 572 fish from 30 species (Table 2-7). Catch per unit effort (CPUE) was 11.00 ± 1.63 fish/run (mean ± SE) and was dominated by Smallmouth Bass, Gizzard Shad, Common Carp, and Alewife (Table 2-8). Fourteen of the 33 species were unique to this sampling method.

Fyke nets and mini-fyke nets were deployed in the harbors and extensive sand flats. The total catch from 12 paired fyke nets was 245 fish from 12 species; CPUE was 20.42 ± 5.64 (mean ± SE) fish/paired net night and dominated by Rock Bass and Round Goby (Tables 2-7 and 2-8). No unique species were captured with paired fyke nets. Twelve mini-fyke nets yielded 1,375 fish from nine species with a CPUE of 114.50 ± 71.40 (mean ± SE) fish/paired net-night. Catch was dominated by Yellow Perch (69%) and Round Goby (24%) (Tables 2-7 and 2-8). Banded Killifish was unique to this gear.

Large-mesh and micromesh gillnets were set throughout the study area primarily on sandy substrates. Large-mesh gillnets (N=13) captured 22 fish of six species with a CPUE of 1.69 ± 0.57 fish/set (mean ± SE; tables 2-7 and 2-8). The only Flathead Catfish of the survey was captured in a large-mesh gillnet. Freshwater Drum were the most commonly captured species when using large-mesh gillnets. Micromesh experimental gillnets (N=14) captured 10.29 ± 1.65 fish/set (mean ± SE) for a total of 144 fish comprised of seven species (Tables 2-7 and 2-8). Nearly all fish captured with micromesh gillnets were either Alewives or Spottail Shiners and no unique species were captured with this gear.

Twelve minnow trap arrays collected 119 fish comprised of five species with a CPUE of 9.92 ± 3.83 fish/array (mean ± SE; Tables 2-7 and 2-8). Round Goby represented 82% of the total catch. Other species collected included: Emerald Shiner, Rock Bass, Spottail Shiner, and Yellow Perch.

During the week of September 14th – 17th, 20 rock bags were deployed in shallow sandy areas and rip-rap break walls (< 4 m) throughout the eastern half of the study area. Rock bags were deployed for approximately one month then retrieved on October 22nd. Only two rock bag samples were recovered due to burial by sediment during high wave events. Rock bag samples were dominated by Zebra and Quagga Mussels, Amphipods, and Chironomids.

31

Using 115 combined units of traditional gear effort, a total of 2,477 fish from 33 species were captured (Table 2-7). Sixteen light traps were deployed; although, failed to capture any fish. Non-native species accounted for 9 of the 33 species present; no new non-native species were caught in the Calumet Harbor sampling area. Size distribution of the ten most commonly encountered species are shown for all gears combined (Figure 2-11).

Using data from all traditional fish sampling gears, we calculated a species accumulation curve with associated 95% confidence intervals using the package vegan in R version 3.1.2. The order in which data was added to the analysis was randomized and 100 unique permutations were performed. The combined species accumulation curve for all gears indicates that our sampling most likely encountered the majority of species present in the study area (Figure 2-12). Extrapolated species richness in the Calumet Harbor study area was estimated to be 37.2 ± 1.9 species (mean ± SE) using bootstrapping techniques, indicating that we likely collected 88.7% of the species present in the Calumet Harbor study area. To capture the total predicted number of species present, we estimate that 223 total units of effort would be required.

Figure 2-10. Map of the Calumet Harbor sampling area with locations of all sampling gears used during September 14 - 17, 2015.

32

33

Table 2-7. Total catch of 33 species collected using six gears in the Calumet Harbor sampling area during September 14 - 17, 2015.

Gear type

Electro- Large-mesh Micromesh Paired Paired mini- Minnow Grand Species fishing gillnet gillnet fyke net fyke net trap Total

Alewife 66 1 59 4 2 - 132

Banded Killifish - - - - 1 - 1

Black Buffalo 5 - - - - - 5

Black Crappie - - - 13 3 - 16

Bluegill 1 - - 17 13 - 31

Bluntnose Minnow 5 - - - - - 5

Brown Trout 2 - - - - - 2

Brown Bullhead 1 - - 3 - - 4

Brook Silverside 1 - - - - - 1

Channel Catfish 3 1 - - - - 4

Chestnut Lamprey 1 - - - - - 1

Chinook Salmon 1 - - - - - 1

Common Carp 74 - - - - - 74

Emerald Shiner 1 - - - - 1 2

Flathead Catfish - 1 - - - - 1

Freshwater Drum 3 13 - - - - 16

Gizzard Shad 62 5 - 5 - - 72

Goldfish 3 - - 1 - - 4

Green Sunfish 4 - - - - - 4

Largemouth Bass 2 - - - - - 2

Pumpkinseed 7 - 1 19 7 - 34

Rainbow Trout 2 - - - - - 2

Rock Bass 25 - - 126 45 2 198

Round Goby 30 - 3 48 331 98 510

Shorthead Redhorse 15 - - - - - 15

Smallmouth Buffalo 2 - - - - - 2

Smallmouth Bass 198 - 1 2 25 - 226

Spottail Shiner 32 - 75 - - 14 121

Walleye 1 - - - - - 1

White Bass 1 - - - - - 1

White Perch 1 - 1 - - - 2

White Sucker 1 1 - 2 - - 4

Yellow Perch 22 - 4 5 948 4 983

Grand Total

572

22

144

245

1375

119

2477

34

Table 2-8. Catch per unit effort (mean ± SE) for 33 fish species sampled with six gears in the Calumet Harbor study sampling area during September 14 - 17, 2015.

