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Early Detection and Monitoring for New Aquatic Invasive Species in Lake Michigan: 2016

Report Number: 2017-008

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Harris, B.S., J. T. Richter, M. Shaffer, B.J. Smith and C-A. Hayer. 2016. Early detection and monitoring for new aquatic invasive species in Lake Michigan. U.S. Fish and Wildlife Service, Green Bay Fish and Wildlife Conservation Office, Green Bay, WI. Report Number 2017-008.

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Table of Contents

List of Figures ......................................................................................................................v

List of Tables .................................................................................................................... vii

Executive Summary ......................................................................................................... viii

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

Objectives ............................................................................................................................4

Methods................................................................................................................................4

Study area........................................................................................................................4

Sampling gears ................................................................................................................5

Fish community sampling ...............................................................................................5

Benthic macroinvertebrate sampling ..............................................................................6

Ichthyoplankton sampling ...............................................................................................7

Analysis and Interpretation .............................................................................................8

Results ..................................................................................................................................9

Environmental DNA .......................................................................................................9

Fish community sampling ...............................................................................................9

Benthic macroinvertebrate sampling ............................................................................10

Ichthyoplankton sampling .............................................................................................11

Discussion ..........................................................................................................................11

Conclusions ........................................................................................................................14

Future Work .......................................................................................................................14

Acknowledgements ............................................................................................................14

References ..........................................................................................................................15

Figures................................................................................................................................21

Tables .................................................................................................................................36

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List of Figures

Figure 1. Lake Michigan study areas (hotspots) sampled by the Aquatic Invasive Species (AIS) program during 2016. All hotspot study sites are outlined in red while study rivers for eDNA sampling are drawn in black and labeled.

Figure 2. Study area of rivers flowing into Lake Michigan for eDNA analysis.

Figure 3. Lower Green Bay, WI traditional gear study area with sample locations for each gear type. Sampling was performed during May-October 2016. Five additional electrofishing runs were performed upstream in the Fox River near the De Pere Dam but are not depicted.

Figure 4. Map of the Milwaukee, WI traditional gear study area with sample locations for each gear type. Sampling was performed during September 2016.

Figure 5. Map of the Chicago, IL traditional gear study area with sample locations for each gear type. Sampling was performed during August 2016.

Figure 6. Map of the Calumet Harbor, IL traditional gear study area with sample locations for each gear type. Sampling was performed during August 2016.

Figure 7. Map of the Burns Harbor, IN traditional gear study area with sample locations for each gear type. Sampling was performed during August 2016.

Figure 8. Map of light trap sampling locations in the Milwaukee River and Milwaukee Harbor, WI during 2016 ichthyoplankton surveys.

Figure 9. Map of light trap sampling locations in the Lower Fox River, Green Bay, WI during 2016 ichthyoplankton surveys.

Figure 10. Length-frequency histograms for nine species collected across all gear types and sites during 2016 Lake Michigan sampling. Note that the X and Y axes differ in scale across species.

Figure 11. Dendrogram of fish communities at hotspot sampling sites calculated using average-linkage hierarchical cluster analysis in 2016.

Figure 12. Sample size based rarefaction (solid lines) and extrapolation (dashed lines, up to double the reference sample size) of species richness for all gears used in 2016 with the five hotspots combined. The reference samples are denoted by the solid dots. The 95% confidence intervals (shaded regions) were obtained using a bootstrap method based on 200 replications. The rarefaction/extrapolation curves along with the asymptotic species richness for each gear were calculated by the Chao et al. 2014 estimator functions within the iNEXT package using the software program R.

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Figure 13. Sample size based rarefaction (solid lines) and extrapolation (dashed lines) of species richness for all traditional gears used in 2016 at the five hotspot locations. The reference samples are denoted by the solid dots. The 95% confidence intervals (shaded regions) were obtained using a bootstrap method based on 200 replications. The rarefaction/extrapolation curves along with the asymptotic species richness for each location were calculated using the Chao et al. 2014 estimator functions within the iNEXT package run in the software program R. Within the figure; y-axis levels vary, n = units of effort, S = # of species caught, and E = Sampling efficiency.

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List of Tables

Table 1. River name, state, number of reaches, number of water samples taken, and dates sampled for environmental DNA analysis in nine hotspot rivers for Bighead and Silver Carp presence during 2016.

Table 2. Total catch from all gears combined at five hotspot sampling locations in Lake Michigan during 2016 including catches from an early season (May) paired fyke net gear comparison in Green Bay. Names of invasive species are bolded.

Table 3. Gear and units of effort allocations expended during 2016 at the five hotspot locations in Lake Michigan using three traditional fish sampling gears.

Table 4. Environmental conditions and sampling site characteristics at light trap locations in the Milwaukee River and Milwaukee Harbor during 2016 ichthyoplankton surveys.

Table 5. Environmental conditions and sampling site characteristics at light trap locations in the Lower Fox River – Green Bay during 2016 ichthyoplankton surveys.

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Executive Summary

For nearly two centuries, continual introductions of non-native species in the Laurentian Great Lakes have altered the ecosystem and cost millions of dollars in damage to fisheries and infrastructure. In recent decades, there has been increasing emphasis on prevention and early detection of new invasive species in the Laurentian Great Lakes. The aquatic invasive species (AIS) program at the Green Bay Fish and Wildlife Conservation Office performed early detection and monitoring throughout Lake Michigan in 2016. Water samples were collected to detect the presence of Bighead and Silver Carp DNA from the Fox and Milwaukee rivers in Wisconsin and the St. Joseph, Kalamazoo, Macatawa, Galien, Black, Grand and Muskegon rivers in Michigan. Fish and macroinvertebrates communities were sampled from the bay of Green Bay, Calumet Harbor, Burns Harbor, and nearshore waters of near Milwaukee and Chicago, five locations identified as hotspots for invasion of new invasive species.

