Methods

Summary

Bioacoustic data collection: Opportunistic recording of acoustic communication will be made with a Roland R-05 WAVE/MP3 Recorder and a directional microphone. For all vocalizations recorded, the location, sex, call context, and accompanying behaviors will also be logged (Geissmann & Parsons, 2011). When possible, the eye color and presence/absence of ear tufts will be noted. Calls will be analyzed for distinguishing vocal patterns, structural features, and frequencies (maximum, format, fundamental) using AviSoft Bioacoustics software. Calls collected here will be compared to calls collected from allopatric populations of E. flavifrons and E. macaco during other field seasons. Inter- and intraspecies comparisons will be made across the sampled populations.

Playback experiments will provide the opportunity to test whether calls are being used for species recognition at the contact zone (Fisher et al., 2013; Fichtel, 2014). A 5-10 meter minimum distance between researcher and lemur will be maintained and all equipment will be concealed. Focal animals (adults) will be identified. A playback period will be defined by the 5 minutes prior to the recording, the length of playback vocalization, and a 5 minutes post-playback lag (ZuberbĂĽhler & Wittig, 2011). Response behaviors, frequency of occurrence, and duration of event will be recorded. Contact calls, agonistic calls, and calls emitted during attempted mating bouts from conspecific, heterospecific, and hybrid lemurs will be used; there will be three versions of each call type in rotation. Local bird calls will serve as controls. The trials will be experimenter-blind, the order of calls played will be randomized, and the number of playbacks to each focal animal limited and spaced out over time to avoid habituation.

Behavioral data collection: Behavioral data will be collected through a combination of all occurrence, ad libitum, and scan sampling (Lehner, 1979). All occurrence sampling of social behaviors and interactions will occur from dusk to dawn. Categories scored will include: approaching an individual, allogrooming, reciprocal grooming between two or more lemurs, resting (when touching or near other), mating, scent marking, vocalizing, aggression, and playing. For bouts of grooming, the initiator and length of time will be recorded. Mating activities will be recorded ad libitum. Additionally, 5 minute scan samples detailing proximity of individuals to other group members will be taken, determining didactic partner preferences. Individuals will be recorded as either being together (touching), near (within 1 meter of each other), or apart (further than 1 meter

Genomic data collection: Fecal samples will be collected through sterile measures and stored in RNAlater (Perry et al., 2010; Goossens et al., 2011). The GPS location (using a Garmin eTrex 20) and the individual’s sex and eye color will also be recorded for each sample. J. Tinsman will assess nuclear genomic DNA using massively-parallel sequencing techniques. The reference genomes for both E. flavifrons and E. macaco have recently been sequenced (Meyer et al., 2015), allowing for easier identification of loci under selection. Genomic results will be used to support both the bioacoustic and behavioral findings from this study.

Protocols

This project has not yet shared any protocols.