Gear type

Electro- Large-mesh Micromesh Paired Paired mini- Minnow fishing gillnet gillnet fyke net fyke net trap

Species

(fish/run)

(fish/set)

(fish/set) (fish/net night)

(fish/net night)

(fish/array)

Alewife 1.27 ± 0.60 0.08 ± 0.07 4.21 ± 1.40 0.33 ± 0.32 0.17 ± 0.16 -

Banded Killifish - - - - 0.08 ± 0.08 -

Black Buffalo 0.10 ± 0.05 - - - - -

Black Crappie - - - 1.08 ± 0.81 0.25 ± 0.17 -

Bluegill 0.02 ± 0.02 - - 1.42 ± 1.04 1.08 ± 0.48 -

Bluntnose Minnow 0.10 ± 0.08 - - - - -

Brown Trout 0.04 ± 0.03 - - - - -

Brown Bullhead 0.02 ± 0.02 - - 0.25 ± 0.17 - -

Brook Silverside 0.02 ± 0.02 - - - - -

Channel Catfish 0.06 ± 0.03 0.08 ± 0.07 - - - -

Chestnut Lamprey 0.02 ± 0.02 - - - - -

Chinook Salmon 0.02 ± 0.02 - - - - -

Common Carp 1.42 ± 0.34 - - - - -

Emerald Shiner 0.02 ± 0.02 - - - - 0.08 ± 0.08

Flathead Catfish - 0.08 ± 0.07 - - - -

Freshwater Drum 0.06 ± 0.03 1.00 ± 0.60 - - - -

Gizzard Shad 1.19 ± 0.32 0.38 ± 0.23 - 0.42 ± 0.25 - -

Goldfish 0.06 ± 0.03 - - 0.08 ± 0.08 - -

Green Sunfish 0.08 ± 0.08 - - - - -

Largemouth Bass 0.04 ± 0.03 - - - - -

Pumpkinseed 0.13 ± 0.06 - 0.07 ± 0.07 1.58 ± 1.07 0.58 ± 0.32 -

Rainbow Trout 0.04 ± 0.03 - - - - -

Rock Bass 0.48 ± 0.20 - - 10.50 ± 3.29 3.75 ± 1.83 0.17 ± 0.11

Round Goby 0.58 ± 0.20 - 0.21 ± 0.11 4.00 ± 1.66 27.58 ± 15.50 8.17 ± 3.65

Shorthead Redhorse 0.29 ± 0.11 - - - - -

Smallmouth Buffalo 0.04 ± 0.04 - - - - -

Smallmouth Bass 3.81 ± 0.63 - 0.07 ± 0.07 0.17 ± 0.11 2.08 ± 0.36 -

Spottail Shiner 0.62 ± 0.53 - 5.36 ± 0.84 - - 1.17 ± 0.55

Walleye 0.02 ± 0.02 - - - - -

White Bass 0.02 ± 0.02 - - - - -

White Perch 0.02 ± 0.02 - 0.07 ± 0.07 - - -

White Sucker 0.02 ± 0.02 0.08 ± 0.07 - 0.17 ± 0.11 - - Yellow Perch 0.42 ± 0.38 - 0.29 ± 0.16 0.42 ± 0.18 79.00 ± 56.88 0.33 ± 0.18

35

Figure 2-11. Histograms of length-frequency for the ten most abundant species collected across all gear types at Calumet Harbor during September 14 - 17, 2015. Note that axes are different across species.

36

Figure 2-12. Species accumulation curve (solid line) for six gears used to sample the near-shore zone of Lake Michigan at Calumet Harbor during 2015 shown with 95% confidence intervals (dashed lines). Site data was added in a random order and subjected to 100 unique permutations. The extrapolated species richness (Asymptote; mean ± SE) at Calumet Harbor (dotted line) was estimated using bootstrapping techniques. We estimate that 88.7% of fish species present at Calumet Harbor were collected during traditional gear sampling.

37

Burns Harbor

The Port of Indiana - Burns Harbor, hereafter referred to as Burns Harbor, was sampled for fishes during August 24th- 28th as part of the 2015 AIS early detection and monitoring program. Sampling encompassed a 7.5 km shoreline reach from Indiana Dunes National Park to just west of the Portage-Burns Waterway and centered on Burns Harbor (Figure 2-13).

Fifty-one electrofishing runs (≈ 10 min each) were conducted in diverse habitats including sand flats, rip-rap break walls, and riverine habitat. Electrofishing yielded 2,429 fish from 43 species (Table 2-9). Catch per unit effort (CPUE) was 47.60 ± 5.40 fish/run (mean ± SE) and was dominated by Gizzard Shad and juvenile Largemouth and Smallmouth Bass (Table 2-10). Twenty of the 51 species were unique to this sampling method. Two Rainbow Trout captured using electrofishing were found to have adipose, left pelvic, and right pelvic fin clips (Appendix 2).

Fyke net and mini-fyke net sets were deployed in the harbor, main lake shoreline, and river areas. Total catch from 14 paired fyke nets was 230 fish from 20 species; CPUE was 16.40 ± 11.27 (mean ± SE) fish per paired net night and dominated by Bluegill and Black Crappie (Tables 2-9 and 2-10). One species, White Sucker, was unique to this sampling method. Thirteen mini-fyke nets yielded 1,075 fish from 17 species with a CPUE of 82.70 ± 27.47 (mean ± SE) fish/paired net-night (Tables 2-9 and 2-10). Mini-fyke net samples were dominated by Bluegill, Round Goby, Spottail Shiner, and Yellow Perch. Johnny darter, a unique species, was only captured with this gear.

Short-term benthic sets of large-mesh gillnets (N=11) and experimental micromesh gillnets (N=12) were performed throughout the study area. Large-mesh gillnets captured 223 fish with a CPUE of 20.30 ± 6.51 (mean ± SE) fish/set and included 12 species, two of which, Brown Trout and Silver Redhorse, were not captured with any other gear (Tables 2-9 and 2-10). Large-mesh gillnet catches were dominated by Freshwater Drum and Gizzard Shad. Micromesh experimental gillnets captured 49.00 ± 10.75 (mean ± SE) fish/set for a total of 588 fish comprised of 10 species, including Bloater and Rainbow Smelt which were not captured with other gears (Tables 2-9 and 2-10). Micromesh gillnet catches were primarily comprised of Alewife and Spottail Shiner.

We deployed 20 rock bags in sheltered shallow areas (< 4 m) throughout the study area on August 25th including the Portage-Burns waterway and along the rip-rap inner break wall of Burns Harbor. Rock bags were left to be colonized for approximately two months then retrieved on October 22nd. Fifteen rock bag samples were recovered. Rock bag samples were dominated by Zebra and Quagga Mussels, Amphipods, and Chironomids.

38

Thirteen light trap deployments were made throughout the study area but concentrated in the calmer water of the Portage-Burns waterway and Burns Harbor itself. Only three fish were captured including: a 24 mm Bluegill, 16 mm Bluegill, and 52 mm Yellow Perch.