Sampling focused on detection of bigheaded carps (i.e., Bighead and Silver Carp) in rivers, monitoring fish and macroinvertebrate communities in five hotspot locations associated with densely populated port cities on Lake Michigan, and developing a monitoring protocol for ichthyoplankton. We found no evidence of Bighead or Silver Carp presence using environmental DNA sampling techniques on nine river systems in Michigan and Wisconsin. No new invasive or non-native species were detected in 2016 at the five hotspot locations in Lake Michigan. A total of 51,864 individual fish representing 73 species were collected with 411 units of sampling effort. Species accumulation curves indicated that fish communities were effectively sampled using nighttime boat electrofishing, experimental gill nets, and paired modified fyke nets that when combined, provided a representative sample of the fish community at each location (90-95% of species collected). Gears and methods for sampling fish were improved during 2016, allowing for reduced effort with similar, or better, efficiency compared to previous years. At each hotspot, benthic macroinvertebrate sampling took place where introduction and establishment would be most likely; including boat ramps, river mouths, and ship docking areas. No new non-native benthic macroinvertebrate species were detected, but invasive Bloody Red Shrimp Hemimysis anomala and amphipod Echinogammarus ischnus were found in new areas within Lake Michigan. Ichthyoplankton sampling was conducted in nearshore waters of Milwaukee and Green Bay. Sampling during 2016 was spatially broad with intensive sampling effort and adaptively incorporated results from previous years. A suite of sampling techniques was used to effectively detect new AIS in Lake Michigan, if present.

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Introduction

Invasions by aquatic invasive species (AIS) are a problem in both marine and freshwater systems and are occurring on a global scale (Mills et al. 1993; Molnar et al. 2008). The Laurentian Great Lakes, the largest freshwater system in the world, have long been a case study for the potential negative consequences of AIS on an ecosystem. As a result of increased connectivity in the Great Lakes, non-native species introductions have been dominated by Ponto-Caspian species (Ricciardi and MacIsaac 2000). Given the importance of the Great Lakes basin and its resources, it is imperative that further invasions are detected early or prevented before introduction.

Comprehensive early detection and monitoring (EDM) programs for AIS are critical for identifying new threats, addressing them quickly before AIS become established, (Vander Zanden et al. 2010) and potentially preventing further invasions (Ricciardi 2001). Realizing that species invasions were likely to continue, Great Lakes researchers and managers increasingly supported development of more vigorous and coordinated AIS monitoring efforts throughout the Great Lakes basin (Vander Zanden et al. 2010).

Early detection and monitoring programs for AIS were began as a result of many different executive orders and international agreements. For example, in 2009, the Great Lakes Restoration Initiative (GLRI) was established which, among other objectives, provided funding for early detection and monitoring for AIS within the Great Lakes. Under the terms of GLRI, the U.S. Fish and Wildlife Service (USFWS) was given responsibility and resources for developing a monitoring program for AIS in U.S. waters of the Great Lakes. The long-term goal of the GLRI Action Plan (GLRI 2010) was developed to guide restoration efforts with a goal to provide “A comprehensive program for detection and tracking newly identified invasive species in the Great Lakes is developed and provides up-to-date critical information needed by decision markers for evaluation potential rapid response actions.”

Additionally, the Great Lakes Water Quality Agreement (GLWQA), called for the establishment of lake wide management plans to assess the state of each Great Lake. In 2012, Canada and the United States amended the GLWQA (GLWQU 2012) to include Annex 6 on Aquatic Invasive Species. The goals of Annex 6 are to 1) develop species watch lists, 2) identify priority locations for surveillance, 3) develop monitoring protocols for surveillance, 4) establish protocols for sharing information, 5) identify new AIS, and 6) coordinate effective and timely domestic and when necessary binational response actions to prevent the establishment of newly detected AIS. Under the terms of GLRI, the Fish and Wildlife Service (USFWS) was given the responsibility and resources for developing a monitoring program for AIS in U.S. waters of the Great Lakes.

In 2013, the AIS early detection and monitoring program was established at the Green Bay Fish and Wildlife Conservation Office (GBFWCO) to monitor for AIS in the near-shore zone of Lake Michigan and its rivers. Primary species targeted during GBFWCO monitoring efforts include Bighead Carp Hypophthalmichthys nobilis and Silver Carp Hypophthalmichthys molitrix, herein

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referred to as bigheaded carps, which threaten to invade the Great Lakes via the Chicago Area Waterway System (CAWS). This canal system artificially connects the Mississippi and Great Lakes drainages. Bigheaded carps have not successfully invaded Lake Michigan, but the invasion front is now within several dozen kilometers of an electric barrier designed to prevent fish from moving between the two drainage basins. Early detection and monitoring is a vital part of managing AIS, however, using traditional gears to capture bigheaded carps at low densities has provided limited utility. Alternatively, one of the AIS early detection tools used to monitor for bigheaded carps is environmental DNA (eDNA) surveillance. Early detection of AIS by eDNA is a new technique undergoing extensive development as it is applied in the field for monitoring efforts. Environmental DNA sampling is a genetic tool that can indicate the presence/absence of species-specific DNA in the aquatic environment.

Beyond bigheaded carps, there are many other potential AIS threats from vectors such as ballast water discharge, pet/aquarium trade releases, and recreational boat traffic (Ricciardi 2006; Howeth et al. 2016; Hirsch et al. 2016). These threats are highest at nodes of water connection networks and are associated with international shipping ports, industrialization, and high human population density, such as Chicago and Milwaukee waters. Early in the development of the AIS detection and monitoring program, analyses were performed to identify the highest risk areas for new species introductions. In Lake Michigan, five sites present the highest risk for new species invasions and include lower Green Bay, Milwaukee, Chicago, Calumet Harbor, and Burns Harbor, and we refer to these five sites as “hotspot” sampling locations. Monitoring the fish and benthic macroinvertebrate communities at these locations annually is a critical component of AIS monitoring in Lake Michigan.

Objectives

According to the Fish and Wildlife strategic framework, the GBFWCO AIS program had four objectives in 2016:

1. Collect eDNA from targeted river systems within the Lake Michigan basin that would provide suitable spawning and environmental conditions for bigheaded carps.

