EXHIBIT H NEEDLE COLLECTION PROTOCOL with list Wyoming.pdf

EXHIBIT H - Needle Collection Protocol for Identification of Whitebark and Limber Pine

Overview:

Whitebark and limber pine are ecologically important species that are impossible to distinguish in the field when cones (male or female) are not present. We have access to an inexpensive genetic test that can distinguish between these two species (and only these two species) using needle samples. We will be collecting needles from whitebark and limber pine in areas where they are known to co-occur. Because this test only works to determine whitebark pine from limber pine, it is important that crews do not mistakenly send in other similar species (e.g., western white pine).

Where are we collecting needle samples?

These needle collection protocols should be followed by all crews working in counties listed at the end of this document (also shown in the GREEN Area of interest map, below). This green area represents the known overlap of the area where limber and whitebark pines can potentially co-occur. If a plot is in one of the counties that intersects the green Area Of Interest shown below, the PDR should prompt you for needle collection when the species for limber pine (113) or whitebark pine (101) are entered for a seedling or sapling. All these counties fall within Montana, Nevada, Idaho, and Wyoming.

1: The known ranges of whitebark (red), limber (blue) with a 35K buffer. The green area is the intersection of these buffered ranges. This green area is where mandatory needle collection for whitebark and limber pine seedlings, saplings, and trees without cones will occur.

It may be easier for you to note that needle sample collection is required on the front of your plot packets that are in these Area-of-Interest counties.

If you are outside of this area but suspect you might have a sample that may suggest a range expansion for whitebark pine or limber pine, please collect the sample and send it to the lab.

How do we know which trees (seedlings/saplings) to collect from on plots in these counties?

NEW AS OF 2023: You can use the lists linked below to get an idea of how many seedlings, saplings, and trees you should anticipate collecting needle samples for on the plot. Seedling and sapling estimates will be closer to what you end up collecting. Trees >5” dbh will be harder to accurately predict because collection is based on presence/absence of cones (see protocol below).

2024_control_list_all_anticipated_PIAL-PIFL.xlsx

Seedlings: Collect samples for five seedlings of each species encountered on every microplot that is either whitebark or limber pine. For example, if you think you may have 8 whitebark seedlings and 10 limber seedlings on the subplot 4 microplot, collect samples from five of the seedlings you think are whitebarks, then collect five more samples from those seedlings you think are limber pines. Try to collect samples evenly throughout the microplot (i.e. not from one cluster of seedlings).

Saplings: Collect needles from all live whitebark or limber saplings on the microplot.

Trees: Collect needles from all live mature (tally) whitebark or limber trees on plot when cones (male or female) are not present to determine the exact species. Read the attached PDF “Field Identification of Whitebark versus Limber Pine” (also in the RMRS FIA FIELD Reference Library here) to find out more about how to tell these species apart.

Keep in mind that whitebark and limber can co-occur on the same plot, and they likely co-occur more frequently than we detect it. This means trees need to be identified on an individual basis; a definitive identification of one tree does not indicate a definitive ID for other trees on the subplot.

The genetic test can only differentiate between whitebark pine and limber pine.

Needle samples from other species may produce a false ID. Because of this, please be careful to send in only samples from whitebark or limber, when possible. Once again, if working in an area with other five-needle pine species, and you are unsure what 5-needle species a tree may be, you should still collect a sample, but make a note on the packet of what other species you think it may be.

What does a needle sample collection look like?

For healthy trees and saplings with relatively abundant needles: Collect a sample of six (6) intact needle fascicles (groups).

For seedlings and/or saplings with sparse foliage: Two full intact fascicle is preferred. If the seedling is extremely sparse, this sample size can be reduced to just half of a needle. Use professional judgement to not impact the health of the seedling.

Enter what you think the species is in the PDR (whitebark or limber pine). We will correct the data later, if necessary, when we get results back from the lab.

Guidelines for collecting samples:

• Use your best judgement and collect as many needles as you can (up to six intact fascicles) without damaging the individual.

https://usfs.app.box.com/file/1488245295726 https://usfs.box.com/s/s7la32e5p9czrhhtc7ngy8fqp9z2btov

• Try to keep the fascicles intact when collecting.

• If less than one fascicle is collected, please note this on the packet.

• Collect the greenest needles possible (e.g., with minimal non-green spots or fungal infections).

Put the needle sample in a small manila envelope, like the one shown on the right. Jim Sandall will mail these out to crews if you don’t already have them.

Pre-printed labels should be coming out soon, but in case you don’t have them, label the envelope as follows for each individual tree sample collected:

State – county – location – subplot - tree number (or "seed" if seedling) – species

If you collect multiple seedlings from a subplot, please identify how many were collected by labelling the envelopes ‘1 of X’, ‘2 of X’, etc.

