Assessing the Use of Social Calls to Attract Bats to Artificial

ASSESSING THE USE OF SOCIAL CALLS TO ATTRACT BATS TO ARTIFICIAL

ROOST SITES

Alyson Frances Brokaw

A Thesis Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Science in Biology

Committee Membership

Dr. Joseph Szewczak, Committee Chair

Dr. Jeffrey Black, Committee Member

Dr. Sharyn Marks, Committee Member

Dr. Elizabeth Whitchurch, Committee Member

Theodore Weller, Committee Member

Dr. Michael Mesler, Graduate Coordinator

May 2015

ABSTRACT

ASSESSING THE USE OF SOCIAL CALLS TO ATTRACT BATS TO ARTIFICIAL ROOST SITES

Alyson Frances Brokaw

Many conservation strategies for bats focus on supporting or enhancing their

roosting and foraging needs. With increasing urbanization and loss of natural habitat,

many species have adapted to roost in anthropogenic structures, resulting in increased

human-wildlife conflict. Bat boxes can provide alternate housing for bats displaced due to exclusions from anthropogenic structures or loss of natural roosts. Researchers and conservationists have begun to investigate the variety of cues bats use to locate and select

possible roost locations, such as visual, olfactory or auditory cues. In this study, I

describe the call structure of social calls emitted by Yuma myotis (Myotis yumanensis) at roost sites. I investigated if free-ranging Yuma myotis react to social calls of conspecifics and other bat species at roost sites. I also evaluated the effects of age, sex and reproductive status on behavioral responses to social calls. In the summers of 2013 and

2014, I recorded calls from Yuma myotis using bat detectors mounted outside of roost exits. The recorded social calls divided into two distinct types that I could isolate and identify. Type 1 calls consist of a single frequency modulation (FM) syllable, while Type

2 calls consist of a descending FM sweep, finishing on a hook-shaped component. I broadcast social call and echolocation calls of Yuma myotis and Mexican free-tailed bats

(Tadarida brasiliensis mexicana) from newly erected artificial roost sites. Bat activity ii

was significantly higher during playbacks of myotis social calls compared to other playback treatments or silent control nights. Additionally, bat activity remained elevated after playback treatments, indicating a latent effect of playbacks at roosting sites. To test individual responses, bats were placed in a field flight tent and exposed to the same broadcast calls used in the field experiment. Individuals in a flight cage displayed no significant response to social calls, regardless of age, sex or reproductive status. This study provides the first description of social calls in a North American myotis species and suggests that understanding the social relationships of bats at roosting and foraging sites may be useful for informing conservation and management decisions.

iii

ACKNOWLEDGEMENTS

This study would not have been possible without the support of many people. I would like to thank my advisor, Joseph M. Szewczak, for making this research possible, providing guidance and input throughout the length of my study and allowing access to detectors and other equipment. I’d also like to thank all the members of my committee, including Dr. Jeffrey Black, Dr. Sharyn Marks, Dr. Elizabeth Whitchurch and Theodore

Weller for their advice and editing support. I would especially like to thank Ted Weller for providing training in bat capture and handling techniques. Thanks to The Benbow Inn for allowing me to install bat boxes and giving access to their property for my research, as well as a having a genuine interest in bat conservation. I’d also like to thank Erinn

Trujillo of Umpqua National Forest, U.S. Forest Service; Patrick Doyle of Van Duzen

County Park, Humboldt County Department of Public Works; Jay Harris of California

State Parks, North Coast Redwoods District; David Anthon of Headwaters Forest

Reserve, Bureau of Land Management; and Desiree Early of Green Diamond Resource

Company for allowing me access to field sites for field playback and bat captures.

Thanks to Jeffrey Clerc for countless hours of discussion, field assistance, statistics advice and moral support throughout the past year. Additional thanks for field assistants and volunteers Bern Fahey, Matthew Scott, Christen Long and Cari Zourdos Williams.

This entire journey would not have been possible without a lifetime of support from my parents William and Patricia Brokaw, who encouraged me to reach my full potential and instilled a love for the environment, wildlife and science at an early age. Also, thanks to my sister Julia Brokaw, for always being my biggest cheerleader in everything. Finally, iv

infinite thanks to my husband Bradley Bogdan, for putting up with my being away for long days and late nights in the field, keeping the house running while I holed away writing and always being there for me.