2. Effectively monitor fish communities at select hotspot locations, identified in 2014, using various passive and active sampling gears and methods suitable for each location to identify new non-native species and develop baseline assemblage data should a comparison be needed if a new invasive species should be introduced. Gears selected in 2016 were based on efficiency of gears from previous years.

3. Develop and implement benthic macroinvertebrate and ichthyoplankton sampling programs at hotspot locations to identify new non-native species and develop baseline assemblage data should a comparison be needed if a new invasive species should be

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introduced. A suite of gears were used to identify the most efficient and appropriate sampling gears.

4. Communicate results of our monitoring efforts to agency partners and the general public.

Methods

Study area— Lake Michigan tributary sites for eDNA sampling of bigheaded carps were selected based on tributaries that initially met the preferred spawning habitat criteria (e.g., water temperature, length of river, hydrograph) for Asian Carp and proximity to the CAWS (2014 Report). These included the Fox and Milwaukee rivers in Wisconsin and the St Joseph, Kalamazoo, Macatawa, Galien, Black, Grand and Muskegon rivers in Michigan (Figure 1; Table 1).

Environmental DNA sampling— The USFWS Fish and Wildlife Conservation Offices (FWCO) is responsible for field sampling and lab processing of eDNA samples. Each FWCO handles field sampling and the Whitney Genetics Lab at the Midwest Fisheries Center processes eDNA samples (USFWS 2017). Current eDNA methods are focused on detecting the presence/absence of bigheaded carps and the GBFWCO eDNA processing followed the standards set forth in the USFWS’s Quality Assurance Project Plan (USFWS 2017). The QAPP provides detailed procedures for eDNA sample collection, sample processing (e.g., centrifuging, DNA extraction), data reporting, and quality control measures. Additionally, to confirm that our sampling is relevant to the States, we consulted the states to gain their concurrence.

Identified hotspot tributaries were divided into 1-10 sampling reaches (~2.5 km) with 20 water samples collected within each reach (Figure 1). Three rounds of eDNA samples were collected at each river; once during the spring when river discharge may fluctuate dramatically (i.e., corresponding to fish movement), and twice more during the summer period when warmer water temperatures were at preferred bigheaded carps spawning temperatures (≥ 17° C; Kolar et al. 2011). Water samples were taken near the surface (no deeper than ~5 cm) where we targeted the most probable places of eDNA accumulation; backwaters, confluences, eddies, bays, slack-water and structure impediments. Using a mobile laboratory, the water samples were centrifuged to concentrate the eDNA and a concentrated “pellet” was fixed in ethanol. The preserved samples were sent to the USFWS Whitney Genetics Laboratory in Onalaska, WI where the eDNA was isolated and amplified using Polymerase Chain Reaction techniques to detect bigheaded carp DNA. All results of eDNA sampling were performed and disseminated by the Whitney Genetics Lab.

Fish community sampling— Sample site selection for traditional sampling gears are described in the Lake Michigan Implementation Plan (King et al. 2015). Risk vectors were identified, such as maritime commerce, hydrologic connections, organisms in trade and illegal activities. The GBFWCO EDM focused sampling efforts on areas that were most vulnerable to invasion by

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non-native species. Vector analyses were conducted and five “hotspot” locations were identified: Milwaukee Harbor and lower Green Bay, WI, Chicago Harbor, IL, Calumet Harbor, IN, IL, Burns Harbor, IN (Figure1). Hotspot locations are heavily industrialized or urbanized and have international shipping ports that are a key vector for potential introduction of new AIS (Figure 2).

Fish community sampling was performed at each hotspot with a goal of achieving 70-80 units of sampling effort using nighttime boat electrofishing, experimental gill nets, and modified fyke nets combined in a 40%, 35%, and 25% effort allocation, respectively. Based on previous monitoring from 2013 - 2015, this level of effort and proportional allocation has been demonstrated to collect 90-95% of the available fish community at our sampling locations. We used a targeted sampling design for all gears following results of previous monitoring (Smith USFWS unpublished data). For our purposes, targeted sampling refers to choosing sampling locations for each gear which will maximize catch and diversity; typically we targeted the most structurally complex habitats because they were likely to provide habitat for multiple species. Sampling was performed during late August-October at all locations. Electrofishing was conducted during nighttime hours (0.5 hr after sunset to 0.5 hr before sunrise) with a 6.1 m long 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 pulses per second, 118-325 volts, 10-37.1 amps, and power of approximately 5,000-5,500 watts. Catch per unit effort was reported as the number of fish captured per minute (i.e., fish/min). Monofilament experimental gillnets used were 1.8 m tall by 40 m long and comprised of 13 randomly ordered panels (3.05 m)with bar-mesh sizes from 6-76 mm. Paired modified fyke nets had frames that were 0.9 m tall by 1.8 m wide with four 0.9 m hoops all constructed with 13 mm mesh. Experimental gill nets and paired modified fyke nets were set in the morning, fished overnight and retrieved the next morning. Catch per unit effort for both passive gears was reported as the number of fish collected per net set. All fish captured were identified to species, and the first 50 individuals from each species were measured (TL; mm) for each unit of effort.

Benthic macroinvertebrate sampling— Benthos sampling was conducted concurrently with fish community sampling from August through October, but was confined to boat launches, river mouths, and shipping channels, locations of highest-risk vectors for species introductions within each of the five hotspot locations. Two passive gears (e.g., rock bags, modified minnow traps) and one active gear (e.g., petite PONAR grabs) was used to target amphipods, decapods (crayfish and mysid shrimp), bivalves, and gastropods. At each hotspot location (excluding Chicago Harbor), 2 to 3 units of effort were allocated to each gear type at each of the three high-risk vectors. In Chicago Harbor, two units of effort were shifted to the boat launch vector (i.e., four units of effort per gear type), because Chicago lacks a shipping channel. We considered the entrance to the Chicago River to be a river mouth vector, although this does not represent a true river mouth due to the reversed flow of the Chicago River.