Using 101 combined units of traditional gear effort, we captured a total of 4,545 fish from 51 species and an additional 3 fish from two species using light traps (Table 2-9). Non-native species accounted for 11 of the 51 species present; no new non-native species were caught in the Port of Indiana Burns Harbor sampling area. A single, female Grass Carp (1154-mm TL, 21 kg), an invasive species of concern, was captured in Burns Harbor using electrofishing (location: 41.64180, -087.15295). This fish had no visible sign of eggs and was determined to be triploid by USFWS at the LaCrosse Fish Health Center. Size distributions of the ten most commonly encountered species are shown for all gears combined (Figure 2-14).

Using data from all traditional fish sampling gears, we calculated a species accumulation curve with associated 95% confidence intervals using the package vegan in R version 3.1.2. The order in which data was added to the analysis was randomized and 100 unique permutations were performed. The combined species accumulation curve for all gears indicates that our sampling most likely encountered the majority of species present in the study area (Figure 2-15). Extrapolated species richness in the Burns Harbor study area was estimated to be 55.90 ± 2.10 species (mean ± SE) using bootstrapping techniques, indicating that we collected 91.2% of the species present in the Burns Harbor study area. To capture the total predicted number of species present, we estimate that 159 total units of effort would be required.

Figure 2-13. Map of the Burns Harbor sampling area with locations of all sampling gears used during August 24 – 28, 2015.

39

40

Table 2-9. Total catch of 51 species collected using six gears in the Burns Harbor sampling area during August 24 – 28, 2015. Light trap deployments did not count towards total effort.

Gear type

Electro- Large-mesh Micromesh Paired Paired mini- Light Grand

Species

fishing

gill net

gill net fyke net

fyke net

trap

Total

Alewife 211 1 278 - - - 490

Banded Killifish 17 - - - - - 17

Black Bullhead 1 - - - - - 1

Black Crappie 7 - - 67 31 - 105

Bluegill 114 - - 105 173 2 394

Bloater - - 3 - - - 3

Bluntnose Minnow 2 - 1 - 8 - 11

Brown Trout - 2 - - - - 2

Bowfin 3 - - - - - 3

Brook Silverside 18 - - - - 18

Channel Catfish 11 9 - - - - 20

Chinook Salmon 1 - - - - - 1

Common Shiner 1 - - - - 1

Common Carp 79 1 - 1 - - 81

Coho Salmon 1 - - - - - 1

Emerald Shiner 14 - - - - 14

Flathead Catfish 1 1 - 1 1 - 4

Freshwater Drum 18 129 1 1 - - 149

Gizzard Shad 318 59 - 1 2 - 380

Goldfish 13 - - - - - 13

Golden Redhorse 14 - - - - - 14

Golden Shiner 73 - - - - - 73

Grass Carp 1 - - - - - 1

Grass Pickerel 18 - - - - - 18

Unknown Catostomidae 3 - - - - - 3

Green Sunfish 49 - - - 2 - 51

Johnny Darter - - - - 1 - 1

Largemouth Bass 455 - - 3 18 - 476

Longnose Sucker - 2 1 - - - 3

Logperch 33 - - - - - 33

Northern Pike 20 - - 2 - - 22

Orangespotted Sunfish - - - 1 2 - 3

Pumpkinseed 32 - - 9 5 - 46

Quillback 11 - - - - - 11

Rainbow Smelt - - 1 - - - 1 Rainbow Trout 7 - - - - - 7

41

Rock Bass 11 - - 3 2 - 16 Round Goby 75 - 8 4 148 - 235

Sand Shiner 3 - - - - - 3

Shorthead Redhorse 135 6 - - - - 141

Silver Redhorse - 1 - - - - 1

Smallmouth Buffalo 8 - - - - - 8

Smallmouth Bass 532 2 - 4 25 - 563

Spottail Shiner 94 - 258 5 373 - 730

Walleye 6 10 1 - - - 17

Warmouth 8 - - 3 9 - 20

White Bass 2 - - 1 - - 3

White Crappie 3 - - 3 18 - 24

White Sucker - - - 1 - - 1

Yellow Bullhead 1 - - - - - 1

Yellow Perch 5 - 36 15 257 1 314

Grand Total

2429

223

588

230

1075

3

4548

*Table continued from previous page

42

Table 2-10. Catch per unit (mean ± SE) for 51 fish species sampled with six gears in the Burns Harbor study sampling area during August 24 – 28, 2015.

Gear type

Electro- Large-mesh Micromesh Paired Paired mini- Light fishing gill net gill net fyke net fyke net trap

Species (fish/run) (fish/set) (fish/set) (fish/net night) (fish/net night) (fish/set)