This study would not have been possible without funding from several local and national organizations, including Humboldt State University Master’s Grant, Bat

Conservation International Student Scholarship, American Society of Mammalogists

Grant-in-Aid of Research and California North Coast Chapter of The Wildlife Society.

TABLE OF CONTENTS

ABSTRACT ...... ii

ACKNOWLEDGEMENTS ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 6

Study Species ...... 6

Study Sites ...... 7

Focal Roosts ...... 7

Capture Locations ...... 9

Collecting and Isolating Social Calls ...... 9

Field Playback Experiment ...... 13

Statistical Analysis ...... 16

Individual Playback Experiment ...... 17

Behavioral Responses ...... 20

Statistical Analysis ...... 21

RESULTS ...... 22

Social Calls in Myotis yumanensis ...... 22

Field Playback Experiment ...... 25

Individual Playback Experiment ...... 34

DISCUSSION ...... 39

Social Calls in Myotis yumanensis ...... 39

Field Playback Experiment ...... 43

Individual Playback Experiment ...... 48

CONCLUSION ...... 51

Literature Cited ...... 52

vii

LIST OF TABLES

viii

LIST OF FIGURES

Figure 1. Top, Example sonogram of a high quality recording of echolocation and social calls recorded from outside a bat roost. Social calls immediately follow a clean sequence of echolocation calls identified as Yuma myotis. Bottom, Real time display of recorded calls, demonstrating temporal patterning of call pulses. Call frequency in kilohertz (kHz) is indicated on the y-axes and time (in milliseconds) on the x-axes for both graphs. Recordings were done with a Pettersson D500x bat detector with extendable microphone and visualized with the program Sonobat 3.0 (Arcata, CA)...... 10

Figure 2. Sonograms of selected and isolated playback calls. (A) Yuma myotis social call, (B) Yuma myotis echolocation call and, (C) Mexican free-tailed bat echolocation call. The call frequency (in kilohertz) is given on the y-axes and the time (in milliseconds) is given on the x-axes. Recordings were done with a Pettersson D500x bat detector with extendable microphone and visualized with the program Sonobat 3.0 (Arcata, CA)...... 13

Figure 3. Schematic diagram of flight tent for individual playback trials. The orientation of the flight tent and location of the playback loudspeaker was randomized each night. 19

Figure 4. Sonograms of isolated Yuma myotis social calls. (A) Type 1 social call, (B) Type 2 social call. The call frequency (in kilohertz) is given on the y-axes and the time (in milliseconds) is given on the x-axes. Recordings were done with a Pettersson D500x bat detector with extendable microphone and visualized with the program Sonobat 3.0 (Arcata, CA)...... 23

Figure 5. Number of bats approaching experimental boxes summed up over all 9 replicates. Boxplots show the median, 25% and the 75% quartiles, and outliers. Friedman tests indicate significant differences between numbers of bats approaches across all treatments (Q =14.98, df = 4, P = 0.004, N = 9)...... 27

Figure 6. Number of bats approaching bat boxes summed up over all 9 replicates and given separately for each behavior type. The approaches were grouped into four types of approaches. Boxplots show the median, 25% and the 75% quartiles, and outliers. Note that y-axes are on different scales...... 29

Figure 7. Number of bats approaching experimental boxes summed up over all 9 replicates, combining MYYU playback treatments. Friedman tests indicate significant differences between numbers of bats approaches across all treatments (Q = 9.61, df = 3, P = 0.02, N = 9)...... 32

Figure 8. Proportion of time all bats spent performing three types of behaviors during the 30 seconds of playback compared between sexes, regardless of treatment type...... 34

Figure 9. Proportion of time spent performing each of 6 different behaviors during the 30 seconds of playback, compared between juveniles (n=7) and adults (n=22), regardless of treatment type...... 35

Figure 10. Proportion of time bats spent performing each of six different behaviors during each playback type, during the 5 minutes post-playback observation period. Proportion of time bats spent performing each behavior was statistically significant (MANOVA: F = 1.85, df = 3, P = 0.04, N = 29)...... 38

INTRODUCTION

Human disturbance and habitat encroachment have had a drastic effect on numerous wildlife species. Recent conservation and mitigation efforts have focused on improving available habitat for species that have not fared well within human-altered landscapes.

