coverage of aquatic invasive risk assessment in usfs region 6 · 2016-07-19 · coverage of aquatic...

Coverage of Aquatic Invasive Risk Assessment in USFS Region 6

Rebecca Flitcroft1, Jim Capurso2, Kelly Christiansen1, and Bruce Hansen1

1U.S. Forest Service Pacific Northwest Research Station

2U.S. Forest Service Region 6

[email protected] (541) 750-7346




New Zealand Mudsnails

Potamopyrgus antipodarum

Purple Loosestrife

Lythrum salicaria

Rusty Crayfish

Orconectes rusticus

Red Swamp Crayfish

Procambarus clarkii

Yellow Flag Iris

Iris pseudacorus

mailto:[email protected]




Citation: Flitcroft, R., J. Capurso, K. Christiansen, and B. Hansen. 2016. Coverage of aquatic invasive risk assessment in USFS Region 6. Report to U.S. Forest Service Region 6. Corvallis, OR: U.S. Department of Agriculture, Forest Service, Pacific Northwest Research Station.

Available from: Rebecca Flitcroft, USFS Pacific Northwest Research Station, Corvallis Forestry Sciences Laboratory, 3200 SW Jefferson Way, Corvallis OR, 97331; [email protected] (541) 750-7346

Cartography by Kelly Christiansen; Graphic design and layout by Kathryn Ronnenberg, USFS PNW Research Station.

All cover photos from Wiki Commons. New Zealand Mudsnail courtesy of U.S. Geological Survey; Purple Loosestrife courtesy of Magnus Manske; Rusty Crayfish courtesy of Carthage College of Wisconsin; Red Swamp Crayfish courtesy of Natuur12; Quagga Mussels courtesy of National Oceanic and Atmospheric Administration; Yellow Flag Iris courtesy of Joost J Bakker.


Coverage of Aquatic Invasive Risk Assessment in USFS Region 6—Flitcroft et al. 2016 3

Executive Summary

The management of public lands in the western US strives toward ecological sustainability and the well-

being of local communities. The restoration and conservation of economically and culturally important fishes

and other aquatic species is often focused on federally managed lands. A critical element of the stewardship

of aquatic species is the identification and eradication of aquatic invasive species. Aquatic invasive species are

known to have devastating ecological (Vitousek et al. 1997) and economic impacts (Pimentel et al. 2000) if they

are allowed to develop strongholds outside their native range.

As part of their overall aquatic monitoring program, Region 6 of the USDA Forest Service (USFS) and

the Pacific Northwest Research Station sought to evaluate how well existing, statistically rigorous monitoring

programs may represent monitoring for aquatic invasive species. To this end, watersheds in Region 6 with at least

5% USFS ownership were identified, and risk factors for aquatic invasive species were mapped. The sampling

strategy and survey watersheds visited by the Aquatic and Riparian Effectiveness Monitoring Program (AREMP)

west of the Cascade crest and the PACFISH/INFISH Biological Opinion Effectiveness Monitoring Program

(PIBO) east of the Cascade crest were utilized within the framework of USFS watersheds and aquatic invasive

risk factors. This report represents a new application of existing monitoring programs and is meant to leverage

their efforts with respect to aquatic invasive species. It is not a critique of the utility of these programs to achieve

their mandated goals.

Our evaluation found that wadeable stream sections are well-represented in the current surveys, particularly

west of the Cascade crest. Non-wadeable stream sections, which may be at highest risk for invasion by aquatic

species, are not currently well-represented by either the AREMP or PIBO survey programs. This result is not

surprising, given the focus on wadeable stream reaches by both of these survey programs.

Five recommendations that would enhance the ability of Region 6 to monitor aquatic invasive species that

emerge from this analysis are to:

1) Expand a statistically rigorous sampling program to the Fremont-Winema National Forest.

2) Augment existing statistically rigorous field sampling of the wadeable portion of river systems by using

next-generation e-DNA sampling techniques in non-wadeable stream segments.

3) Develop a regional genomic library of invasive aquatic species in Region 6 to facilitate next-generation

e-DNA sampling in non-wadeable stream segments.

4) Expand aquatic invasive species monitoring to lentic environments.

5) Explore the probability of detection of aquatic invasives with existing survey protocols.


Introduction

Public lands in the western U.S. are managed for ecological sustainability and the well-being of local

communities. An important part of this mandate for managers of national forests in the Pacific Northwest is the

conservation, restoration, and recovery of aquatic habitats that support native fishes. One element of this work is

vigilance in the detection and eradication of aquatic invasive species (AIS) that have the potential to compromise

local habitats and aquatic and riparian ecosystems. Aquatic invasive species are known to have devastating

ecological (Vitousek et al. 1997) and economic impacts (Pimentel et al. 2000) if they are allowed to develop

strongholds outside their native range.