Alewife 4.14 ± 1.38 0.09 ± 0.09 23.17 ± 6.08 - - -

Banded Killifish 0.33 ± 0.22 - - - - -

Black Bullhead 0.02 ± 0.02 - - - - -

Black Crappie 0.14 ± 0.06 - - 4.79 ± 3.67 2.38 ± 1.69 -

Bluegill 2.24 ± 1.03 - - 7.50 ± 6.93 13.31 ± 4.91 0.15 ± 0.10

Bloater - - 0.25 ± 0.13 - - -

Bluntnose Minnow 0.04 ± 0.04 - 0.08 ± 0.08 - 0.62 ± 0.46 -

Brown Trout - 0.18 ± 0.17 - - - -

Bowfin 0.06 ± 0.03 - - - - -

Brook Silverside 0.35 ± 0.15 - - - - -

Channel Catfish 0.22 ± 0.07 0.82 ± 0.31 - - - -

Chinook Salmon 0.02 ± 0.02 - - - - -

Common Shiner 0.02 ± 0.02 - - - - -

Common Carp 1.55 ± 0.37 0.09 ± 0.09 - 0.07 ± 0.07 - -

Coho Salmon 0.02 ± 0.02 - - - - -

Emerald Shiner 0.27 ± 0.14 - - - - -

Flathead Catfish 0.02 ± 0.02 0.09 ± 0.09 - 0.07 ± 0.07 0.08 ± 0.07 -

Freshwater Drum 0.35 ± 0.13 11.73 ± 3.42 0.08 ± 0.08 0.07 ± 0.07 - -

Gizzard Shad 6.24 ± 2.40 5.36 ± 4.55 - 0.07 ± 0.07 0.15 ± 0.10 -

Goldfish 0.25 ± 0.12 - - - - -

Golden Redhorse 0.27 ± 0.09 - - - - -

Golden Shiner 1.43 ± 0.53 - - - - -

Grass Carp 0.02 ± 0.02 - - - - -

Grass Pickerel 0.35 ± 0.12 - - - - -

Unknown Catostomidae 0.06 ± 0.06 - - - - -

Green Sunfish 0.96 ± 0.30 - - - 0.15 ± 0.10 -

Johnny Darter - - - - 0.08 ± 0.07 -

Largemouth Bass 8.92 ± 1.57 - - 0.21 ± 0.11 1.38 ± 0.80 -

Longnose Sucker - 0.18 ± 0.12 0.08 ± 0.08 - - -

Logperch 0.65 ± 0.22 - - - - -

Northern Pike 0.39 ± 0.12 - - 0.14 ± 0.09 - -

Orangespotted Sunfish - - - 0.07 ± 0.07 0.15 ± 0.15 -

Pumpkinseed 0.63 ± 0.23 - - 0.64 ± 0.37 0.38 ± 0.23 -

Quillback 0.22 ± 0.11 - - - - - Rainbow Smelt - - 0.08 ± 0.08 - - -

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Rainbow Trout 0.14 ± 0.05 - - - - - Rock Bass 0.22 ± 0.07 - - 0.21 ± 0.23 0.15 ± 0.10 -

Round Goby 1.47 ± 0.36 - 0.67 ± 0.34 0.29 ± 0.21 11.38 ± 5.37 -

Sand Shiner 0.06 ± 0.04 - - - - -

Shorthead Redhorse 2.65 ± 0.86 0.55 ± 0.27 - - - -

Silver Redhorse - 0.09 ± 0.09 - - - -

Smallmouth Buffalo 0.16 ± 0.06 - - - - -

Smallmouth Bass 10.43 ± 1.55 0.18 ± 0.17 - 0.29 ± 0.28 1.92 ± 1.62 -

Spottail Shiner 1.84 ± 0.72 - 21.50 ± 9.11 0.36 ± 0.34 28.69 ± 27.57 -

Walleye 0.12 ± 0.06 0.45 ± 0.47 0.08 ± 0.08 - - -

Warmouth 0.16 ± 0.08 - - 0.21 ± 0.21 0.69 ± 0.45 -

White Bass 0.04 ± 0.03 - - 0.07 ± 0.07 - -

White Crappie 0.06 ± 0.03 - - 0.21 ± 0.11 1.38 ± 1.33 -

White Sucker - - - 0.07 ± 0.07 - -

Yellow Bullhead 0.02 ± 0.02 - - - - - Yellow Perch 0.10 ± 0.06 - 1.07 ± 0.63 19.77 ± 10.36 0.08 ± 0.07

*Table continued from previous page

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Figure 2-14. Histograms of length-frequency for the ten most abundant species collected across all gear types at Burns Harbor during August 24 - 28, 2015. Note that axes are different across species.

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Figure 2-15. Species accumulation curve (solid line) for six gears used to sample the near-shore zone of Lake Michigan at Burns Harbor during 2015 shown with 95% confidence intervals (dashed lines). Site data was added in a random order and subjected to 100 unique permutations. The extrapolated species richness (Asymptote; mean ± SE) at Burns Harbor (dotted line) was estimated using bootstrapping techniques. We estimate that 91.2% of fish species present at Burns Harbor were collected during traditional gear sampling.

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Macroinvertebrate sampling Some of the most dramatic ecological changes in Lake Michigan during the last few decades have been concentrated in the benthic community of the near shore zone. First Zebra Mussel Dreissena polymorpha then Quagga Mussel Dreissena bugensis invaded the lake transforming water quality, decimating the historic spring phytoplankton bloom and concentrating nutrients in the benthos (Vanderploeg et al. 2010; Turschak et al. 2014; Rowe et al. 2015). Invasive amphipods are widespread and a new invasive mysid, Bloody Red Shrimp Hemimysis anomala, discovered in Muskegon Lake in 2006 continues to spread (Pothoven et al. 2007; Pritzker and Sullivan 2016). For early detection and monitoring of AIS to be effective and informative in Lake Michigan the macroinvertebrate community, particularly the benthic community, must be sampled.

During 2015 the AIS Program conducted a pilot study to develop sampling and processing strategies for benthic macroinvertebrates. Sampling was performed at each hotspot and CSMI location sampled during 2015 (N=13). This was accomplished using rock bags, a passive, colonization gear constructed with netting and filled with 1 L of large gravel and a coarse sponge-like material. Sampling effort varied between hotspot (N=20) and CSMI (N=10) locations. Rock bags were deployed on hard substrate in shallow (<3 m) areas along break walls, river banks, and harbor areas and allowed to colonize for approximately 1 month before retrieval was attempted.

Approximately 50% of rock bags were recovered. Some rock bags were washed out and buried during storm events while others were removed by boaters and others from the public. When rock bags were retrieved, all contents were rinsed with lake water through a 500 µm sieve and the contents were placed into a labeled, 500 mL bottle and preserved in 95% ethanol.

In the laboratory, retained contents were viewed under dissecting microscopes and all invertebrates were picked and placed into labeled vials with 95% ethanol. All large macroinvertebrates (>10 mm), amphipods, snails, crayfish and mussels were identified to species and enumerated as many watch list species belong to these taxa (e.g., Killer Shrimp Dikerogammarus villosus, Golden Mussel Limnoperna fortunei, Red Swamp Crayfish Procambarus clarkii). Species assemblages of the remaining invertebrates were characterized using 10% sub-samples or 200 individuals, whichever was larger.

Initial results indicate that Quagga Mussels, Amphipods, and Chironomids were the most abundant taxa collected with this gear. On average, rock bags captured 2,129 ± 1,707 (mean ± SD) invertebrates. Crayfish were sampled infrequently and Bloody Red Shrimp were absent from samples, indicating a strong gear bias against these taxa. Identification of samples is on- going; however, this method appears to be the most efficient for colonizing benthic species.

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Environmental DNA (eDNA) sampling New AIS typically occur at low abundance making them difficult to detect (Dejean et al. 2012; Takahara et al. 2013). Some species, including Bighead and Silver Carp, are particularly difficult to collect with traditional sampling gears when they occur at low abundance (Jerde et al. 2011). The advance of bigheaded carps towards Lake Michigan has heightened concern over the ability of managers to detect and monitor these species, should they successfully invade the lake. To address these limitations, a newly developed scientific method, based on detection of environmental DNA (eDNA), was adopted by USFWS specifically for early detection of bigheaded and Grass carps.