Some strategies include relocating problem animals and providing artificial alternatives for nesting, roosting and dens. Some efforts have become conservation success stories.

For example, the eastern bluebird (Sialia sialis) experienced sharp declines due to loss of sparsely wooded habitats and dead tree snags required for nesting (Robbins et al. 1989), until conservation organizations began promoting the implementation of artificial nest boxes. This resulted in a 3 percent population increase per year between 1980 and 2001.

However, most relocation efforts fail, particularly those that were aimed at reducing human-wildlife conflicts (Fischer and Lindenmayer 2000)

Despite some species’ urban successes, many North American bat species are listed as endangered, threatened, or are in general population decline. A variety of factors contribute to these declines such as increased human disturbance, loss of habitat from urbanization, disease (Blehert et al. 2009), climate change (Adams 2010), agricultural pesticides (Agosta 2002), and wind energy development (Kunz et al. 2007).

Many conservation strategies for bats in North America have focused on roosting and foraging needs for bats throughout the year. The number and diversity of bats can be limited by availability of suitable roosts (Humphrey 1975, Pierson 1998). In addition, bat distribution and population can be affected by interspecific competition for appropriate roost sites (Perkins 1996). In temperate bats, the types of roosts used differ temporally

2 and seasonally. Day roosts are used to raise young and provide protection during the day from diurnal predators. Night roosts provide a place to conserve energy, social interaction and information transfer and consumption of prey, and are generally different locations from day roosts (Adam and Hayes 2000). The need for a range of roost sites is increased in bat species that frequently switch between roost sites. This roost switching helps bats reduce parasite loads (Reckardt and Kerth 2007), avoid predation, and select ideal roosting temperatures in response to changing weather conditions and the bats’ reproductive status (Willis and Brigham 2004, Pretzlaff et al. 2010).

Temperate bats roost in a wide diversity of locations, including caves, tree cavities, amongst foliage or under exfoliated bark. During the summer, bats rely on day roosts with appropriate microclimatic conditions for raising young, typically preferring roost temperatures between 27 and 39 degrees C (Hamilton and Barclay 1994, Kunz and

Robson 1995, Kunz and Lumsden 2003). Historically, bats found these roost conditions in sun-exposed natural locations such as hollow trees, cavities, and rock crevices. With increasing urbanization and loss of natural habitat through land alterations, many species have adapted to roost in anthropogenic structures.

Bats roosting in bridges and dwellings are perceived as a nuisance and risk for disease

(e.g., histoplasmosis and rabies, De Serres et al. 2008). Bat boxes erected in backyards and parks can support bat ecosystem services and conservation by providing roost resources in areas where natural habitat has been lost due to development (Flaquer et al.

2006). As roost alternatives away from nuisance areas, bat boxes can reduce human- wildlife conflict (Brittingham and Williams 2000) and provide eco-friendly alternatives

3 to exclusions and roost destruction.

Though the roost characteristics of many species are fairly well known (e.g.,

Humphrey 1975, Brigham et al. 1997, Kunz and Lumsden 2003, Evelyn et al. 2004), the exact mechanisms by which bats find suitable roosts, particularly bat boxes, remains unclear. In general, total bat occupancy rates of artificial roosts average between 40 and

70% in North America (Tuttle and Hensley 1993). It has been proposed that the use of guano-treated wood and associated olfactory cues may decrease the time to occupancy of bat boxes (Tuttle and Hensley 1993), though in some instances this did not improve overall occupancy rates (Neilson and Fenton 1994). There is some evidence of insectivorous bats using olfactory cues to identify individual roost mates (Bouchard 2001,

Bloss et al. 2002, Engelhart and Greene 2009), however, most insectivorous bats do not have a particularly keen sense of smell (Bhatnagar and Kallen 1974) and it is unlikely that olfactory cues would be effective at roost-searching distances (Ruczyński et al.

2007).