In freshwater settings, one primary agent that distributes aquatic invasive species is the boots and boats of

recreationalists (Johnson et al. 2001). This means that some of the highest-risk locations for potential invasion

by AIS are the places most frequented by anglers, boaters, and campers. This includes many locations on U.S.

national forest lands. To promote management of moving waters under the jurisdiction of the USFS, Region

6 and the Pacific Northwest Research Station have evaluated how well existing, statistically rigorous sampling

programs represent lotic environments in all Region 6 watersheds (national forests in Region 6, Figure 1), in

relation to invasion risk factors for aquatic invasive species. This evaluation includes six elements:

1. Description of current Focal Aquatic Invasive Species in Region 6, and current documentation of aquatic

invasive species in the region.

2. Description of the two statistically rigorous sampling programs in Region 6 (AREMP and PIBO). This is

not a comparison of the effectiveness of these programs; rather, it is an evaluation of how they might be

extended to answer a new monitoring question.

3. Identification of the population of interest for monitoring of aquatic invasive species.

4. Mapping risk factors for aquatic invasive species, including high-use locations (boat launches and

campgrounds) and wadeable/non-wadeable classification of streams.

5. Evaluation of the representativeness of existing, statistically rigorous monitoring activities (AREMP and

PIBO) relative to watershed characteristics that measure risk factors for aquatic invasive species.

6. Recommendations for the monitoring of areas not currently included in systematic monitoring programs,

or areas of high risk that are not adequately covered by ongoing systematic monitoring programs.

7. Recomendations for additional work necessary for applied monitoring, including probability of invasive

species detection using various methods.

Aquatic Invasive Species and Region 6

Region 6 of the USFS encompasses 17 National Forests and a National Scenic Area distributed across

the states of Oregon and Washington (Figure 1). These national forests provide recreational opportunities,

critical habitat for a variety of threatened and endangered species, and serve as the major source of municipal

and agricultural water for the Pacific Northwest (USDA Forest Service 1990). These resource demands make

monitoring of invasive species of critical management interest.

An AIS Focal Species List was established by Region 6 in 2010 and updated annually with the assistance of

regional AIS experts in state agencies and universities. The intent of the list is to be a basic catalog of invasive


species with high probability of invasion and high potential for significant environmental and economic impacts.

Twenty-four focal species of interest are currently listed for Region 6, including both lotic and lentic flora and

fauna (Table 1). The environmental characteristics necessary for the establishment and spread of each species are

varied, but all have been known to become invasive outside their natural range.

Figure 1. National forests in USDA Forest Service Region 6.


Table 1. Region 6 focal aquatic invasive species list for 2016.

Type Common name Scientific name Species code

Aquatic animals

Chinese Mystery Snail Cipangopaludina chinensis CICH

Asian Clam Corbicula fl ina COFL

Zebra Mussel Dreissena polymorpha DRPO

Quagga Mussel Dreissena rostriformis bugensis DRRO

Nutria Myocaster coypus MYCO

Ringed Crayfish Orconectes neglectus ORNE

Rusty Crayfish Orconectes rusticus ORRU

Northern Crayfish Orconectes virilis ORVI

New Zealand Mudsnail Potamopyrgus antipodarum POAN

Red Swamp Crayfish Procambarus clarkii PRCL

Big Eared Radix Radix auricularia RAAU

American Bullfrog Rana catesbeiana RACA

(now Lithobates catesbeianus)

Aquatic plants

Terrestrial

Animal

Flowering Rush Butomus umbellatus BUUM

Didymo Didymosphenia geminata DIGE

Brazilian Elodea Egeria densa EGDE

Hydrilla Hydrilla verticillata HYVE

Yellow Flag Iris Iris pseudacorus IRPS

Water Primrose Ludwigia spp. LU

Garden Loosestrife Lysimachia vulgaris LYVU

Purple Loosestrife Lythrum salicaria LYSA

Nonnative milfoils Myriophyllum spp. MYSP

Yellow Floating Heart Nymphoides peltata NYPE

Common Reed Phragmites australis PHAU

Curly-leaf Pondweed Potamogeton crispus POCR

Feral swine Sus scrofa SUSC

Terrestrial plants

Garlic Mustard Alliaria petiolata ALPE

Old Man’s Beard Clematis vitalba CLVI

Japanese Knotweed Fallopia japonica FAJA

English Ivy Hedera helix HEHE

Giant Hogweed Heracleum mantegazzianum HEMA

Orange Hawkweed Hieracium aurantiacum HIAU

Yellow Archangel Lamiastrum galebdolon LAGA

Hybrid Bohemian Knotweed Polygonum x bohemicum POBO

Giant Knotweed Polygonum sachalinese POSA


H

i

m

alayan Blackberry Rubus discolor RUDI

Salt Cedar Tamarisk ramosissima TARA


Aquatic invasive species present in lotic and riparian environments have already been identified in some

locations in, or in close proximity to, USFS lands in Region 6. We mapped documented invasive aquatic animal

species in Oregon and Washington present in riparian areas or lotic environments using databases available from

the U.S. Geological Survey (USGS).1 To facilitate a comparison among species, we divided the species into two

categories (based on documented observation) and displayed them in map form. The categories were “rare” (<30

occurrences), and “common” (>30 occurrences) (Figure 2). Although they are not widely distributed among the

forests of Region 6, awareness of the presence of these species offers managers an opportunity to take the lead in

combatting their spread through early detection and rapid response with partner agencies, organizations, and

Tribes.

