DOI: 10.5061/dryad.bnzs7h47g
title: Translocation experiment reveals capacity for mountain pine beetle persistence under climate warming

description: Predicting species response to climate change is a central challenge in
 ecology, particularly for species that inhabit large geographic areas. The
 mountain pine beetle (MPB) is a significant tree mortality agent in
 western North America with a distribution limited by climate. Recent
 warming has caused large-scale MPB population outbreaks within its
 historical distribution, in addition to migration northward in western
 Canada. The relative roles of genetic and environmental sources of
 variation governing MPB capacity to persist-in-place in a changing
 climate, and the migratory potential at its southern range edge in the
 United States, have not been investigated. We reciprocally translocated
 MPB populations taken from the core and southern edge of their range, and
 simultaneously translocated both populations to a warmer, low-elevation
 site near the southern range boundary where MPB activity has historically
 been absent despite suitable hosts. We found genetic variability and
 extensive plasticity in multiple fitness traits that would allow both
 populations to persist in a warming climate that resembles the thermal
 regime of our low-elevation site. We demonstrate, for the first time, that
 supercooling points in MPBs are influenced both by genetic and
 environmental factors. Both populations reproduced with seasonally
 appropriate univoltine generation times at all translocated sites, and
 bivoltinism was not observed. The highest reproductive success occurred at
 the warmest, out-of-range low-elevation site, suggesting that southward
 migration may not be temperature-limited.
description: <strong>MPB
 collection, tree harvest, and bolt infestation</strong>
 For both field and
 laboratory experiments, we felled MPB-infested trees on 21 June 2016 from
 the S<sub>UT</sub> site and on 4 May 2016 from the
 S<sub>AZ-high</sub> site (Table 1). Cut bolts (~46 cm long)
 were harvested from one tree at each of the S<sub>UT</sub> and
 S<sub>AZ-high</sub> sites. Bolt ends were sealed with paraffin
 wax and transported to the Rocky Mountain Research Station (RMRS)
 Laboratory in Logan, UT where they were placed at ambient room temperature
 to allow adults to emerge naturally. Adult beetles were collected daily
 and stored in Petri dishes lined with distilled water-moistened filter
 paper at 4°C for up to approximately 10 days. To rear the next generation
 of beetles we also harvested three live, healthy trees of the same species
 at each site, cut them into ~46 cm long bolts, and sealed the cut ends
 with paraffin wax to retain moisture and deter fungal contamination. Bolts
 were stored at 4°C for up to 3 weeks. The uninfested experimental bolts
 from each site were randomized among the three field sites and the two
 temperatures in the laboratory study. We determined the sex of emerged
 adult beetles using the morphologically distinct
 7<sup>th</sup> tergite (Lyon 1958). To avoid potential genetic
 differences in development time among emerging adults, and to standardize
 for cohort density, we used beetles that emerged during the time beginning
 just before and throughout peak emergence from natal bolts. Experimental
 bolts of the same species were infested by drilling a small hole
 vertically into the phloem at the anatomical bottom of the bolt, inserting
 first a female then a male beetle, and stapling a mesh screen over each
 hole to prevent beetle escape. To minimize potential maternal effects due
 to host species (Burke and Carroll 2017), P<sub>UT</sub> were
 reared in <i>P. flexilis</i> and P<sub>AZ</sub> in
 <i>P. flexilis/ strobiformis</i> hybrids that were harvested
 from the same locations as infested bolts (Table 1). Individuals were
 randomized by sex and mating pairs were infested 6 cm apart, with 10 to 13
 pairs per bolt depending on bolt circumference. Following infestation,
 bolts were inverted to allow for natural upward gallery excavation.
 Infested bolts were either transported to field sites or placed in
 laboratory incubators as described below. <b>Field
 Experiments</b> <i>Experiment
 set-up</i> For the field reciprocal translocation
 experiment, we enclosed infested bolts individually in escape-proof
 netting (Rothco, MPN 8088) and within 24 hours of infestation, suspended
 each bolt ~ one meter above the ground in wooden A-frame structures with
 metal covers at each location (Table 1; Appendix S1: Fig. S1). Prior to
 infestation bolts were randomly distributed to their respective treatment
 groupings. Nine bolts infested with the P<sub>UT</sub>
 population and nine bolts infested with the P<sub>AZ</sub>
 population (18 bolts total per site) were placed at each of the three
 sites (54 bolts total). Bolt location was randomized among three A-frame
 structures at each site such that there was an equal number of
 P<sub>UT</sub> and P<sub>AZ</sub> infested bolts
 per A-frame (6 bolts per structure) (Appendix S1: Fig. S1). Field
 experiments were initiated as follows: S<sub>UT</sub>: on 30
 July 2016; S<sub>AZ-high</sub>: on 10 August 2016;
 S<sub>AZ-low</sub>: on 11 August 2016. To
 capture thermal conditions at each field site, temperature probes were
 inserted into the phloem on the south aspect of each infested bolt and
 temperatures were recorded hourly over the duration of MPB development and
 emergence (i.e., August 2016 to August 2017) (CR1000, Campbell Scientific,
 Logan, UT). To describe environmental effects, growing degree hours (GDH)
 &gt;10°C and &lt;0°C (i.e., cumulative heat and cold units) and
 weekly maximum and minimum temperatures were calculated for each bolt
 beginning on 12 August 2016. <i>MPB collection and trait
 measurements</i> Adult brood emergence at each
 site was monitored at least twice weekly throughout emergence and daily
 during the weeks of peak emergence. We collected individuals by bolt and
 transported them on ice to either Northern Arizona University, Flagstaff,
 AZ, or the RMRS Laboratory in Logan, UT. Generation time for each
 individual was calculated as the time difference between bolt infestation