Recent studies have focused on the acoustic cues bats may use to signal roost locations (Ruczyński et al. 2009). Calls emitted from roosts may play a crucial role in maintaining group cohesion and contact among individuals in fission-fusion dynamic societies (Furmankiewicz et al. 2011, Chaverri et al. 2010). Fission-fusion societies consist of individuals spread among different roosts/subgroups at the same time, with frequent changes of subgroups (Kerth 2008). Many North American bat species adhere to the fission-fusion model of sociality (Willis and Brigham 2004), suggesting that they must have some mechanism for recognizing familiar conspecifics at roost sites.

Bats produce two broad types of vocalizations: echolocation calls and social calls.

Bats use echolocation calls primarily for close range spatial orientation and prey

detection while foraging, although recent investigations suggest they can also convey

social information about the caller, such as age and sex (Kazial et al. 2001, Kazial and

Masters 2004), group association (Voigt-Heucke et al. 2010) or individual identity

(Masters et al. 1995, Knörnschild et al. 2012) in a lab setting. Playback of echolocation

calls has been demonstrated to attract free-ranging little brown bats (Myotis lucifugus) to

roosting sites (Barclay 1982). The relationship between frequency and echolocation

resolution constrains bats to use high frequencies (Siemers and Kerth 2006). However,

high frequencies attenuate over short distances, limiting the broadcast range of social communication. As a result, bats often use lower frequencies for vocalizations intended just for social communication (Fenton 2003).

Social calls have the potential to convey a wider variety of information than echolocation calls. These calls can be used as contact calls to locate group members while foraging (Wilkinson and Boughman 1998), mother-pup communication via isolation calls (Balcombe 1990, Bohn et al. 2007), and social cues for locating conspecifics at roosting sites (Ruczyński et al. 2009, Arnold and Wilkinson 2011). The conspicuousness of such signals can allow for unintentional communication, or social eavesdropping by con- and heterospecifics. Schöner et al. (2010) demonstrated that social calls can be used to attract bats to artificial roost sites and that at least some bats eavesdrop on the social calls of other species. This indicates potential for eavesdropping between species, particularly when species share a similar resource such as roost location

(Avargues-Weber et al. 2013).

Since social calls are understudied in North American insectivorous bats, I describe the social call characteristics of a common bat species, the Yuma myotis (Myotis yumanensis). I also present an experimental field study that investigated if free-ranging bats are attracted to social calls or other acoustic cues broadcast from potential roost sites

(e.g., bat boxes). In the summers of 2013 and 2014, I conducted a playback experiment to assess the potential for conspecific social and echolocation calls and heterospecific echolocation calls to attract Yuma myotis to artificial roost sites. I predicted that if these bats use acoustic cues (echolocation or social calls) to locate roost locations, then the number of approaching bats will be greater on playback nights than during nights where there is no playback. I predicted more bat approaches during playback of conspecific calls compared to Mexican free-tailed bat (Tadarida brasiliensis mexicana) echolocation calls. I also captured and presented Yuma myotis individuals with social call and echolocation call playbacks to evaluate the effects of age, sex and reproductive status on behavioral responses to these acoustic cues.

MATERIALS AND METHODS

Study Species

Yuma myotis are widespread and common throughout California, with their distribution closely tied to bodies of water used for foraging. Roosting habitat includes old growth tree cavities and rock crevices, but they will also readily roost in buildings, mines and under bridges. Like many North American Myotis species, Yuma myotis mate primarily in the fall, giving birth to a single pup sometime between May and July

(Dalquest 1947). Females form maternity colonies, separate from males, during the summer. Yuma myotis and the closely related little brown bat (M. lucifugus) ecologically similar and morphologically cryptic species where their ranges overlap, often making them difficult to identify in the field (Weller et al. 2007). Additionally, Yuma myotis and other bat species such as Mexican free-tailed bats sometimes roost together (J. Szewczak, pers. comm., 2013), providing the potential for cross-species cue recognition (Ubernickel et al. 2013). Mexican free-tailed bats are also common throughout California, and together with Yuma myotis comprise a large proportion of urban bat populations (D.

Johnston, pers. comm., 2013).

Study Sites

Focal Roosts

To describe social calls in Yuma myotis, four focal roosts of the target species

were selected between 2013 and 2014. Two of these focal roosts were used to acquire

social call recordings needed for analysis and playback. Additionally, I tested the

response of free-ranging bats to calls broadcast from bat boxes at each focal roost site.