Aquatic Monitoring Programs—AREMP and PIBO

In much of Region 6, ad-hoc level-2 surveys2 and two statistically rigorous watershed sampling programs are

tasked to monitor stream condition. Because we are interested in how well surveys capture stream conditions

relevant for monitoring aquatic invasive species, we will focus on the two statistically rigorous sampling

programs (AREMP and PIBO). However, level-2 stream surveys may also play an important role in monitoring

for aquatic invasive species. The ad-hoc nature of the survey locations, and the absence of a consistent return

interval, makes these surveys potentially informative in finding random occurrences of aquatic invasive species,

but is not compatible with a probability-based long-term monitoring survey design or monitoring strategy.

West of the Cascade crest, in the area covered by the Northwest Forest Plan (NWFP), the Aquatic and

Riparian Effectiveness Monitoring Program (AREMP) is responsible for monitoring aquatic conditions at the

scale of the 6th-field hydrologic unit (sub-watershed), as part of an interagency forest management plan (Miller

et al. in press). East of the Cascade crest, in the drainage of the Interior Columbia River basin, the USFS’s

PACFISH/INFISH Biological Opinion Effectiveness Monitoring Program (PIBO) is responsible for assessing the

integrity of riparian and instream habitats (Kershner et al. 2004). South-central of Oregon, where the Fremont-

Winema National Forest is located, is the only portion of Region 6 in which a statistically rigorous sampling

program is not currently active. There are currently discussions about expanding PIBO sampling points onto the

Fremont-Winema National Forest. Characteristics of AREMP and PIBO are different in terms of the population

of watersheds of interest, and sample site identification.

The differences between AREMP and PIBO are relevant because we are interested in how they currently

monitor aquatic invasive species under our definitions of areas of risk. This is an assessment for the purpose of

determining the adequacy of repurposing these existing monitoring programs for detection of aquatic invasive

species. Therefore, we must be clear about what AREMP and PIBO are intended to monitor in the context of

aquatic invasive species detection using the existing designs of the respective programs.

1Note: these data are preliminary or provisional and are subject to revision. They were provided to meet the need for timely best science. The data have not received final approval by the USGS and are provided on the condition that neither the USGS nor the U.S. Government shall be held liable for any damages resulting from the authorized or unauthorized use of the data.

2Level-2 stream surveys involve field measurements of characteristics of aquatic conditions (such as substrate, gradient, water depth, etc.) along a designated length of stream (sensu Hankin and Reeves 1988).


Figure 2. Aquatic invasive species in close proximity to, or found on, USFS land. Common species were defined as having more than 30 occurrences and rare species as having fewer than 30 occurrences in data sets acquired from the U.S. Geological Survey.


Although AREMP and PIBO are both intended to monitor watersheds, each program defines this monitoring

in different ways. Both programs use a spatially random sampling design (Miller et al. in press, Kershner et al.

2004, respectively) to select monitored watersheds. Both AREMP and PIBO are intended to monitor HUCs at the

6th-field scale (subwatersheds) (Miller et al. in press, Kershner et al. 2004, respectively) through the collection of

in-stream survey data that describe river geomorphology, riparian vegetation, and macroinvertebrates. Within a

selected watershed, AREMP subsamples multiple river reaches and uses the composite of conditions across

reaches to describe the watershed. In contrast, PIBO selects the reach at the lowest wadeable point in a watershed

(generally with a gradient of <4%), which is intended to be a depositional reach (characterized as low-gradient),

the idea being that the characteristics of the deposited material and geomorphology of the reach will capture

conditions throughout the watershed (Table 2). In the PIBO program, additional sites are sometimes selected in a

watershed, generally in response to the presence of grazing. However, the full suite of field sampling is not

generally done, and macroinvertebrate sampling may be omitted at these additional sites. One important mandate

of the PIBO survey program is the monitoring of federal grazing lands. To this end, this program has a strong

method for riparian vegetation inventory and a proven track record of detecting invasive terrestrial plants (Archer

et al. 2012). Likewise, AREMP has also been successful in identifying some terrestrial invasive species (Pennell

and Raggon 2016).

Table 2. Comparison of AREMP and PIBO aquatic sampling programs.

AREMP PIBO

Spatial extent Area of NW Forest Plan Interior Columbia River basin

Sample watershed 6th-field HUCs with 25% federal ownership of stream km

6th-field HUCs with 50% of watershed upstream of the study federally owned

Watershed selection Spatially random Spatially balanced random sampling design (Al-Chokhachy et al. 2013)

Sample site selection 2–9 sites randomly distributed within HUCs of interest

Lowest reach in watershed that is wadeable (gradient <4%). Additional sites may be added ad-hoc.