Fish are continuously sloughing DNA into the environment through multiple pathways and persists in aquatic environments for several weeks to a month, making it a useful proxy for species presence (Dejean et al. 2011). Since 2013, the Midwest Region of USFWS (Region 3) has used eDNA sampling methods developed by the U.S. Army Corps of Engineers and the University of Notre Dame to sample for Asian Carp in the Chicago Area Waterway System (CAWS) and other waterways. Current sampling and processing methodologies are written in the Quality Assurance Project Plan (QAPP 2017 available at https://www.fws.gov/midwest/fisheries/eDNA/documents/QAPP.pd f).

Water samples are collected, centrifuged, and shipped by UWFWS field staff to the Whitney Genetics Lab (WGL) at the LaCrosse Fish Health Center where eDNA is extracted and Polymerase Chain Reactions (PCR) are performed. At all stages, great care is

Timothy Strakosh (USFWS) performing the duties of a sampling lead collecting eDNA water samples from the Fox River, WI.

taken to maintain sterile work environments and chain of custody for water samples and data is strictly controlled.

Sampling of eDNA was designed to be spatially and temporally robust to accommodate periods of fish movement, feeding, and spawning. The AIS Program collected eDNA from six tributaries to Lake Michigan, three times each (Figures 3-1 to 3-6). These tributaries were chosen based on their likelihood to provide suitable feeding and spawning habitat for Bigheaded carps (Kocovsky et al. 2012; Murphy and Jackson 2013). Twenty eDNA samples were collected per 2.5 km long site and the number of sample sites for each tributary ranged from 4-10. Total samples per river ranged from 80-200 during each sampling period. Overall, 240-600 total eDNA samples were collected from each river during 2015.

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Water collection sampling methods for lotic systems can be either targeted or systematic. Targeted sampling focuses on collecting water samples in eDNA accumulation areas (e.g., eddies, slackwater, effluent areas). Systematic sampling requires collection of three samples (i.e., left descending bank, mid-channel, and right descending bank) at predetermined transects in each sample site. The AIS Program collected eDNA using both methods depending on habitat complexity in each river. At sites with abundant accumulation areas, a targeted approach was preferred. In areas with slower water movement or few areas of potential accumulation, a systematic transect method was used.

During 2015, the AIS Program collected 2,400 total eDNA samples from six tributaries to Lake Michigan and there were no detections of bigheaded carp DNA. Given the sensitivity of eDNA methods, it is unlikely that bigheaded carps were present in these systems. These results support the hypothesis that they have not spread beyond the CAWS into Lake Michigan and its tributaries.

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Figure 3-1. Environmental DNA sample points collected on the Fox River, WI during three sampling rounds in 2015.

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Figure 3-2. Environmental DNA sample points collected in the Milwaukee River Estaury, WI during three sampling rounds in 2015.

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Figure 3-3. Environmental DNA sample points collected on the St. Joseph River, MI during three sampling rounds in 2015.

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Figure 3-4. Environmental DNA sample points collected on the Kalamazoo River, MI during three sampling rounds in 2015.

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Figure 3-5. Environmental DNA sample points collected on the Grand River, MI during three sampling rounds in 2015.

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Figure 3-6. Environmental DNA sample points collected on the Muskegon River, MI during three sampling rounds in 2015.

55

Ichthyoplankton sampling Green Bay is a highly productive embayment of Lake Michigan and an AIS hotspot. Intensive ichthyoplankton sampling was concentrated in Green Bay as part of a cooperative research project between USFWS and the University of Wisconsin-Green Bay investigating spatiotemporal distribution of larval fish. This project also functioned as an early detection program for larval stages of AIS. Monitoring for larval AIS enhances early detection and monitoring capabilities by focusing on a largely overlooked and under-sampled life stage for most fishes.

Beginning in April, immediately after ice-out, routine sampling events were conducted every two weeks through the end of summer. In total, six sampling events were performed on Green Bay during 2015. During each sampling event, bongo net tows (N=7) were used to sample deeper areas > 2 m and quatrefoil light traps (N=28) were used in shallow areas < 2 m. Water quality parameters were measured at each sample site during each sampling event. Total effort for ichthyoplankton sampling during 2015 was 35 bongo tows and 168 quatrefoil light traps.

During 2015 we collected 19,227 larval fish in lower Green Bay. The predominant fish species captured were Yellow Perch, Common Carp, Spottail and Emerald Shiners, Gizzard Shad, and Freshwater Drum. Most larval fish captured were native to Lake Michigan.

The only AIS encountered was the Round Goby. Beginning in June/July, larval Round Goby began appearing in bongo

Larval fish captured in a bongo net on Green Bay.

tows and light traps. Peak abundance occurred in the Fox River and the east/southeast shoreline of lower Green Bay during July/August.

Abundance of larval fish varied spatially and temporally. The highest abundance of larval fish occurred during late-May/early-June. Early in the season, sample sites in the Fox River and its confluence had the highest densities of larval fish. During midsummer, the highest densities were found in the central-outer bay, and by the end of summer the distribution became patchier; though, the highest densities were still observed in the central-outer bay.

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Cooperative Science and Monitoring Initiative (CSMI) Large tributaries have a significant impact on the receiving waters they flow into. River mouths from large tributaries have unique chemical, biological, and physical attributes that make them appealing habitat to many native and potentially invasive fish species. Lake Michigan is fed by numerous large tributaries; however, little is known about their localized effects on fish communities in the highly productive, near-shore zone.

On a rotating five year basis, each of the Great Lakes is intensively sampled through a program referred to as the Cooperative Science and Monitoring Initiative (CSMI). During 2015, Lake Michigan was sampled with the goal of investigating linkages between near- shore and offshore habitats. The U.S. Geological Survey (USGS), National Oceanic and Atmospheric Administration (NOAA), and U.S. Environmental Protection Agency (EPA) planned to assess spatiotemporal patterns in water quality, plankton, and fishes in offshore, deep-water (≥ 18 m) habitats that are either adjacent to – or unassociated with large tributaries in Lake Michigan. The Green Bay Fish and Wildlife Conservation Office (GBFWCO) expanded these efforts to near-shore (< 18 m) areas in the vicinity of river mouths. Our objective was to investigate the influence of large tributaries on near-shore fish communities with an emphasis on invasive species.

Carolyn Malecha (USFWS) with a Burbot collected using a large-mesh gill net.