The Benbow Inn was used as a focal roost in 2013 for social call recording and experimental field playback of bat social calls. This historic inn, located in southern

Humboldt country, California, holds a maternity colony of close to 1000 adult females and juvenile Yuma myotis that roost each summer in one corner of the 4th floor,

rendering them easily accessible and accustomed to human disturbance. These bats exit

primarily from crevices and window openings on the east and west sides of the building.

A smaller maternity colony of Yuma myotis also roost within an open shaft in a historic

stone bridge located immediately to the west of the inn. A small colony of between 30

and 100 Mexican free-tailed bats also roost within The Benbow Inn structure. The inn

borders Lake Benbow State Recreation Area, which consists of approximately 1200 acres

alongside the South Fork Eel River. Each night, bats exit from the east and west sides of

the inn to forage over the river and lawn of the inn. Riparian areas are sparsely vegetated, dominated by annual grasses and mixed shrub covers. The river is sided by redwood- dominated forest and maintained lawn and golf courses.

Three additional sites were selected as focal roosts for the field playback experiments in the summer of 2014. One focal roost was located within the Smith River

National Recreation Area in northeastern Del Norte County, California. Several bat boxes were erected as mitigation for structural changes at Big Flat Campground along

Hurdygurdy Creek, a small tributary creek to the South Fork Smith River. A maternity colony of approximately 300 Yuma myotis have taken up residence in the new structures.

Located adjacent to a seasonal campground, these individuals are also accustomed to human presence. Forest cover there consists mainly of coastal redwood, Douglas-fir, white fir, sugar pine and Port-Orford-cedar, with a high diversity of rare and endemic plant species due to unique serpentine soils.

A second focal roost was located at the Steamboat Compound along the North

Umpqua River, within Umpqua National Forest, Douglas County, approximately 30 miles east of Roseburg, Oregon. Bats roost in current Oregon Department of

Transportation (ODOT) office buildings and the converted tree cooler and bunkhouses used by the U.S. Forest Service. Approximately 200 Yuma myotis bats roost in the attic of the ODOT office buildings each year, entering through small cracks and openings at the base of the roof. It is unknown if these bats are a maternity colony or a mixed roost.

Forests are a mix of Douglas-fir, maple and hemlock, with several old-growth Douglas- fir groves.

A third focal roost was located in central Humboldt County, at the Swimmer’s

Delight County Park. Swimmer’s Delight is located about 20 miles southeast of Fortuna,

California. Swimmer’s Delight is a popular camping and recreation area located along the

Van Duzen River. The vegetation is dominated by several old growth redwood groves.

Each summer, approximately 150 bats can be found roosting under the roof of the park

9 ranger’s station and are commonly seen foraging along the Van Duzen river.

Capture Locations

For individual playback trials, bats were captured from three locations in 2014:

Camp Bauer, Headwater Forest Reserve, and Benbow Lake State Recreation Area. Camp

Bauer, owned by Green Diamond Resource Company, is located in second-growth redwood forest on the North Fork of the Mad River, near Korbel, CA. Riparian vegetation consists of a mix of redwood, alder and cottonwood trees. Headwater Forest

Reserve is located 6 miles southeast of Eureka, CA. The reserve consists of nearly 7500 acres of redwood forest, including old growth redwood groves and the headwaters for the

South Fork Elk River. Bats were also captured along the South Fork Eel River in Benbow

Lake State Recreation Area, adjacent to The Benbow Inn focal roost.

Collecting and Isolating Social Calls

I recorded bat calls from just outside selected focal roosts prior to the anticipated beginning of parturition, thus ensuring only adult calls were recorded. During the 2013 field season, bat calls were recorded from the roof of The Benbow Inn from June 14 to

June 16, 2013. Only calls isolated from The Benbow Inn were used for field and individual playback, to maintain quality and consistency between field locations.

Recordings were also obtained from Smith River from June 17 to June 21, 2013 and from May 2 – May 5, 2014. Additional recordings were obtained from the Steamboat

Compound from April 19 - April 20, 2014 to confirm species identification. No social calls were recorded at the Steamboat Compound, so this site was not included in social call analysis. All recordings were acquired as 500 kHz 16 bit wave files using a

Pettersson D500X full spectrum bat detector (Pettersson Electronik, Uppsala, Sweden).