Return interval Varied 5 years

AIS monitoring protocol Field survey training— macroinvertebrate samples taken

Field survey training— macroinvertebrate samples taken


Sample Population and Risk Factors

Sample Population:

Statistically rigorous monitoring of lotic environments for aquatic invasive species begins by defining the

sample population. Throughout USFS Region 6, all 6th-field HUCs (USGS http://www.nrcs.usda.gov/wps/

portal/nrcs/main/national/water/watersheds/dataset/), technically defined as subwatersheds, but hereafter

referred to as watersheds, having at least 5% USFS ownership were our population of interest (this differs from

the AREMP and PIBO survey frames). A total of 2035 watersheds were identified. Of these, 1052 watersheds

were found west of the Cascade crest in the region of the NWFP, and under the monitoring aegis of AREMP

(Figure 3). Of these watersheds, 909 are part of the sample population in the AREMP program, with a total of

138 individual watersheds visited (Figure 4). A total of 983 watersheds were identified east of the Cascade crest.

Of these, 138 watersheds are found in the southernmost portion of Oregon, outside of the interior Columbia

River basin drainage, and therefore outside of the range of the PIBO survey program. The PIBO program surveys

290 watersheds in the Interior Columbia River drainage area of Region 6 (Figure 5).

High-risk locations:

The probability of colonization is different for each of the Region 6 focal invasive species, but some elements

of risk for the spread of these species are universal. In particular, we focused on the presence or absence of

high-use locations such as boat ramps, campgrounds, and trailheads. Of particular concern are locations where

individuals might be expected to directly interact with water, perhaps moving from one stream to another. This

is the case for anglers who might fish in multiple river systems, but may not sterilize their equipment between

rivers, potentially transferring invasive species from one location to another. Boats are known vectors for the

distribution of aquatic invasive species due to their easy transportability between waters by trailer. The state of

Oregon has a mandatory Aquatic Invasive Species Prevention Permit, required for all boats over a certain size

(http://www.dfw.state.or.us/conservationstrategy/invasive_species/quagga_zebra_mussel.asp).

For the purposes of this assessment, we acquired and mapped the highest-risk locations identified by

the national forests in Region 6 (source: J. Capurso, Region 6). We mapped a total of 791 high-risk locations

throughout Region 6 (141 high-use locations, 581 campgrounds, and 69 boating sites) (Figures 6, 7).

http://www.nrcs.usda.gov/wps/

http://www.dfw.state.or.us/conservationstrategy/invasive_species/quagga_zebra_mussel.asp)


Figure 3. All 6th-field HUs in Region 6 with at least 5% USFS ownership west of the Cascade crest (in the area of the NWFP), and HUs in the AREMP survey program, with other HUs containing federal ownership shown for comparison.


Figure 4. All 6th-field HUs in Region 6 with at least 5% USFS ownership west of the Cascade crest, and those surveyed by AREMP.

Coverage of Aquatic Invasive Risk Assessment in USFS Region 6-Flitcroft et al. 2016 13

N

WWE s

IDA HO

Legend

- HU with FS lands

HUs surveyed

by PIBO

Area east of

Cascade crest

o i s :o 60 M iles

I I I I I I I I 0 25 50 100 Kilometers

C A LIFOR NIA NEVA DA

Figure 5. All 6th-field HUs in Region 6 with at least 5% USFS ownership east of the Cascade crest, and those

surveyed by PIBO.


s

Nanaimo Vancouver

N

w-&w\E

Kennewick

Legend

o High-use sites

O Boating sites

- HU with FS lands

D Area west of

Cascade crest

0 25 50 100 Miles

I I

I I I I I 0 :iO GO 1 20 Kilomet ers

Figure 6. All 6th-field HUs in Region 6 with at least 5% USPS ownership west of the Cascade crest (in the area of

the NWFP), with high-use and boating sites.


N

WE

s

J' r

Legend

D High-use sites

o Recreation sites

- HU with FS lands

[ Area east of

Cascade crest

() 20 40 80 Miles

I I I I I I

I I I I I I

0 :>o GO 120 Kilometers

Figure 7. All 6th-field HUs in Region 6 with at least 5% USPS ownership east of the Cascade crest, with high-use

and recreation sites.


Wadeable and Non-wadeable Stream Reaches:

We summarized wadeable and non-wadeable streams (length in km) for each watershed at the scale of the

entire watershed. The stream data layer that we used for this analysis was generated by NetMAP/Terrainworks

(http://www.terrainworks.com/terrainworks) and represents a uniform density of stream linework, making

stream km summary information comparable across Region 6. Summarizing wadeable and non-wadeable

streams was an important step because existing monitoring programs are conducted on wadeable streams, while

some aquatic invasive risk factors (in particular, boating) generally occur in non-wadeable segments of streams.

Because of the strong relationship between drainage area and river discharge (Clarke et al. 2008), we accessed

the field survey records for AREMP to define the drainage area at which we would expect rivers to shift from

being wadeable to non-wadeable. The AREMP program is responsible for monitoring of watersheds within the

area of the Northwest Forest Plan, and surveys stream segments throughout the wadeable range (Miller et al. in

press). We found that stream reaches with upstream drainages between 0.08 km2 and 399 km2 could generally be

considered wadeable.