We sampled the near shore zone (<18 m) at eight locations around Lake Michigan at the terminal ends of four transects (Figure 4-1). Sampling locations typically occurred at a large tributary confluence with Lake Michigan. The Winthrop Harbor, Sturgeon Bay, and Arcadia study areas lacked large tributaries and acted as overall control sites for other CSMI sampling areas. Sampling was performed during June, August, and September – November.

Sampling at each location was divided into two sites that were sampled with equivalent amounts of effort; an experimental block, centered on a river mouth and/or harbor entrance, and a control block approximately 2 km north or south of the experimental block in the opposite direction of lake currents (Figure 4-2). Each block was divided into four zones with two corresponding to shallower depths (≈ 6 m) and two in deeper areas (≈ 12 m). The order in which zones were sampled was randomized for all gear types. All sampling was performed during nighttime hours (i.e., 30 minutes after sunset, 30 minutes before sunrise).

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At each location, water quality parameters were measured to help differentiate between control and experimental sites. Measurements of Chlorophyll a, temperature, pH, and turbidity were taken with a YSI 650 MDS. Dissolved oxygen, conductivity, and salinity were measured with a YSI model 85.

Fish community sampling was performed using benthic large-mesh and micromesh gillnets. During each sampling period (i.e., spring, summer, fall) at both blocks (i.e., experimental, control) large-mesh nets were deployed only at the 12 m depth zones (N=4) and micromesh gillnets were deployed at all depth zones (N=8). Total catch from gillnet sampling is presented in Table 4-1. Bongo nets were used to collect ichthyoplankton at each zone within each block (N=8). After each five minute tow, nets were retrieved and all contents rinsed into a 500 mL bottle and preserved with 95% ethanol for further processing in the laboratory. Larval fish were identified to the lowest possible taxonomic level and measured to the nearest mm (TL).

We found that locations near large tributaries had greater abundances of Rainbow Smelt, Spottail Shiner, and White Sucker and fewer Lake Trout and Round Goby than locations lacking large tributaries. The impact of confluences was more localized for small-bodied species such as Rainbow Smelt and Spottail Shiner that were found immediately adjacent to the river mouth. Impacts were broader for species like White Sucker where abundance was higher throughout the entire 2 km study area. River mouth study areas had the most distinct fish communities sampled during this study. Only one established invasive species, Rainbow Smelt, was more abundant at river mouths than nearby control areas. These current observations of the highly productive littoral zone of Lake Michigan will contribute to our evolving understanding of lake-wide patterns in this dynamic Great Lakes ecosystem.

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Figure 4-1. Near-shore sampling locations corresponding to CSMI transects.

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Figure 4-2. Experimental blocks were at the mouth of Lake Michigan tributaries or centralized at the end of a CSMI transect if no tributaries were in the area. Control blocks were located 2 km in the opposite direction of lake currents and away from the river plume, if present.

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Table 4-1. Total number of fish collected using short-term sets of large-mesh and micromesh gillnets in the near shore zone of Lake Michigan during June-November 2015.

Gear

Large-mesh Micromesh Grand Species gillnet gillnet total Alewife 3 1803 1806 Bloater 0 54 54 Brown Trout 5 1 6 Burbot 9 0 9 Channel Catfish 8 0 8 Chinook Salmon 4 4 8 Emerald Shiner 0 8 8 Freshwater Drum 3 0 3 Gizzard Shad 13 25 38 Lake Herring 0 4 4 Lake Sturgeon 4 0 4 Lake Trout 102 4 106 Lake Whitefish 44 0 44 Longnose Sucker 42 1 43 Ninespine Stickleback 0 4 4 Rainbow Smelt 0 64 64 Round Goby 0 1903 1903 Round Whitefish 15 3 18 Slimy Sculpin 0 1 1 Spottail Shiner 0 722 722 Trout-perch 0 14 14 Walleye 13 1 14 White Perch 0 1 1 White Sucker 142 2 144 Yellow Perch 2 49 51

Grand Total 409 4668 5077

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Summary

During 2015 the AIS Program executed the most intensive and extensive sampling season since the beginning of the program in 2013. We sampled all five hotspots, began the development of a benthic macroinvertebrate sampling program, collected eDNA from six tributaries three times each, surveyed ichthyoplankton communities in Green Bay, and aided in a lake-wide research study. In total, the AIS Program visited 19 primary sample sites using 3,792 units of effort between all sampling methods and handled at least 37,810 fish of 76 species. We identified 13 existing non-native and invasive fish species, but did not find any new AIS. A single Grass Carp was found in each of two locations (i.e., Milwaukee Harbor, Burns Harbor) while White Perch were found mostly in lower Green Bay. No positive detections for Silver or Bighead Carp were detected after taking 2,400 total eDNA samples. At traditional gear sites, we estimate that 90- 95% of the fish species present were collected.

Sampling gears and methods will continue to be refined, allowing for improved targeting of AIS while increasing sampling efficiency. During traditional gear sampling we identified a particular suite of gears that provided robust species diversity information including electrofishing, gillnets, and paired modified fyke nets. In the future, we plan to improve efficiency by excluding tandem set minnow traps and using one comprehensive experimental gillnet to replace our two current designs. For electrofishing, we identified targeted sampling designs as particularly effective for collecting species richness data. Our participation in CSMI sampling highlighted the importance of targeting confluences to Lake Michigan when sampling for AIS.

Moving forward the AIS Program aims to broaden the scope of AIS sampling in Lake Michigan. Inclusion of larval fish sampling will increase the sensitivity of our hotspot sampling for invasive fish. A robust benthic macroinvertebrate sampling design will extend AIS sampling into the important near-shore benthic community. Given the level of effort used at locations likely to harbor new AIS, we find it unlikely that any new invasive fish species populations are being established in the near-shore zone of Lake Michigan at this time.

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Literature cited Cooke, S. L. and W. R. Hill. 2010. Can filter‐feeding Asian carp invade the Laurentian Great

Lakes? A bioenergetic modelling exercise. Freshwater Biology 55:2138-2152. Dejean, T., A. Valentini, A. Duparc, S. Pellier-Cuit, F. Pompanon, P. Taberlet, and C. Miaud.

2011. Persistence of environmental DNA in freshwater ecosystems. PloS one 6:e23398. Dejean, T., A. Valentini, C. Miquel, P. Taberlet, E. Bellemain, and C. Miaud. 2012. Improved

detection of an alien invasive species through environmental DNA barcoding: the example of the American bullfrog Lithobates catesbeianus. Journal of Applied Ecology 49:953-959.