Calls were analyzed and isolated using Sonobat 3 (Arcata, CA). On each call recording,

echolocation calls and social calls were isolated from background noise. Echolocation

calls of the target species (Yuma myotis) were identified based on temporal patterning of

sequences, known call characteristics (Szewczak et al. 2011a) and comparison with

reference calls for Yuma myotis. Social calls were identified as bat vocalizations distinct

from, but associated with echolocation call sequences and were compared to social calls

obtained from other myotis species (Pfalzer and Kusch 2003). To minimize uncertainty

of species origin, I only selected social calls from single species call files. Furthermore, I

only selected social calls with good apparent signal quality, that is, those that separated

clearly from other ambient noise and displayed clean tonal trends (Figure 1).

All isolated social calls were quantitatively measured for a variety of parameters, using Sonobat 3.0. Selected parameters include call shape, duration of social call components, characteristic frequency (Fc), minimum call frequency (Fmin), maximum call frequency (Fmax), call bandwidth, frequency of maximum energy (FmaxE; the frequency of the call where the most energy is concentrated), and start and end

frequencies (Fstart and Fend). If distinct differences were found in call structure, I used a

t- test to compare call parameters.

Two types of playback files were prepared for Yuma myotis: Yuma myotis social

call (Figure 2A) and Yuma myotis echolocation call (Figure 2B). Four final playback

files were prepared combining one to three repetitions of the target call (social or

echolocation), with a one second break of no calls at the end of each recording. Specific

files were randomly selected and varied throughout each treatment night to avoid

habituation.

To test bat responses to cohabitating heterospecifics, I also prepared playback recordings of Mexican free-tailed bat echolocation calls (Figure 2C). Prepared files were a mix of reference files recorded from around northern California. These calls were prepared in the same way as Yuma myotis echolocation and social calls. Although these calls were not all obtained from the study sites, it is unlikely that this had an effect on responses. A previous study in Germany used recordings obtained from bats in the United

Kingdom but was still able to demonstrate a measureable response (Schöner et al. 2010).

Field

Playback

Figure 1. Sonograms of selected and isolated playback calls. (A) Yuma myotis social call, (B) Yuma myotis echolocation call and, (C) Mexican free-tailed bat echolocation call. The call frequency (in kilohertz) is given on the y-axes and the time (in milliseconds) is given on the x-axes. Recordings were done with a Pettersson D500x bat detector with extendable microphone and visualized with the program Sonobat 3.0 (Arcata, CA).

Experiment

All playback replicates in 2013 were carried out at one focal roost, The Benbow Inn.

Seven wooden three chambered bat boxes (Bat Conservation and Management, Carlisle,

PA) were constructed and installed surrounding the roost site. Each box was 47

centimeters wide and 61 centimeters tall, with three, 3 centimeter wide chambers, painted

black and mounted a minimum of 4 meters off the ground. All seven boxes were oriented

towards the southeast (120 to 130 degree azimuth) to maximize sun exposure and internal

temperature. Boxes were installed between 20 and 150 meters from the roost site. All

areas had little to no undergrowth to ensure uninhibited bat access to the box. Four boxes

were located immediately adjacent to riparian habitat (less than 10 meters from the

stream bank) while the other three were located greater than 50 meters away from

riparian areas. Each bat box was used as an experimental replicate once.

The loudspeaker (Pettersson L400) was mounted just under the bat box, without

blocking access to the roost entrance. One call type per night was broadcast, beginning at

sunset and running continuously until sunrise. Four different files of each type were

broadcast, with each file being played for 1 hour before switching to a new file. Each file

was played a maximum of three hours each night. Call broadcast were played through a

laptop using a 96 kHz, 24-bit conversion USB sound card (Focusrite Scarlet 2i2, United

Kingdom), managed using Live 9 sound software (Ableton, Berlin, Germany).