We wanted to use a consistent definition of wadeable and non-wadeable streams throughout watersheds

in our population of interest. The AREMP program is limited to the west side of the Cascade crest, and does

not include east-side streams. To ensure that the definition of wadeable and non-wadeable applied to east-side

streams as well, we compared our definition of wadeable and non-wadeable in east side streams to the surveys

conducted by PIBO. This program also conducts field work using surveyors on foot. We found strong overlap

between the locations surveyed by PIBO and areas we identified as wadeable. Therefore, we were comfortable

using our definition of wadeable and non-wadeable streams based on drainage area throughout Region 6

(Figures 8, 9).

http://www.terrainworks.com/terrainworks)


Legend

D High-use sites

o Boating sites

- - Wadeable streams

- Non-wadeable streams

DHU with FS lands

CJ Area west of

Cascade crest

o :ns 7..r, I I I I

I S Miles

I I I

I I I I I I I I

0 5 I 0 20 Ki lomelers

Figure 8. Example of wadeable and non-wadeable stream segments of streams in the AREMP survey area west of

the Cascade crest.


N D W E

s

Legend

I I I

1 0

7..'J

I I

I 20 Kilometers

D High-use sites

o Recreation sites

Non-wadeable

-- Wadeable

DHU with FS lands

DArea east of

Cascade crest

Figure 9. Example of wadeable and non-wadeable stream segments of streams in the PIBO survey area east of

the Cascade crest.


Alignment of Watersheds Currently Surveyed by AREMP and PIBO with Risk Factors for Aquatic Invasive Species

In this evaluation, we wanted to know how well surveys conducted by AREMP and PIBO align with risk

factors for aquatic invasive species in Region 6. We split the region into two areas along the Cascade crest

(Figures 3, 5). To the west, we compared AREMP survey watersheds with all watersheds having at least 5%

federal ownership. To the east, we compared PIBO survey watersheds with all watersheds having at least 5%

federal ownership.

Analysis Methods—West of the Cascade Crest

The AREMP survey program sample population encompasses 909 of 1052 watersheds (including 138

watersheds with surveyed sites) in the range of the Northwest Forest Plan in western Oregon and Washington.

All survey sites occur in wadeable stream reaches. We conducted three watershed-scale analyses using the

AREMP data set. In the first, we compared watersheds surveyed by the AREMP program (n = 138) to all USFS

watersheds with at least 5% federal ownership. In the second, we compared all 909 AREMP watersheds to the

143 USFS watersheds that are not part of the AREMP sample population. For our third comparison, we sought

to ensure that we were comparing the 143 watersheds not part of the AREMP population to similar AREMP

watersheds. To accomplish this, we sub-selected 183 watersheds having USFS ownership of 54% or greater (one

standard deviation around the average amount of USFS ownership in AREMP watersheds; average of 79% ±

25%). This reduced the number of AREMP watersheds for comparison to 183 and was more consistent with the

average federal ownership in non-AREMP watersheds (average USFS ownership of 20%).

We compared AREMP and non-AREMP survey watersheds for each of the three data sets (the 138 surveyed

AREMP watersheds, the full AREMP data set compared to non-AREMP watersheds, and the reduced AREMP

sample population compared to non-AREMP watersheds) using t-tests of 6 watershed-scale variables: area

(acres); USFS ownership (%); wadeable stream length (m); non-wadeable stream length (m); wadeable stream

length on USFS land (m); and non-wadeable stream length on USFS land (m). High-risk locations were not

evenly distributed across watersheds west of the Cascade crest. Further, many watersheds did not contain high-

risk locations. The abundance of 0 counts precludes simple statistical comparison tests. Thus, we summarized in

tabular form the number of high risk locations in both sample data sets for comparison only.

Results—West of the Cascade Crest

Results of comparative t-tests showed a lack of statistically significant differences in most variables,

comparing watersheds surveyed by AREMP with all watersheds with 5% USFS ownership. However, there were

statistically significant differences between most characteristics of AREMP streams and non-AREMP streams

for the full data set (Table 3). The only variable that was not statistically significant between these two sets of

watersheds was the length of wadeable streams (Satterthwaite p-value = 0.8981). However, the reduced AREMP

and non-AREMP comparative t-tests suggested that the non-AREMP sites are indistinguishable from AREMP

sites for the variables of wadeable stream length, non-wadeable stream length, and non-wadeable USFS-owned

stream length (Table 3).


Summaries comparing the number of USFS high-use sites, campgrounds, and boating sites in the sampled,

population-scale, and reduced AREMP watersheds with the non-AREMP watersheds show that AREMP

generally surveys more watersheds with specific risks for invasive species introduction than are present in non-

AREMP watersheds (Table 4). Likewise, when all risks in a watershed were tallied, we found that the AREMP

survey program generally visits watersheds with similar numbers of risks compared to non-AREMP watersheds

(Table 5).