 and brood adult emergence. Generation time includes the time duration for
 mating, oviposition, egg, larval, pupal and teneral adult (pre-emergent)
 development, in addition to a facultative prepupal diapause. We considered
 generation time resulting in seasonally appropriate (i.e., summer) adult
 emergence to represent higher relative fitness than a generation time
 resulting in aseasonal emergence. Adult emergence synchrony is important
 to successful mass attacks and colonization of new host trees (Logan and
 Bentz 1999). We define emergence synchrony as the standard deviation in
 generation time across all individuals of a population at a site, where a
 lower standard deviation suggests greater emergence synchrony and
 therefore greater fitness (see section <i>Statistical
 Analyses</i>). Reproductive success, a direct measure of fitness
 representing number of offspring produced, was calculated as the number of
 emerged brood adults per bolt divided by the number of successful
 galleries within the bolt, thereby compensating for uneven mating success
 and subsequent brood production among bolts. A parent gallery was
 considered successful (and therefore included in the count) if the gallery
 length was greater than 10 cm (Eidson et al. 2018), assuming that
 galleries less than 10 cm were the result of failed copulation by the
 inserted mating pair. The subset of bolts sampled for cold-hardening (see
 below) were not included in the determination of reproductive success, as
 the removal of larvae altered the number of emerged brood. To measure larval cold-hardening,
 individual larvae were collected from three infested bolts per population
 at each field site three times throughout the annual generation: (1) late
 November/early December 2016, (2) late January/early February 2017, and
 (3) late March/early April 2017. To account for temperature variability
 due to bolt aspect, we randomly sampled MPB larvae on three aspects (N,
 SW, and SE) along the bolt circumference, with each population at each
 site sampled from all three aspects (one aspect per bolt) each sampling
 period. To extract larvae, the outer bark and phloem were removed using a
 15 cm hole-saw, and the wound was sealed with paraffin wax. Larvae were
 placed in Petri dishes with filter paper and transported directly or
 overnight-shipped on ice to the RMRS Laboratory in Logan, UT. Larval
 instar was determined based on head capsule width
 (P<sub>UT</sub>: Logan et al. 1998;
 P<sub>AZ</sub>: Bentz unpublished). Supercooling points (i.e., the
 temperature of hemolymph crystallization) (Lee 1989) of larvae were
 analyzed within 24 hours of collection. Supercooling points of collected
 larvae were measured following the protocol of Bentz and Mullins (1999).
 Briefly, the temperature of individual larvae was monitored while the
 environmental temperature was lowered at a rate of ~1.5°C
 min<sup>-1</sup>. The supercooling point of each larvae was
 estimated as the lowest recorded temperature prior to tissue freezing,
 which was observed as an increase in temperature (≥ 0.5°C) caused by the
 exothermic latent heat of crystallization. MPB typically has four larval
 instars prior to pupation, and we observed some combination of larval
 instars 2, 3, and 4 in cold-hardening samples taken in the fall, winter
 and spring at each site. No other life stages were observed during
 sampling for this study. To assess population source and environmental
 differences in life stage development, we calculated a ‘developmental
 index’ by averaging instar number (i.e., instars 2, 3, 4) across all
 observed individuals at each field site and seasonal sampling
 period. Adult
 pronotal width was measured and sex determined (Lyon 1958) for 6,251
 individuals (65% of total emerged brood adults). Individuals were
 collected for sex determination and size measurement at least every 4
 days, and every 2 days during peak emergence. We measured pronotal width
 as a proxy for size (Kozol et al. 1988) using an ocular micrometer to the
 nearest 0.01 mm. <b>Laboratory
 Experiments</b> We reared each population in
 laboratory incubators at a constant 18°C and 25°C with a 12:12 hr.
 photoperiod (Appendix S1: Fig. S2). Optimal larval development in the
 laboratory occurs at ~25°C for P<sub>UT</sub> (Régnière et
 al. 2012) and ~27°C for P<sub>AZ</sub> (McManis et al. 2018).
 18°C was used because it is the lowest temperature, in either population,
 where the majority of individuals can develop directly to the adult stage
 without induction of a facultative prepupal diapause (Bentz and Hansen
 2017). Induction of the prepupal diapause would delay development, and
 because the two populations differ in diapause intensity (Bentz and Hansen
 2017), the developmental delay would generate confounding differences
 between the populations. Four infested bolts of each population were
 reared at 18°C and three bolts for each population were reared at 25°C.
 Adult emergence from individual bolts was monitored daily. Generation time
 for each individual brood adult and reproductive success per bolt were
 measured as described in the section <i>Field Experiments</i>.
 Sex and pronotal size (mm) of 1,532 individuals (31% of total emerged
 brood adults) were measured as described above, collected from a weekly
 random population subsample.
description: Contents of data files are briefly described below. Each data
 file has a 'metadata' tab with column header
 descriptions dryad_data_field_temp.xlsx - phloem
 temperature data (south aspect) from field sites 
 dryad_data_field.xlsx - mountain pine beetle data from field
 experiment, excluding supercooling and offspring success data (see
 below) dryad_data_field_supercooling.xlsx - mountain
 pine beetle data from field experiment, supercooling data
 dryad_data_field_offspringsuccess.xlsx - mountain pine beetle
 data from field experiment, offspring success data
 dryad_data_laboratory.xlsx - mountain pine beetle data from
 laboratory experiment, excluding size and sex data
 dryad_data_laboratory_size_sex.xlsx - mountain pine beetle data
 from laboratory experiment, size and sex data