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Rock bags were constructed by enclosing 2.5-7.6 cm beach stones (standardized by filling a 0.8 L bottle with these stones) in nylon mesh (mesh size = 1 cm) bags; rock bags were deployed in shallow areas (i.e., 2 mm to species, yet some specimens were only identified to family or genus due to minor damage from sample processing or unclear morphological characteristics preventing species level identification. All decapods were identified to species. All mussels ≥5 mm in length and all gastropods with ≥3.5 whorls were identified to species (if distinguishing characteristics were present), but smaller, less developed specimens were identified to lowest possible taxonomic level.

Ichthyoplankton sampling— Ichthyoplankton were collected during the last week of June at two sites in Lake Michigan- Green Bay (Fox River; Figure 9) and Milwaukee River and Harbor (Figure 8). Most larval freshwater fishes exhibit diel vertical migration behavior, which involves swimming up toward the surface at nighttime to seek zooplankton prey while avoiding predation (Hensler and Jude 2007) and some fish including bigheaded carps are phototactic (Kawamura

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and Shinoda 1980; Bulowski and Meade 1983; George and Chapman 2015). Quatrefoil light traps (0.3-m-diameter, 0.25-m-tall, gap width 5 mm) equipped with waterproof flashlights and light diffusers were used to passively collect ichthyoplankton. Larval fish are attracted to the light, follow the outside curvature into the funnel gap (Brogan 1994), and are collected in a 2-L bottle threaded to the bottom of the trap. Light traps were set in areas ranging from 0.7 to 9.0 m in depth and were placed in high traffic shipping channels and ports and off-channel backwater habitats. Light traps were deployed at dusk, left to fish overnight, and retrieved early the following morning (2000 – 0800).

Sampling site information (e.g., location, depth) and surface water quality measurements including water temperature (˚C), dissolved oxygen (mg/L), pH, chlorophyll a (mg/L), and turbidity (NTU; YSI EXO2 sonde) were recorded at each light trap sampling site. Samples were preserved in a 95% ethyl alcohol solution and brought to the GBFWCO laboratory for processing. In the laboratory, larval fish were separated from the rest of the sample debris and enumerated. Larval fish were measured to the nearest mm from the mouth to the tip of the tail and were digitally imaged using a stereoscope (Nikon SMZ-1270) equipped with a camera (Nikon NIS-Elements software). Larval fish were then morphologically identified to the lowest possible taxonomic level. Specimen vouchers were stored in vials and labeled with sampling site ID, date of collection, gear type used, total specimen count, and length.

Analysis and interpretation— Only fish data were analyzed and interpreted because eDNA data is are analyzed and reported by the Whitney Genetics Lab (https://www.fws.gov/midwest/WGL/); ichthyoplankton data was limited in 2016 and not included in this report, and benthic invertebrate data will be summarized in an additional report. Analyses for fish data focused on community analyses and the ability of sampling to encounter the full diversity of species at a location, including rare species. Analysis began by assessing similarity of fish communities between hotspot locations. We suspected that locations close together would be more similar and that Green Bay would be the most distinct due to its unique geography and distance from the other sampling locations. For this task, we used average-linkage hierarchical cluster analysis based on Euclidean distances to visualize differences in fish communities between hotspot locations. Additionally, we provided length-frequency distributions for the most abundant species captured lake wide.

To describe the relationship between fish sampling effort and estimates of species richness, species accumulation curves were constructed. Sample size based rarefaction/extrapolation curves (R/E curves; species accumulation methodology) were created within the iNEXT package using the program R version 3.2.2 through RStudio 1.0.44 (Hsieh et al. 2016, R Core Team 2016). The iNEXT package contains functions to create a single, smooth sampling curve, derived from a reference sample that can be interpolated (rarefied) to smaller sample sizes or extrapolated to a larger sample size, guided by an estimate of asymptotic richness (Chao et al. 2014). First, the sample-based abundance data was converted to sample-based incidence data following the iNEXT incidence-frequency data format. Diversity estimates were computed for

https://www.fws.gov/midwest/WGL/

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rarefied and extrapolated samples up to 200 units of effort or double the reference sample size, resulting in diversity estimated sampling curve plots with respect to sample size. Sample size based R/E curves were generated for each of the five hotspot locations with all three traditional gear types combined (i.e., fyke nets, gill nets, and night electrofishing). Additionally, all five locations were pooled to generate R/E curves for each of the three traditional gear types, as well as all three gears combined. Asymptotic species richness estimates were calculated using the ChaoRichness function within the iNEXT package, which calculates a lower bound for species richness, based largely on the lower-order frequency counts. A bootstrap method was used to construct 95% confidence intervals based on 200 replications.

Results

Environmental DNA sampling— A total of 2,760 water samples were collected from nine Lake Michigan tributaries during 2016, yielding zero positive detections for bigheaded carps DNA (Table 1). A comprehensive list of eDNA results for Lake Michigan can be found at the U.S. Fish & Wildlife Service Midwest Region Fisheries website: https://www.fws.gov/midwest/fisheries/eDNA/Results-michigan.htmlhttps://www.fws.gov/midwest/fisheries/eDNA/Results-michigan.html

Fish community sampling— No new invasive fishes were collected at the five hotspot sampling locations during 2016 (Table 2). The most common invasive species collected were Alewife Alosa pseudoharengus, Common Carp Cyprinus carpio, Rainbow Smelt Osmerus mordax, Round Goby Neogobius melanostomus, and White Perch Morone Americana (Table 2). Seventy-three fish species were captured in Lake Michigan using the three traditional gears. Sixty-five species were captured by night electrofishing (15 unique to this gear), 48 species by gill nets (five unique to this gear), and 38 species by fyke nets (one unique to this gear). Of the five hotspot locations, the fish community of Green Bay was distinct from the remaining four sites, with Calumet and Chicago Harbor being most similar (Figure 12). Length-frequencies for the nine most abundant species show the size ranges of species commonly encountered throughout all hotspot locations (Figure 11).