The results of these comparisons show that watershed-scale variability in non-AREMP watersheds is

fairly well represented within the range of variability that is captured by the AREMP data set.

Table 3. T-test results comparing watersheds in statistically rigorous sampling programs with unsurveyed watersheds in Region 6. All USFS watersheds with at least 5% federal ownership that occur west of the Cascade crest are compared against the AREMP survey program, whereas east of the Cascade crest, comparisons are made with the PIBO survey program. In this case, high p-values (bold face) signify similarity between sets of areas compared, thus more representative sampling, whereas low p-values suggest that sets of areas compared are not similar.

USFS

ownership Wadeable

stream length Non-

wadeable stream

Wadeable,

Non-wadeable,

Comparison Area (m2) (%) (m) length (m) USFS (m) USFS (m)

- - - - - - - - - - - - - - - - - - - - - - - Satterthwaite p-value - - - - - - - - - - - - - - - - - - - - - -

AREMP surveys vs. non-AREMP 0.0018 <0.0001 0.1404 0.4146 <0.0001 0.5505 (n = 138, 914) AREMP sample population vs. 0.0265 <0.0001 0.8981 0.0021 <0.0001 0.0001 non-AREMP (n = 909, 143) Reduced AREMP vs. non-AREMP 0.0439 <0.0001 0.1650 0.3721 <0.0001 0.9610 (n = 183, 143) PIBO vs. non-PIBO 0.6663 <0.0001 0.0024 <0.0001 <0.0001 <0.0001 (n = 290, 693) PIBO vs. non-PIBO (north region) 0.0313 <0.0001 0.0056 0.0146 <0.0001 <0.0001 (n = 290, 555) Non-PIBO (north) vs. non-PIBO <0.0001 <0.0001 0.5672 <0.0001 0.0110 <0.0001 (south) (n = 555, 137) PIBO vs. non-PIBO (south) 0.0035 0.0023 0.0700 <0.0001 <0.0001 <0.0001 (n = 290, 131)


Table 4. Summary of risk factors for aquatic invasive species invasion that occur in watersheds surveyed by the AREMP and PIBO survey programs compared to unsurveyed watersheds in their area of responsibility in Region 6. All USFS watersheds with at least 5% USFS ownership that occur west of the Cascade crest are compared against the AREMP survey program, whereas east of the Cascade crest, comparisons are made with the PIBO survey program.

High-use Campgrounds Boating sites* Total

- - - - - - - - - - - - Number of risk factors - - - - - - - - - - - -

All Region 6 141 581 69 791

All USFS west side 92 53 8 72

AREMP surveys only 11 53 8 72

AREMP full sample population 83 315 41 439

AREMP sample population with <54% USFS ownership

10 26 4 40

All USFS east side 49 251 20 320

PIBO 18 114 2 134

Non-PIBO (all) 31 137 18 186

Non-PIBO (north region) 27 117 16 160

Non-PIBO (south region) 4 20 2 26

*In AREMP area, “boating sites” is boat launch sites from State of Oregon data; in PIBO area, “boating sites” derives from the recreational site database.

Table 5. Summary of all risk factors for aquatic invasive species invasion that occur in watersheds surveyed by the AREMP and PIBO survey programs compared to unsurveyed watersheds in their area of responsibility in Region 6. All USFS watersheds with at least 5% USFS ownership that occur west of the Cascade crest, are compared using the AREMP survey program, whereas east of the Cascade crest, comparisons are made with the PIBO survey program.

Number of risks

1 2 3 4 5 6 7 * 11

All USFS west side 280 67 19 0 0 0 0 - 0

AREMP surveys only 45 9 3 0 0 0 0 - 0

AREMP full sample population 267 62 16 0 0 0 0 - 0

AREMP sample population with <54% USFS ownership

29 4 1 0 0 0 0 - 0

All USFS east side 108 41 15 6 4 2 3 - 1

PIBO 38 24 7 1 2 1 1 - 0

Non-PIBO (all) 70 17 8 5 2 1 2 - 1

Non-PIBO (north region) 59 15 5 5 1 1 2 - 1

Non-PIBO (south region) 11 2 2 0 1 0 0 - 0

*No summarized data had a total number of risks equal to 8, 9, or 10, hence the break in the data shown.


Analysis Methods—East of the Cascade Crest

The PIBO survey program encompasses 290 of the 983 watersheds east of the Cascade crest having at least

5% USFS ownership. In these sampled watersheds, 351 field survey reaches are present. Of these reaches, 337

surveys occur in wadeable stream segments and 13 in non-wadeable stream segments, most of which are

clustered in the John Day River basin. Because the range of inference for the PIBO survey program is the Lower

Columbia River drainage, we conducted three sets of analyses with these data. In the first, we compared the

PIBO survey sites to all 983 watersheds east of the Cascade crest having USFS ownership. In the second analysis,

we compared the PIBO survey sites to the northern watersheds in the lower Columbia River drainage (effectively

excluding the southern watersheds in the Fremont-Winema National Forest). In the third analysis, we compared

the USFS watersheds not surveyed by PIBO in the north with the USFS watersheds in the south to see if they

were similar or different with respect to the variables that we are interested in for aquatic invasive monitoring.