Playbacks took place from June 28 through September 4, 2013, organized into seven replicates. Each replicate consisted of three broadcast nights with two silent controls on each end of the broadcast nights (Control A and Control B). Broadcast nights allowed for

one night for each call type: Yuma myotis echolocation calls (MYYU echolocation),

Yuma myotis social calls (MYYU social), and Mexican free-tailed bat echolocation calls

(TABR). The order of broadcast treatment was randomized for each replicate. During

playback trials, an observation point was set up approximately 3 meters from the bat box.

An infrared video camera (PC177IRHR-6, Super Circuits, Austin, Texas) was directed towards the roost to monitor flight responses to playbacks. The camera was mounted at a height equal to the bat box, allowing about 1 - 2 meters on either side of the box and set to record throughout the entire night.

On each video recording, every bat approach was registered and flight response classified into four categories: (1) passing (flying through the visual field of the camera but not inspecting), (2) circling (flying circles within 0.5 meters of roost), (3) direct approach (direct flight towards box within 0.5 m), and (4) landing on roost. To avoid counting a bat twice when it left the visible area while circling, a new bat approach was defined when at least 30 seconds passed after a bat was first recorded on the video.

During control nights, a bat detector (Pettersson D500X) was mounted on the camera at box height, set to record during the entire night. During playback nights, a detector was set up approximately 50 meters away from the box location in the opposite direction of broadcasts to determine a baseline for bat activity and species diversity each night. The detector was mounted far enough away to ensure it did not pick up playback calls, but close enough to accurately represent local bat activity.

In 2014, four replicates were carried out at three different field sites between June 6 and August 2, 2014. Two replicates were carried out at Smith River, with one replicate

16 each at the Steamboat Compound and Swimmer's Delight County Park. I used the same protocol as used in 2013, using smaller, more portable bat boxes (Audubon Society single chambered bat boxes). Bat boxes were temporarily installed at each field site, placed between 20 and 50 meters from roost sites, oriented towards the southeast. Each replicate consisted of five days, during which control and broadcast nights were carried out as previously described.

Statistical Analysis

I used a non-parametric Friedman test, matched per box, for comparison between the five different observation nights for both 2013 and 2014 playback box data. This test allowed me to control for a possible influence of the location of the experimental boxes on the number of approaching bats. Playbacks were conducted at each box location under similar conditions and could hence be considered related groups. I tested if the minimum nightly temperatures or other environmental factors differed between observation nights to exclude the possibility that bat activity was affected by environmental factors and not treatment type. To measure if treatment order per replicate had an effect on the number of approaching bats, I compared the number of bat approaches over all observation nights.

I tested for the effect of the different call types by comparing the number of approaching bats together and for each type of approach (passing, circling, direct approach and landing). I also combined the two Yuma myotis call types (MYYU social and MYYU echolocation) into a single treatment to compare conspecific calls versus heterospecific (TABR) calls and control nights. To compare this treatment to the two other treatments, I calculated the average number of bat approaches during the two Yuma

myotis bat calls per replicate (total number/2). If the Friedman tests detected significant

(P ≤ 0.05) differences between treatments, I used Wilcoxon matched pairs test to explore

pair-wise differences post-hoc.

Individual Playback Experiment

Yuma myotis were captured between July 24th and September 11, 2014. Bats were

captured on July 23, July 24, August 19, and August 24 at Camp Bauer, on September 4

and 11 at Benbow Lake State Recreation Area, and on August 2 at Headwaters Forest

Reserve. Individuals were caught using single high mist nets placed over streams or other water sources at each site. One to three nets were used at each location, depending on vegetation coverage and water depths. Nets were checked every 5 to 10 minutes depending on the general bat activity for the night. Nets were opened at sunset and left open a maximum of 2 hours.

Upon capture I determined sex, age and reproductive status of each bat, and only adult non-lactating individuals were kept for flight trials. Age was determined by

evaluating the epiphyseal-diaphyseal fusion of the long phalanges (Brunet-Rossinni and

Wilkinson 2009). I measured body mass using a digital scale and forearm length with

calipers. Only individuals with forearms lengths less than 35 mm were used for stimulus

testing, minimizing overlap with little brown bats to ensure only Yuma myotis were

tested (Weller et al. 2007). Species identification was further confirmed using acoustic

data obtained during each flight trial (Szewczak et al. 2011a). Bats were temporarily

marked using non-toxic marker on the wing to avoid replication, since multiple capture

nights were carried out at each location. Bats were held in soft cloth bags for a minimum

of 1 hour prior to playback trials and released immediately following each flight trial. All

experimental protocols were carried out under permits from California Department of

Fish and Wildlife and Humboldt State University Institutional Animal Care and Use

Committee protocols (13/14.B.110-A).