Great Lakes Commission. 1999. Counterattack: Great Lakes Panel Targets Aquatic Nuisance

Species. Hayer, C-A., K. King, M. Petasek, A. Rieth, M. Shaffer and T. Strakosh. 2017. Lake Michigan:

Implementation plan for the early detection of non-native fishes and benthic macroinvertebrates. U.S. Fish and Wildlife Service, Green Bay Fish and Wildlife Conservation Office, New Franken, WI. Report Number 2017-013.

Hulme, P. E. 2009. Trade, transport and trouble: managing invasive species pathways in an era

of globalization. Journal of Applied Ecology, 46:10-18. Jerde, C. L., W. L. Chadderton, A. R. Mahon, M. A. Renshaw, J. Corush, M. L. Budny, S.

Mysorekar and D. M. Lodge. 2013. Detection of Asian carp DNA as part of a Great Lakes basin-wide surveillance program. Canadian Journal of Fisheries and Aquatic Sciences 70:522-526.

Jerde, C. L., A. R. Mahon, W. L. Chadderton and D. M. Lodge. 2011. “Sight‐unseen” detection

of rare aquatic species using environmental DNA. Conservation Letters 4:150-157. Kocovsky, P. M., D. C. Chapman and J. E. McKenna. 2012. Thermal and hydrologic suitability

of Lake Erie and its major tributaries for spawning of Asian carps. Journal of Great Lakes Research, 38:159-166.

Mills, E. L., J. H. Leach, J. T. Carlton and C. L. Secor. 1993. Exotic species in the Great Lakes:

a history of biotic crises and anthropogenic introductions. Journal of Great Lakes

63

Research 19:1-54. Murphy, E. A., and P. R. Jackson. 2013. Hydraulic and water-quality data collection for the

investigation of Great Lakes tributaries for Asian carp spawning and egg-transport suitability. Urbana 51:61801-2347.

National Invasive Species Council. (2008). 2008-2012 National Invasive Species Management

Plan. 35 pp. Padilla, D. K. and S. L. Williams. 2004. Beyond ballast water: aquarium and ornamental trades

as sources of invasive species in aquatic ecosystems. Frontiers in Ecology and the Environment 2:131-138.

Pimental, D. 2005. Aquatic Nuisance Species in the New York State Canal and Hudson River

Systems and the Great Lakes Basin: An Economic and Environmental Assessment. Environmental Management 35(5). 692-701.

Pothoven, S. A., I. A. Grigorovich, G. L. Fahnenstiel and M. D. Balcer. 2007. Introduction of

the Ponto-Caspian bloody-red mysid Hemimysis anomala into the Lake Michigan basin. Journal of Great Lakes Research 33:285-292.

Rahel, F. J. and J. D. Olden. 2008. Assessing the effects of climate change on aquatic invasive

species. Conservation Biology 22:521-533. Ricciardi, A. 2006. Patterns of invasion in the Laurentian Great Lakes in relation to changes in

vector activity. Diversity and Distributions 12:425-433. Rixon, C. A., I. C. Duggan, N. M. Bergeron, A. Ricciardi, and H. J. Macisaac. 2005. Invasion

risks posed by the aquarium trade and live fish markets on the Laurentian Great Lakes. Biodiversity & Conservation 14:1365-1381.

Rosaen, A.L., E.A. Grover, and C.W. Spencer. 2012. The Cost of Aquatic Invasive Species to

Great Lakes States. Retrieved from http://www.aquaticnuisance.org/wordpress/wp- content/uploads/2010/06/The_Costs_of_Aquatic_Invasive_Species_Nature_Conservancy _2012.pdf

Rowe, M. D., E. J. Anderson, J. Wang, and H. A. Vanderploeg. 2015. Modeling the effect of

invasive quagga mussels on the spring phytoplankton bloom in Lake Michigan. Journal of Great Lakes Research 41:49-65.

64

Sturtevant, R., L. Berent, T. Makled, W. Conard, A. Fusaro, and E. Rutherford. 2016. An overview of the management of established nonindigenous species in the Great Lakes. NOAA Technical Memorandum GLERL-168.

Takahara, T., T. Minamoto and H. Doi. 2013. Using environmental DNA to estimate the

distribution of an invasive fish species in ponds. PloS one 8:e56584. Turschak, B. A., D. Bunnell, S. Czesny, T. O. Höök, J. Janssen, D. Warner, and H. A. Bootsma.

2014. Nearshore energy subsidies support Lake Michigan fishes and invertebrates following major changes in food web structure. Ecology 95:1243-1252.

U.S. Fish and Wildlife Service. 1994. Great Lakes Fishery Resources Restoration Study.

Vanderploeg, H. A., J. R. Liebig, T. F. Nalepa, G. L. Fahnenstiel, and S. A. Pothoven. 2010.

Dreissena and the disappearance of the spring phytoplankton bloom in Lake Michigan. Journal of Great Lakes Research 36:50-59.

Vanderploeg, H. A., T. F. Nalepa, D. J. Jude, E. L. Mills, K. T. Holeck, J. R. Liebig, I. A.

Grigorovich and H. Ojaveer. 2002. Dispersal and emerging ecological impacts of Ponto- Caspian species in the Laurentian Great Lakes. Canadian Journal of Fisheries and Aquatic Sciences 59:1209-1228.

Vander Zanden, M. J., G. J. Hansen, S. N. Higgins and M. S. Kornis. 2010. A pound of

prevention, plus a pound of cure: early detection and eradication of invasive species in the Laurentian Great Lakes. Journal of Great Lakes Research 36:199-205.

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Appendix 1 Common and species names of fish captured during sampling in Lake Michigan by the GBFWCO early detection and monitoring program during 2015.