In each of the three analyses that we completed, we compared between groups using t-tests of 6 watershed-

scale variables: area (acres); USFS ownership (%); wadeable stream length (m); non-wadeable stream length

(m); wadeable stream length on USFS land (m); and non-wadeable stream length on USFS land (m). As with

the AREMP data set, high-risk locations were not evenly distributed across watersheds east of the Cascade crest.

Further, many watersheds did not contain high-risk locations. The abundance of 0 counts precludes simple

statistical comparison tests. Thus, we summarized in tabular form the number of high-risk locations in both

sample data sets for comparison only.

Results—East of the Cascade Crest

Results of comparative t-tests showed statistically significant differences between most characteristics of

PIBO streams and non-PIBO streams across the area east of the Cascade crest (Table 3). The only variable that

was not statistically different between these two sets of watersheds was watershed area (Satterthwaite p-value =

0.6663). Similarly, comparing PIBO to non-PIBO sites in the north, there were statistically significant differences

between these two groups for all variables tested (Table 3). We found differences between non-PIBO watersheds

in the north compared to the south in all variables tested except for the number of wadeable stream km

(Satterthwaite p-value = 0.5672) (Table 3).

Summaries of the number of USFS high-use sites, campgrounds, and boating sites comparing PIBO to non-

PIBO watersheds show that PIBO generally surveys a higher proportion of watersheds with risks for invasive

species introduction than are present in non-PIBO watersheds (Table 4). Likewise, when all risks in a watershed

were tallied, we found that PIBO and non-PIBO watersheds generally contained comparable numbers of risks

(Table 5). Further, it appears that PIBO may better reflect the number and type of invasive risk factors in the

non-PIBO watersheds of the north than in the south (Table 4, 5), most likely because there is limited PIBO

presence in the south.

The PIBO watersheds do capture locations with risk factors for introduction of aquatic invasive species.

However, smaller watersheds in the sample frame identified for this analysis may not be well-represented

for aquatic invasive monitoring.


This assessment represents a new monitoring question, thus it is not unexpected that elements of the existing

survey programs are not fully compatible. Because of the purpose of the PIBO program, larger watersheds

tend to be selected (Figure 10). With respect to the new AIS monitoring framework, some additional smaller

watersheds would enhance inference on the east side.

Figure 10. Within the population of watersheds with 5% or greater USFS ownership east of the Cascade crest, non-PIBO watersheds tend to be smaller (a) with larger number of wadeable streams (b) than is characteristic of PIBO watersheds.


Conclusions

1. Wadeable stream sections are most likely well-represented by current monitoring programs, particularly

west of the Cascade crest. However, high-risk watersheds are well-represented east of the Cascade crest.

2. Non-wadeable stream sections, which may also be the highest-risk locations in a watershed, are not

sampled in a systematic way under either the PIBO or AREMP monitoring programs.

3. The Fremont-Winema National Forests currently has no statistically designed sampling program for

freshwater habitat that could be used as a base for aquatic invasive species monitoring.

Recommendations

1. A statistically rigorous sampling program needs to be extended to the Fremont-Winema National Forest.

This is important for overa l l management as well as for the monitoring and tracking of aquatic invasive

species. An extension of the PIBO sampling program may be most effective, as this forest is more

characteristic of other east-side, rather than west-side, forest conditions.

2. Supplemental surveys in smaller watersheds would enhance the PIBO survey program’s ability to address

the new AIS monitoring question. The PIBO survey design tends to select larger watersheds for sampling. The

addition of smaller systems would allow PIBO to represent the broader array of watershed characteristics found

on USFS watersheds when monitoring for invasive species.

3. To augment current statistically rigorous field sampling of the wadeable portion of river systems, we

suggest water sampling using next-generation e-DNA techniques in non-wadeable stream segments (discussion of

techniques in Appendix 1). It may be most beneficial to pair e-DNA sampling with watersheds that are currently

being sampled upstream by field crews in wadeable areas in both the AREMP and PIBO survey programs. This

would leverage current efforts and borrow the statistical rigor from current sampling programs. Interpretation of

this type of additional sampling data will require specialized expertise, as from a bioinformaticist.

4. Develop a regional genomic library for invasive aquatic species in Region 6 to facilitate next-generation

e-DNA sampling in non-wadeable river segments (discussion of currently available markers in Appendix

2). Associated research into detection probabilities for individual species would enhance application and

interpretation of e-DNA results throughout the region.

5. Develop a detection-probability study of existing methods. This needs to be done for each invasive species,

to ensure that monitoring is effective.

6. Additionally, monitoring for aquatic invasive species that occur in lentic environments (lakes, ponds,

reservoirs) is not represented in this report, and may be a critical consideration for lotic (moving water) invasion.