A total of 411 units of effort, divided between nighttime boat electrofishing, experimental gill nets, and modified fyke nets were utilized across all five locations. Lake-wide gear allocation was 47% night electrofishing (n=191), 29% gill nets (n=120), and 24% fyke nets (n=100). Total units of effort and gear allocation amounts varied at each location with a minimum of 72 units of effort expended at any one location (Table 3). The asymptotic species richness was estimated to be 76.5 ± 3.5 fish species for all sites combined, achieving a 95.4% efficiency level (Figure 13). A minimum estimate of 393 units of effort was needed to reach the 95% efficiency level.

By hotspot location, Milwaukee had the highest observed species richness (n=48), followed by Green Bay (n=47), Burns Harbor (n=45), Calumet Harbor (n=38), and Chicago (n=26). Asymptotic species richness estimates varied by location; Burns Harbor (50.9 ± 6.4), Calumet

https://www.fws.gov/midwest/fisheries/eDNA/Results-michigan.htmlhttps://www.fws.gov/midwest/fisheries/eDNA/Results-michigan.htmlhttps://www.fws.gov/midwest/fisheries/eDNA/Results-michigan.html

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Harbor (62.7 ± 23.9), Chicago (32.0 ± 6.0), Green Bay (51.0 ± 4.0), and Milwaukee (58.0 ± 7.9; Figure 13). Based on observed versus expected species richness estimates, our highest detection efficiency was at Green Bay (92%), followed by Burns Harbor (88%), Milwaukee (83%), Chicago (81%), and Calumet Harbor (61%; Figure 14).

Benthic macroinvertebrate sampling— We were unable to identify all of our specimens and provide a full analysis in time for this report; an appendix to this report will be completed upon full analysis of benthos data. Monitoring efforts detected an invasive mysid shrimp (Bloody Red Shrimp Hemimysis anomala) and amphipod (scud Echinogammarus ischnus) at all five hotspot sampling locations; we collected two relic shells of an invasive Asian Clam Corbicula sp. in the lower Fox River in Green Bay. To the best of our knowledge (according to GLANSIS website), our detection of Bloody Red Shrimp is the first documented detection in Burns, Calumet, and Chicago Harbors. They have previously been reported in Milwaukee Harbor and Green Bay, although our detection is the first for lower Green Bay. The presence of the scud is the first documented report of this invasive species in Burns, Chicago, Calumet, and Milwaukee Harbors. The presence of Asian Clam is the first documented report of this invasive species in Green Bay according to GLANSIS. However, we determined the Wisconsin Department of Natural Resources has two verified accounts (in 1999 and 2011) of Asian Clams in this area of Green Bay but it remains unknown whether they were live specimens. Nonetheless, we re-sampled the area where we found Asian Clam shells and collected additional shells and two live specimens in February 2017.

Ichthyoplankton sampling—Twenty-five sites in the Milwaukee River and Milwaukee Harbor were sampled on June 27-28 with larval light traps, and six of the 25 larval light traps yielded larval fish (Figure 7). A total of 69 larval fish were collected representing three species: Round Goby (75% of total catch), Fathead Minnow Pimephales promelas, Smallmouth Bass Micropterus dolomieu, and Notropis sp. Water depth and environmental conditions at light trap sampling locations in Milwaukee were variable (Table 5). Twenty-four sites in the Lower Fox River, Green Bay were sampled June 29-30 with larval light traps, and seven of the 24 larval light traps yielded larval fish (Figure 8). Twenty-two larvae were collected representing four species: Gizzard Shad Dorosoma cepedianum (50% of total catch), Round Goby, White Bass Morone chrysops, White Perch, and Notropis spp. Water depth and environmental conditions at light trap sampling locations in the Fox River were variable (Table 6).

Discussion

The goal of the AIS early detection and monitoring program is to be able to identify new potential invasive species when they are rare and early in the introduction phase, allowing time for a proportional and targeted management response. In the fourth year of monitoring Lake Michigan and its rivers, we have developed an extensive and adaptive sampling approach for identifying potential new invaders at identified hotspots where they are most likely to first invade. Based on species accumulation curves and predicted species richness the Green Bay,

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Burns Harbor, and Milwaukee study locations were sampled most effectively while sampling at Chicago and Calumet study areas has room for improvement. Bigheaded carp DNA was not detected from eDNA water samples in any of the nine rivers sampled in 2016. We found existing and widespread invasive fishes at all five hotspot sampling locations using traditional sampling gears, but no new non-native fishes were detected. Also, we documented two invasive benthos; Scud and Bloody Red Shrimp within Lake Michigan where they had not been previously detected, and confirmed the presence of live Asian Clams in Green Bay.

Our eDNA results provide supporting evidence that our sampling locations are outside the known range of bigheaded carp. However, since several physical connections exist between well-established populations in the Mississippi River basin (e.g., Illinois, Wabash, Maumee rivers) and the Great Lakes, continued eDNA surveillance for bigheaded carps is warranted. Bigheaded carps have the ability to spread quickly, reproduce in large numbers, and become a dominant species in an ecosystem (Peters et al. 2006; Kolar et al. 2007; Chapman and Hoff 2011). Lake Michigan is especially at risk through the CAWS due to the proximity of established and invading bigheaded carp populations, past positive eDNA detections, and the capture of one live Bighead Carp in an area above the electric dispersal barrier (Lake Calumet in 2010; Jerde et al. 2011).

If bigheaded carps enter Lake Michigan, they will likely spread throughout the Great Lakes basin due to the natural and man-made connections and the widespread distribution of suitable habitat and food resources (Cudmore and Mandrak 2011, Currie et al. 2011, Kocovsky et al. 2012). While bigheaded carps may not find all areas of the basin suitable, the Great Lakes contain areas where the fish may become established and cause ecological alterations (Herborg et al. 2007). Models of food consumption by Bighead and Silver Carps indicate that some areas of the Great Lakes have sufficient food to support populations (Cooke and Hill 2010). Regions of particular risk include Green Bay and western Lake Erie due to favorable food resources and environmental conditions, however, all of the Great Lakes could potentially provide habitat for bigheaded carps in rivers and shallower, more productive zones. Risk assessments have shown that nearly two dozen rivers in U.S. Great Lakes waters alone are potentially suitable for bigheaded carp reproduction (Kolar et al. 2007, Kocovsky et al. 2012).