Common name Scientific name Alewife Alosa pseudoharengus Banded Killifish Fundulus diaphanus Bigmouth Buffalo Ictiobus cyprinellus Black Bullhead Ameiurus melas Black Crappie Pomoxis nigromaculatus Bloater Coregonus hoyi Bluegill Lepomis macrochirus Bluntnose Minnow Pimephales notatus Bowfin Amia calva Brook Silverside Labidesthes sicculus Brown Bullhead Ameiurus nebulosus Brown Trout Salmo trutta Burbot Lota lota Channel Catfish Ictalurus punctatus Chestnut Lamprey Ichthyomyzon castaneus Chinook Salmon Oncorhynchus tshawytscha Coho Salmon Oncorhynchus kisutch Common Carp Cyprinus carpio Common Shiner Luxilus cornutus Deepwater Sculpin Myoxocephalus thompsonii Emerald Shiner Notropis atherinoides Flathead Catfish Pylodictis olivaris Freshwater Drum Aplodinotus grunniens Gizzard Shad Dorosoma cepedianum Golden Redhorse Moxostoma erythrurum Golden Shiner Notemigonus crysoleucas Goldfish Carassius auratus Grass Carp Ctenopharyngodon idella Grass Pickerel Esox americanus Greater Redhorse Moxostoma valenciennesi Green Sunfish Lepomis cyanellus Johnny Darter Etheostoma nigrum Lake Chub Couesius plumbeus Lake Herring Coregonus artedi

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Lake Sturgeon Acipenser fulvescens Lake Trout Salvelinus namaycush Lake Whitefish Coregonus clupeaformis Largemouth Bass Micropterus salmoides Logperch Percina caprodes Longnose Dace Rhinichthys cataractae Longnose Gar Lepisosteus osseus Longnose Sucker Catostomus catostomus Mooneye Hiodon tergisus Mottled Sculpin Cottus bairdii Muskellunge Esox masquinongy Ninespine Stickleback Pungitius pungitius Northern Pike Esox lucius Orangespotted Sunfish Lepomis humilis Pumpkinseed Lepomis gibbosus Quillback Carpiodes cyprinus Rainbow Smelt Osmerus mordax Rainbow Trout Oncorhynchus mykiss River Redhorse Moxostoma carinatum Rock Bass Ambloplites rupestris Round Goby Neogobius melanostomus Round Whitefish Prosopium cylindraceum Sand Shiner Notropis stramineus Shorthead Redhorse Moxostoma macrolepidotum Shortnose Gar Lepisosteus platostomus Silver Redhorse Moxostoma anisurum Slimy Sculpin Cottus cognatus Smallmouth Bass Micropterus dolomieu Smallmouth Buffalo Ictiobus bubalus Spotfin Shiner Cyprinella spiloptera Spottail Shiner Notropis hudsonius Threespine Stickleback Gasterosteus aculeatus Trout-Perch Percopsis omiscomaycus Walleye Sander vitreus Warmouth Lepomis gulosus White Bass Morone chrysops White Crappie Pomoxis annularis White Perch Morone americana White Sucker Catostomus commersonii Yellow Bullhead Ameiurus natalis

Yellow Perch Perca flavescens

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Appendix 2 Boat electrofishing - Electrofishing was conducted during nighttime hours (i.e., 30 minutes after sunset, 30 minutes before sunrise) and daytime hours with a Kann, 2-boom electrofishing boat equipped with an Infinity shock box (Midwest Lake Electrofishing Systems). Settings for pulsed DC electrofishing were standardized with duty cycle set at 30%, pulse rate at 60 Hz, 118-325 volts, 10-37.1 amps, and power of approximately 5,000-5,500 watts. Units of effort for electrofishing were approximately 10 min of pedal time per shocking run. Electrofishing was performed with two netters in littoral areas (<3 m) including sand flats, weed beds, break walls, and riverine habitats. Catch per unit of effort is reported as the mean number of fish captured per 10 min run (i.e., fish/run).

Paired fyke-nets - Each set of paired fyke nets consisted of two nets joined together by their leads (15 m long x 0.91 m high). Each net is constructed with two rectangular frames 0.91 m high by 1.22 m wide, followed by four circular frames (rings) 0.91 m in diameter. All netting materials are 2.35 mm bar mesh. This gear was deployed parallel to the shoreline or along macrophytes in water depths < 3.0 m and fished overnight. Catch per unit effort for this gear is reported as the number of fish captured for the combined pair of fyke nets per night (i.e., fish/paired net night).

Paired mini-fyke nets - Each set of paired mini-fyke nets consisted of two nets joined together by their leads (6 m long x 0.45 m high). Each net is constructed with two rectangular frames 0.45 m high by 0.90 m wide, followed by four circular frames (rings) 0.45 m in diameter. All netting materials are 1.59 mm bar mesh. This gear was deployed parallel to the shoreline or along macrophytes in water depths < 3.0 m and fished overnight. Catch per unit effort for this gear is reported as the number of fish captured for the combined pair of fyke nets per night (i.e., fish/paired net night).

Large-mesh gillnets – Monofilament, large-mesh gillnets were constructed using a single panel of 57 mm bar mesh monofilament 2.01 m in height by 91.44 m long. Hanging ratio was 0.5 for all nets. Each net was set on the bottom and fished independently (0.5-2 hours) then retrieved. Sampling occurred during nighttime hours (i.e., 30 minutes after sunset, 30 minutes before sunrise) in depths of 6-12 m. Units of effort are reported as the number of fish captured per net set (i.e., fish/net set).

Micromesh experimental gillnets – Monofilament micromesh experimental gillnets contained six panels of 6.25, 8, 10, 12.5, 16, and 19 mm bar mesh each 1.52 m high by 10.05 m long. Hanging ratio was 0.5 for all nets. Each net was set on the bottom and fished independently (0.5-2.0 hours) then retrieved. Sampling occurred during nighttime hours (i.e., 30 minutes after

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sunset, 30 minutes before sunrise) in depths of 6-12 m. Units of effort are reported as the mean number of fish captured per net set (i.e., fish/net set).

Baited minnow trap arrays – Baited minnow trap arrays are comprised of five minnow traps set in series spaced 7.6 m apart for a total array length of 30.5 m. Minnow traps one, three, and five were always baited with dog food while minnow traps two and four were left un-baited. Minnow traps were 413 mm long with 6 mm bar mesh and a 19 mm diameter throat. Catch per unit effort for this gear was the number of fish captured per array per night (i.e., fish/array/night).

Quatrefoil light traps – Quatrefoil light traps (0.3-m-diameter, 0.25-m-long, gap width 5 mm, 500-micron-mesh) were used to collect larval fish in shallow areas. Light traps were modified to include a waterproof flashlight of at least 105 lumens as a light source with a horizontal light diffuser (conical or other) to control light dispersal direction. Traps were allowed to sit overnight. Catch per unit effort for this gear was the number of fish captured per set (i.e., fish/set).