Within reason, do your best to store collection envelopes out of the heat in a dark, dry place while awaiting shipping to preserve the genetic integrity of the sample.

Please do NOT send these to your Area Leader or QA in the plot packet! Instead, once a month, mail them to Ogden. You can include your tree cores and plant samples as well.

Send all samples to:

Ogden Forestry Sciences Laboratory

4919 S 1500 W, Ste. 200 Riverdale, UT 84405

Will there be follow up to prompt us to mail in samples or let us know whether we had correct species calls?

We plan to!

The plan is to send out notices one to two times throughout the season to crews that had whitebarks and /or limber pine seedlings or saplings on plots in the Area of Interest (aka, the counties listed below), requesting that they mail in their samples.

We will also do our best to get information back to crews individually on where they were correct/incorrect on their identification of these seedlings/saplings.

Why are we doing this?

Identifying 5 needle pines in general, but particularly whitebark pines v. limber pines, is a known problem in botany/ecology. Botanists have determined that there is no way to definitively distinguish whitebark from limber pine when cones or other signifiers are not present (male or female). RMRS - FIA is no different.

Study results from a previous needle sample collection (2010) showed that in the Area of Interest, our species identification between whitebark and limber pine was accurate only about 80% of the time. Our accuracy was worse for seedlings and where both species co-occurred on the same plot.

Sending needles in to get genetically verified doesn’t cost much (about $2.50 per sample) and it increases our data quality for an important species (whitebark pine) that was listed as a threatened species by the US Fish and Wildlife Service in December 2022.

FIA data are playing an important role in the legal status of this species. For example, FIA data were cited in the 2022 US Fish and Wildlife Service’s decision, in the Federal Register, to list whitebark pine as a threatened species. (The “more than half of all standing whitebark pine trees are dead” soundbite, which is entirely based on FIA data, got a LOT of press!). On top of all that, limber pine is having its own struggles that warrant close tracking. Since FIA data is increasingly being used for making management decisions for these species, it only makes sense to provide the best data we can for these important efforts.

Counties with mandatory whitebark /limber seedling/sapling needle collection (also see Area Of Interest map in GREEN, above).

State County State County State County State County 16 3 30 1 32 5 56 3 16 7 30 3 32 7 56 13 16 11 30 7 32 9 56 17 16 13 30 9 32 11 56 23 16 15 30 13 32 19 56 29 16 19 30 15 32 21 56 35 16 23 30 23 32 29 56 37 16 25 30 27 32 31 56 39 16 29 30 29 32 33 16 33 30 31 32 510 16 35 30 35 16 37 30 37 16 39 30 39 16 41 30 41 16 43 30 43 16 49 30 45 16 51 30 47 16 59 30 49 16 63 30 51 16 65 30 53 16 67 30 57 16 73 30 59 16 81 30 63 16 83 30 65 16 85 30 67

30 73 30 77 30 81 30 89 30 93 30 95 30 97 30 99 30 101 30 107 30 111

List of Plots with Possible Needle Sample Collection (Wyoming)

Field Season 2024

County Code

County Name Plot Estimated Number of Samples

23 Lincoln 8 Possibly 1 or more 23 Lincoln 11 Possibly 1 or more 23 Lincoln 15 Possibly 1 or more 23 Lincoln 39 Possibly 1 or more 23 Lincoln 43 Possibly 1 or more 23 Lincoln 67 Possibly 1 or more 23 Lincoln 120 Possibly 1 or more 23 Lincoln 147 2 23 Lincoln 177 Possibly 1 or more 23 Lincoln 195 4 23 Lincoln 3008 1 29 Park 668 7 35 Sublette 15 Possibly 1 or more 35 Sublette 20 7 35 Sublette 50 Possibly 1 or more 35 Sublette 68 2 35 Sublette 73 12 35 Sublette 113 Possibly 1 or more 35 Sublette 124 Possibly 1 or more 35 Sublette 143 6 35 Sublette 190 Possibly 1 or more 35 Sublette 206 8 35 Sublette 230 24 35 Sublette 3039 8 39 Teton 185 Possibly 1 or more 39 Teton 193 1 39 Teton 198 Possibly 1 or more 39 Teton 203 Possibly 1 or more 39 Teton 254 4 39 Teton 267 2 39 Teton 304 1 39 Teton 310 Possibly 1 or more 39 Teton 315 16 39 Teton 318 Possibly 1 or more 39 Teton 366 5 39 Teton 368 Possibly 1 or more

39 Teton 374 4 39 Teton 379 Possibly 1 or more 39 Teton 419 Possibly 1 or more 39 Teton 424 Possibly 1 or more 39 Teton 431 Possibly 1 or more 39 Teton 3045 Possibly 1 or more