Effective management of bigheaded carps on the leading edge of their invasion front requires consistent and intensive monitoring to detect presence while densities may still be low. Consequently, eDNA surveillance remains a valuable tool used by the USFWS to detect the presence/absence of bigheaded carps. Environmental DNA methodology is rapidly advancing with refinements and advances in technology (Farrington et al. 2015) and validation in field studies (Mahon et al. 2013; Smart et al. 2015). Therefore, eDNA sampling will continue to be paired with traditional field gears, as an early indicator of bigheaded carp presence/absence in Lake Michigan. Future eDNA sampling in Lake Michigan will focus on areas that have high likelihood of attracting and retaining bigheaded carps (e.g., rivers, shallow/productive bays, drowned river mouths).

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There are dozens of potential non-native species that have a reasonable likelihood of being introduced into Lake Michigan and establishing self-sustaining populations (Kolar and Lodge 2002; Rixon et al. 2005; Howeth et al. 2016). Many watch list species have an analogous congener already established in Lake Michigan. These watch list species should have similar vulnerabilities to our gears as their congeners, allowing us to gauge the effectiveness of our gears for collecting these species. For example, Monkey Goby Neogobius fluviatilis is a watch list species which is morphologically and ecologically similar to the already established invasive Round Goby routinely collected in our experimental gill nets, therefore, we have reasonable expectation that if present, this gear should detect Monkey Goby.

Based on analysis of species accumulation curves, we were able to effectively sample near-shore fish communities at the current five hotspot locations. The curves indicated that most available species were present, but differed in the quality of information provided. Species accumulation methods typically underestimated species richness thereby overestimating sampling efficiency, a problem common to species extrapolation methods (Gotelli and Colwell 2011). .

Sampling gears were tailored to target near-shore and benthic habitats, areas that typically harbor the highest aquatic diversity in lentic systems (Forbes 1925). Similar to previous years’ sampling events, we found established invasive species widespread at all hotspot locations, especially Round Goby and Alewife. White Perch were most abundant in estuarine habitat of lower Green Bay and were rarely found elsewhere. Additionally, in 2015, we collected a two adult Grass Carp Ctenopharyngodon idella by electrofishing at Milwaukee (N=1; diploid) and Burns Harbor (N=1; triploid), but did not detect any during 2016, likely reflecting their low abundance in Lake Michigan. Currently, efforts are underway to develop eDNA techniques to better monitor for this species.

Bloody Red Shrimp and scud are common and widespread invasive species in the Great Lakes (Witt et al. 1997; Dermott et al. 1998; Nalepa et al. 2001; Grigoravich et al. 2003a; Marty et al. 2010). Nonetheless, our monitoring is the first to detect these invasive species at multiple locations within Lake Michigan according to the GLANSIS website (https://www.glerl.noaa.gov/res/Programs/glansis/glansis.html). We also confirmed the presence of live Asian Clams Corbicula sp. in Green Bay. However, it is unclear whether: 1) we are the first to document these species in those locations, 2) researchers are not sampling benthos communities of nearshore Lake Michigan, and 3) they may not be uploading and documenting their species accounts. We believe few studies currently monitor for benthic invertebrates in nearshore Lake Michigan, but the fact we found Asian Clam species accounts on another website leads us to believe that species accounts are not being uploaded. Documenting the presence of an invasive species is important because early detection is the key to successful eradication measures (Tobin et al. 2014). Early detection and eradication of invasive species are shared goals of invasive species programs, but these are difficult and highly unsuccessful, respectively. To some degree, it is equally as important to document spatial and temporal components as

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invasive species spread across the Great Lakes. This provides additional information that can contribute measures to control their spread.

These findings represent an large-scale investigation of near-shore and river locations most likely to be impacted by new species invasions. Sampling protocols for bigheaded carps using eDNA are well-established and continue to adaptively provide reliable early detection capabilities. The GBFWCO AIS program has been refining methods for sampling fish and benthic macroinvertebrates and will continue to improve efficiency and accuracy. No new invasive species were found at our sampling locations this year; yet early detection and monitoring efforts will continue in Lake Michigan to help protect and conserve the distinctive and highly valuable fish and wildlife resources within the watershed.

Conclusions

● We found no evidence that Bighead or Silver Carp have migrated into Lake Michigan or its tributaries. ● No new non-native fish species were detected at the five hotspot sites. ● We documented the occurrence of several invasive benthic macroinvertebrate species at new locations in Lake Michigan. ● A targeted sampling approach paired with complimentary sampling gears provides for an extensive EDM program, while an adaptive framework allows for continual gains in terms of our AIS programs effectiveness and efficiency.

Future Work

Based on the adaptive framework of the program, our AIS monitoring efforts will continue to progress in 2017. Risk assessments and sampling protocols are continuously being reevaluated and updated to reflect our current understanding of AIS. Both benthos and ichthyoplankton sampling will greatly be expanded to include all hotspot locations. Benthos and ichthyoplankton sampling gears and methodology are being refined to improve sampling capabilities. Additionally, we are implementing a seasonal catch comparison of our night electrofishing efforts, to further evaluate traditional gear sampling protocols. Also, we will be piloting a nearshore zooplankton sampling strategy. All these changes, reflect fine-tune adjustments to our AIS program, based on results from previous field work and by incorporating the latest AIS knowledge, in an effort to provide an effective and efficient early detection and monitoring program for new AIS in Lake Michigan.

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Acknowledgements

This report represents the collective work of the entire Aquatic Invasive Species program at the Green Bay Fish and Wildlife Conservation Office during 2016 and includes: Jessica Finger, Brandon Harris, Tyler Harris, Cari-Ann Hayer, Lisa LaBudde, Matthew Petasek, Anthony Rieth, Jacob Richter, Rachel Richter, Marian Shaffer, Darin Simpkins, Bradley Smith, Matthew Stowe and Touhue Yang. Funding for AIS program is largely provided through the Great Lakes Restoration Initiative, which is administered through the Environmental Protection Agency.

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Figure 1. Lake Michigan study areas (hotspots) sampled by the Aquatic Invasive Species (AIS) program during 2016. All hotspot study sites are outlined in red.

