2.2 Sampling

The elevation gradient included in the sampling sites covers the main distribution ranges of each bark beetle species: 1500–2000 m a.s.l. for D. frontalis and 2000–2500 m a.s.l. for D. mexicanus [8,51]. A total of 147 sampling sites were established on 24 altitudinal gradients. The lowest and highest elevation at which traps were placed was 1342 and 3611 m a.s.l., respectively. Each altitudinal transect had between 4 and 10 traps, placed sequentially every 100 m of altitudinal change (see S01 to S11 Tables in S1 File for details). The length of the altitudinal transects also aimed to cover the full natural altitudinal distribution of the most economically and/or ecologically important pine species in each studied region.

For example, in the case of the State of Michoacán, two transects were placed to cover the natural altitudinal distribution of Pinus pseudostrobus Lind., the most economically and ecologically important pine species of the Mexican Transvolcanic Belt in that State. On each of those two transects, eight trapping sites were placed, covering an altitudinal range of 700 m of elevational difference. The slope of the transects are illustrated for two states with zoom panels in Fig 1.

At each of the 147 sampling sites, 12-unit Lindgren funnel traps were placed and baited with a mixture of frontalin, racemic endo-brevicomin and turpentine from Synergy Semiochemicals Corporation, Canada. Release devices for each semiochemical were as follows: frontalin was dispensed from an Eppendorf tube inside of a plastic sachet (release rate 12 mg / day, chemical purity racemic); endo-brevicomin was dispensed from a flexlure (release rate 0.15 mg/ day, chemical purity racemic); and turpentine was dispensed from a single sealed plastic bag (8 × 19 cm) (release rate 3.6 g / day, chemical purity 70:30 mixture of alpha- and beta-pinene). Semiochemicals were replaced every 45 days in the field. Each Lindgren trap had a collecting cup filled with antifreeze solution (PRESTONA AF EX composed mainly of ethylene glycol) to preserve the beetles [66].

Every two weeks traps were visited, antifreeze was replaced, and trapped insects were collected and stored in 70% ethanol. Insect trapping started on March 1^st, 2015, and ended for most of the states on March 30^th, 2017, for a collection period of two full years. However, some transects in the following states had to end observations earlier: Michoacán (March 30^th 2016, although climate was recorded until March 2017), Chiapas (December 30^th 2016), Mexico State (November 15^th 2016), and Hidalgo (30^th December 2016). Thus the total abundance of trapped insects was estimated separately for each of the two years analyzed, by obtaining the sum of insects trapped by insect species per each trap for each year, where the operative definition of year was as follows. Hereafter, we refer to the “year 2015” as March 1^st 2015 to February 28^th 2016, and “year 2016” as March 1^st 2016 to February 28^th 2017. In Michoacán and Chiapas, only “year 2015” was considered. Referring to the “year” as March through February of the following year corresponds more closely to the actual seasons of ecological activity in Mexican pine and pine-oak temperate forests than the calendar year. The warm dry season occurs from March through May, the rainy season from June to October, and the cold dry season from November to February. Seasonal and annual climate estimates were calculated for these periods. Our operative definition of seasons and start and end of year is in general coincidental with the definition of climatic regions of México by Vidal-Zepeda [67], for the corresponding climatic regions of: North, Northeast, Center, Balsas Depression and Oaxaca Valleys and Southeast. In all those regions, in general, March is a month where temperature increases as a transition from colder winter temperatures to the warmer temperatures of Spring and Summer; also, June to October are months that are included in the rainy season of such regions.

All individuals of D. frontalis and D. mexicanus were identified and counted to estimate the abundance of bark beetle flight activity as a proxy of the abundance of those insects. Taxonomical identification was done following Armendáriz-Toledano et al. [7]. Specimens were collected in accordance with all applicable Mexican regulations, and a sample of the specimens from each locality were deposited at the Colección de Insectos of the Facultad de Biología, Universidad Autónoma de Querétaro. Field experiments were approved by the National Science Research Council (CONACYT) and the Comisión Nacional Forestal (CONAFOR) (Permit number CONACYT-CONAFOR-2014-C01-234547) and by the Dirección General de Vida Silvestre SGPA/DGVS/02594/16. Since we did not worked or interviewed people we did not ask for a informed consent.