Statistically rigorous monitoring programs are not currently conducted in lentic environments. This may be a

good application for e-DNA monitoring.


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Appendix 1: Next-generation eDNA sampling.

Rapid advances in genomic sequencing technology have made it possible to identify organisms by the

residual DNA (feces, urine, or skin cells) they leave behind in the environment (water, soil). This environmental

DNA, or “eDNA”, screening approach has been adapted for screening many rare, special-status, and invasive

species, and it can be used to estimate occupancy of aquatic flora and fauna in water (Goldberg et al. 2015)

The most commonly used eDNA method—quantitative PCR (qPCR)—targets a single focal organism per

sample. Although this method has proven successful and offers a low per-sample cost, it is not cost efficient for

multiple species (because costs scale linearly with species and samples) and it does not provide information on

the amount of genetic diversity for the focal species.

An alternative strategy being developed at the PNW Research Station is a multi-species DNA screen to

enrich DNA sequences from multiple target species across taxa simultaneously, and then directly screen for the

presence, abundance, and genetic variability of target species using massively-parallel DNA sequencing. This is

accomplished via ‘barcode sequencing’, a method pioneered at the PNWRS (Cronn et al. 2008), and it has been

applied to screening animals of conservation concern (Knaus et al. 2011, Alexander et al. 2013), trees species

(Parks et al. 2009), and even the microflora on trees. The advantage of this approach over qPCR is that the

enormous capacity of modern DNA sequencers (~ 60 billion bases per sample, equal to 20 human genomes) is

harnessed to screen multiple species and multiple water samples simultaneously. In preliminary tests, we have

successfully screened for fish (salmonids) and trees from 48 samples simultaneously; we believe that this method

can be scaled to screen 50 aquatic species from 96 water samples simultaneously. The effect of this high-level

‘multiplexing’ is that 10s of species can be screened from a water sample for nearly the same cost as a single-

species qPCR assay.

Collecting and filtering eDNA water sample in the field to be brought back to the lab for processing.


Appendix 2: Development of regional genomic library for invasive aquatic species in Region 6.

To effectively screen for multiple species, a genomic reference library is vital. Genomic libraries are used

to identify which species of interest are present in a sample by referring to a collection or library of genomic

DNA. Although many species and subspecies have had partial or complete sequencing of their DNA, other

species of interest still lack information. On the Region 6 Focal Invasive Species List, of the 12 identified aquatic

animals, 10 have at least preliminary barcoding available. Of the identified aquatic plants, 12 of the 14 genera

have preliminary barcoding available (Table A2-1). Additional barcoding of currently unrepresented species is

necessary to build up a reference library that contains information on all species of interest that may be screened.

Also, there may be a need for additional collection of species for which current barcoding is available to further

enhance identifications in Region 6.

Beyond development of a regional genomic library for invasive aquatic species in Region 6, other research

that improves the interpretation of eDNA results may be necessary. Detectability of eDNA in the water column

varies by season (due to water flow and temperatures) and species abundance. Evaluations of eDNA samples

with field-based methods may be necessary to determine detection frequency and the specific environmental

conditions that may influence detection probability.


Table A2.1. Region 6 focal invasive species list, with identification of species that have preliminary barcoding available for eDNA analysis.

Type

Common name

Scientific name

Species code

Preliminary species barcoding to run eDNA analysis available?

Aquatic Chinese Mystery Snail Cipangopaludina chinensis CICH Yes animals Asian Clam Corbicula fl ina COFL No; only genus

information found

Zebra Mussel Dreissena polymorpha DRPO Yes

Quagga Mussel Dreissena rostriformis bugensis DRRO Yes

Nutria Myocaster coypus MYCO Yes

Ringed Crayfish Orconectes neglectus ORNE Yes

Rusty Crayfish Orconectes rusticus ORRU No; only one relevant entry in Genbank

Northern Crayfish Orconectes virilis ORVI Yes

New Zealand Mudsnail Potamopyrgus antipodarum POAN Yes

Red Swamp Crayfish Procambarus clarkii PRCL Yes

Big Eared Radix Radix auricularia RAAU Yes

American Bullfrog Rana catesbeiana RACA Yes

(now Lithobates catesbeianus)

Aquatic plants

Flowering Rush Butomus umbellatus BUUM Yes

Didymo Didymosphenia geminata DIGE Yes

Brazilian Elodea Egeria densa EGDE Yes

Hydrilla Hydrilla verticillata HYVE Yes

Yellow Flag Iris Iris pseudacorus IRPS Yes

Water Primrose Ludwigia spp. LU No; only genus information found

Garden Loosestrife Lysimachia vulgaris LYVU Yes

Purple Loosestrife Lythrum salicaria LYSA Yes

Nonnative milfoils Myriophyllum spp. MYSP Yes

Yellow Floating Heart Nymphoides peltata NYPE Yes

Common Reed Phragmites australis PHAU No; only one relevant entry in Genbank

Curly-leaf Pondweed Potamogeton crispus POCR Yes

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