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Figure 2. Study area of rivers flowing into Lake Michigan for eDNA analysis.

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Figure 3. Lower Green Bay, WI traditional gear study area with sample locations for each gear type. Sampling was performed during May-October 2016. Five additional electrofishing runs were performed upstream in the Fox River near the DePere Dam but are not depicted.

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Figure 4. Map of the Milwaukee, WI traditional gear study area with sample locations for each gear type. Sampling was performed during September 2016.

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Figure 5. Map of the Chicago, IL traditional gear study area with sample locations for each gear type. Sampling was performed during August 2016.

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Figure 6. Map of the Calumet Harbor, IL traditional gear study area with sample locations for each gear type. Sampling was performed during August 2016.

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Figure 7. Map of the Burns Harbor, IN traditional gear study area with sample locations for each gear type. Sampling was performed during August 2016.

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Figure 8. Map of light trap sampling locations and whether or not larvae were collected at that location in the Milwaukee River and Milwaukee Harbor, WI during 2016 ichthyoplankton surveys (N=25).

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Figure 9. Map of light trap sampling locations in the Lower Fox River, Green Bay, WI during 2016 ichthyoplankton surveys (N=24).

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Figure 10. Length-frequency histograms for nine species collected across all gear types and sites during 2016 Lake Michigan sampling. Note that the X and Y axes differ in scale across species.

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Figure 11. Dendrogram of fish communities at hotspot sampling sites calculated using average-linkage hierarchical cluster analysis in 2016.

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Figure 12. Sample size based rarefaction (solid lines) and extrapolation (dashed lines, up to double the reference sample size) of species richness for all gears used in 2016 with the five hotspots combined. The reference samples are denoted by the solid dots. The 95% confidence intervals (shaded regions) were obtained using a bootstrap method based on 200 replications. The rarefaction/extrapolation curves along with the asymptotic species richness for each gear were calculated by the Chao et al. 2014 estimator functions within the iNEXT package using the software program R.

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Figure 13. Sample size based rarefaction (solid lines) and extrapolation (dashed lines) of species richness for all traditional gears used in 2016 at the five hotspot locations. The reference samples are denoted by the solid dots. The 95% confidence intervals (shaded regions) were obtained using a bootstrap method based on 200 replications. The rarefaction/extrapolation curves along with the asymptotic species richness for each location were calculated using the Chao et al. 2014 estimator functions within the iNEXT package run in the software program R. Within the figure; y-axis levels vary, n = units of effort, S = # of species caught, and E = Sampling efficiency.

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Table 1. River name, state, number of reaches, number of water samples taken, and dates sampled for environmental DNA analysis in nine hotspot rivers for Bighead and Silver Carp presence during 2016.

Location State Reaches Samples Dates Sampled

Fox River WI 4 80 April 28 June 7 June 28

Milwaukee River WI 4 80 April 28 June 8 June 28

St. Joseph River MI 8 160 May 4 June 15 July 7

Kalamazoo River MI 8 160 May 5 June 17 July 8

Macatawa River MI 4 80 May 6 June 16 July 9

Galien River MI 1 20 May 7 June 18 July 10

Black River MI 1 20 May 7 June 18 July 10

Grand River MI 10 200 May 8 & 9 June 19 & 20 July 11 & 12

Muskegon River MI 6 120 May 10 June 21 July 13

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Table 2. Total catch from all gears combined at five hotspot sampling locations in Lake Michigan during 2016 including catches from an early season (May) paired fyke net gear comparison in Green Bay. Names of invasive species are bolded.

Burns Calumet Green Gear Grand Species Harbor Harbor Chicago Bay Comparison Milwaukee Total Alewife* Alosa pseudoharengus 239 216 144 291 0 299 1,189 Banded Killifish Fundulus diaphanus 12 23 0 29 5 2 71 Bigmouth Buffalo Ictiobus cyprinellus 0 0 0 7 0 0 7 Black Buffalo Ictiobus niger 6 1 0 0 0 0 7 Black Bullhead Ameiurus melas 4 0 0 8 0 12 24 Black Crappie Pomoxis nigromaculatus 37 4 0 66 33 1 141 Bluegill Lepomis macrochirus 317 10 5 64 2 87 485 Bluegill hybrid 0 3 0 0 0 0 3 Lepomis macrochirus x Lepomis spp.

Bloater Coregonus hoyi 0 0 0 0 0 8 8 Blacknose Dace Rhinichthys atratulus 0 0 0 0 0 2 2 Bluntnose Minnow Pimephales notatus 18 9 7 4 1 12 49 Brown Trout** Salmo trutta 0 1 4 0 0 51 56 Bowfin Amia calva 1 0 0 10 0 1 12 Brown Bullhead Ameiurus nebulosus 1 5 3 12 2 1 24 Brook Stickleback Culaea inconstans 145 0 1 0 0 5 151 Burbot Lota lota 0 0 0 15 0 0 15 Channel Catfish Ictalurus punctatus 32 15 1 83 4 1 136 Chinook Salmon** Oncorhynchus tshawytscha 3 0 0 0 0 17 20 Central Mudminnow Umbra limi 0 0 0 4 0 0 4 Common Shiner Luxilus cornutus 0 0 0 9 0 182 191 Common Carp* Cyprinus carpio 45 39 28 136 10 18 276 Coho Salmon** Oncorhynchus kisutch 0 0 0 1 0 5 6 Emerald Shiner Notropis 0 1 0 514 1 16 532

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atherinoides Flathead Catfish Pylodictis olivaris 7 0 1 5 0 0 13 Fathead Minnow Pimephales promelas 2 0 0 15 0 27 44 Freshwater Drum Aplodinotus grunniens 31 50 13 544 11 1 650 Gizzard Shad Dorosoma cepedianum 580 176 89 4,435 1,731 191 7,202 Goldfish* Carassius auratus 9 1 0 0 0 1 11 Golden Redhorse Moxostoma erythrurum 11 10 1 0 0 33 55 Golden Shiner Notemigonus crysoleucas 20 0 0 1 0 0 21 Grass Pickerel Esox americanus 1 0 0 1 0 0 2 Green Sunfish Lepomis cyanellus 48 2 0 53 2 10 115 Green Sunfish x Pumpkinseed 0 0 0 1 0 0 1 Lepomis cyanellus x Lepomis gibbosus

Lake Chub Couesius plumbeus 0 0 2 0 0 2 4 Lake Sturgeon Acipenser fulvescens 2 1 0 0 0 9 12 Lake Trout Salvelinus namaycush 4 0 0 0 0 3 7 Largemouth Bass Micropterus salmoides 289 5 0 64 0 58 416 Longnose Dace Rhinichthys cataractae 0 11 0 0 0 19 30 Longnose Gar Lepisosteus osseus 0 0 0 6 2 0 8 Longnose Sucker Catostomus catostomus 6 0 2 0 0 14 22 Logperch Percina caprodes 0 0 0 24 0 0 24 Mimic Shiner Notropis volucellus 0 0 0 0 0 4 4 Mottled Sculpin Cottus bairdii 0 0 0 0 0 2 2 Muskellunge Esox masquinongy 0 0 0 3 0 0 3 Northern Pike Esox lucius 9 4 0 34 0 10 57 Orangespotted Sunfish Lepomis humilis 0 4 0 0 0 0 4 Pumpkinseed Lepomis gibbosus 17 33 2 30 1 22 105 Quillback Carpiodes cyprinus 7 1 0 98 49 0 155 Rainbow Smelt* Osmerus 9 0 0 1 0 449 459

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mordax Rainbow Trout** Oncorhynchus mykiss 0 0 9 0 0 3 12 Rock Bass Ambloplites rupestris 33 158 161 77 8 304 741 Round Goby* Neogobius melanostomus 152 155 143 92 151 660 1,353 Round Whitefish Prosopium cylindraceum 0 0 0 0 0 4 4 Sauger Sander canadensis 0 0 0 6 0 0 6 Sand Shiner Notropis stramineus 26 20 2 0 0 42 90 Spotfin Shiner Cyprinella spiloptera 0 1 0 0 0 0 1 Shorthead Redhorse Moxostoma macrolepidotum 61 37 8 12 0 7 125 Silver Redhorse Moxostoma anisurum 4 5 0 0 0 0 9 Smallmouth Buffalo Ictiobus bubalus 1 1 0 0 0 0 2 Smallmouth Bass Micropterus dolomieu 497 144 207 133 1 178 1,160 Shortnose Gar*** Lepisosteus platostomus 0 0 0 10 1 0 11 Spotted Gar Lepisosteus oculatus 0 0 0 2 0 0 2 Spotted Sucker Minytrema melanops 1 0 0 0 0 1 2 Spottail Shiner Notropis hudsonius 590 327 215 273 22,167 62 23,634 Trout-Perch Percopsis omiscomaycus 0 0 0 49 289 0 338 Three-Spine Stickleback* Gasterosteus aculeatus 0 1 0 0 0 0 1 Walleye Sander vitreus 9 5 0 477 12 4 507 Warmouth Lepomis gulosus 12 0 0 0 0 0 12 White Bass Morone chrysops 0 5 0 176 2 0 183 White Crappie Pomoxis annularis 11 0 0 1 1 0 13 White Perch* Morone americana 6 0 2 1,350 6 1 1,365 White Sucker Catostomus commersonii 37 6 6 62 7 741 859 Unidentified 1 0 0 0 2 1 4 Yellow Bullhead Ameiurus natalis 0 3 0 5 0 0 8

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Yellow Perch Perca flavescens 1,703 892 969 3,596 131 1,291 8,582 Yellow Bass Morone mississippiensis 0 0 1 2 0 0 3

Grand Total 5,056 2,385 2,026 12,891 24,632 4,874 51,864 *indicates an invasive species

**indicates deliberately introduced sport fish

***indicates non-native to Lake Michigan

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Table 3. Gear and units of effort allocations expended during 2016 at the five hotspot locations in Lake Michigan using three traditional fish sampling gears.

Electrofishing Fyke Net Gill Net Total % Effort

Burns Harbor 41 18 28 87 21%

Calumet Harbor 36 18 21 75 18%

Chicago 40 12 20 72 18%

Green Bay 42 28 23 93 23%

Milwaukee 32 24 28 84 20%

Total 191 95 125 411

% Allocation 47% 24% 29%

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Table 4. Environmental conditions and sampling site characteristics at light trap locations in the Milwaukee River and Milwaukee Harbor during 2016 ichthyoplankton surveys.

Range Mean

Site Depth (m) 1.5 – 9.0 4.31 ± 2.01 S.D.

Water Temperature (°C) 19.83 – 25.72 23.26 ± 2.04 S.D.

pH 7.83 – 8.44 8.20 ± 0.18 S.D.

Turbidity (NTU) 0.27 – 3.82 2.09 ± 0.93 S.D.

Chlorophyll-a (mg/L) 2.54 – 27.82 7.81 ± 4.97 S.D.

Dissolved Oxygen (mg/L) 4.52 – 10.19 6.95 ± 1.64 S.D.

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Table 5. Environmental conditions and sampling site characteristics at light trap locations in the Lower Fox River – Green Bay during 2016 ichthyoplankton surveys.

Range Mean

Site Depth (m) 0.7 – 5.7 1.48 ± 0.90 S.D.

Water Temperature (°C) 22.92 – 24.72 23.8 ± 0.45 S.D.

pH 8.47 – 8.81 8.61 ± 0.9 S.D.

Turbidity (NTU) 9.28 – 70.82 14.99 ± 12.89 S.D.

Chlorophyll-a (mg/L) 17.83 – 40.09 28.59 ± 6.71 S.D.

Dissolved Oxygen (mg/L) 9.27 – 11.19 10.27 ± 0.55 S.D.

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On the back cover:

Lisa LaBudde releases a native Lake Sturgeon Acipenser fulvescens captured using a gill net back into Lake Michigan at Burns Harbor, Indiana.

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