One playback trial was conducted on each bat. Following a minimum of 1 hour after

capture, a single bat was transferred into a flight tent. The flight tent was 6.1 meters long

by 3 meters wide by 5 meters tall, made of two backyard canopies placed end to end and

surrounded by mosquito screening walls. Two small bat boxes were mounted on each end

of the flight tent (Figure 3). An infrared camera (Sony DCR-SR45 NightShot Camcorder,

Tokyo, Japan) was placed at both ends of the flight tent to record bat responses. A third infrared camera was placed at the center of the flight tent to record bat responses prior to flight. For the playback, a Peterson L400 loudspeaker was placed under one of the mounted bat boxes, randomly chosen for each trial (Figure 3). An infrared floodlight (Bat

Conservation and Management, Carlisle, PA) was used to provide additional lighting.

The orientation of the flight tent was randomized each night.

Figure 3. Schematic diagram of flight tent for individual playback trials. The orientation of the flight tent and location of the playback loudspeaker was randomized each night.

Each bat was randomly assigned and presented with one of the playback stimuli:

Yuma myotis social calls (MYYU social), Yuma myotis echolocation call (MYYU

echolocation), Mexican free-tailed bat echolocation call (TABR) and no playback

(Control). Playback files used were the same files used for the field playback experiment.

At the beginning of each trial, the bat was placed on a folding table at the center of the

flight tent and covered by a clear plastic box attached to a pulley. The bat remained

covered for a minimum of two minutes or until it remained motionless for 30 seconds.

Once the bat was motionless, the box was lifted and playback began. Playback stimuli

were broadcast for 30 seconds, followed by a 5 minute observation period. Following the

5 minute observation period, one side of the tent was removed to allow bats to exit

naturally. All bats exited within five minutes of the tent opening. Bat behavioral

responses were recorded during the 30 second playback and the following 5 minute

observation period.

Latency time was measured from the time the box was lifted to the time the bat took flight. All bats took flight immediately upon release from the box, so I did not compare latency between treatment types. Behaviors were grouped into three types of responses

(positive, neutral and negative), with six sub-categories (see below). We recorded the

duration of each behavior and frequency (N/30 second and N/300 seconds) for all

behaviors. I coded all video files blindly, that is I assigned a behavioral response code

without knowledge of the type of playback used for that trial.

Behavioral Responses

Positive Response:

(1) Near Circle: Bat is seen circling on the side of the tent where loudspeaker is

located.

(2) Near Landing: Bat is seen approaching and/or landing on the bat box containing

the loudspeaker, or on the wall or ceiling immediately adjacent to the loudspeaker.

Landing includes behaviors that occur following landing such as crawling, hanging or

grooming.

Neutral Response:

(3) Large Circle: Bat is seen entering and leaving the frame of the camera, flying

large circles around the entire space of the flight tent.

(4) Side Landing: Bat is seen approaching and/or landing on the sides of the flight

tent parallel to the bat boxes, or on the center ceiling. Landing includes behaviors that

occur following landing such as crawling, hanging or grooming.

Negative Response:

(5) Far Circle: Bat is seen circling the end of the flight tent farthest away from the

loudspeaker.

(6) Far Landing: Bat is seen approaching and/or landing on the wall or box opposite of the loudspeaker. Landing includes behaviors that occur following landing such as crawling, hanging or grooming.

Statistical Analysis

For behaviors observed during both the 30 seconds of playback and in the following 5

minute observation period, I performed a MANOVA to assess differences in the

proportion of time spent doing each of the 3 response categories (Positive, Neutral or

Negative), controlling for age, sex and reproductive status of each individual. Prior to

analysis, data were transformed used an arcsine transformation to account for use of

proportion data and meet assumptions of a normal distribution. Proportion of time spent

with each specific response variable (1-6) was also analyzed using a MANOVA,

following an arcsine transformation of the data.

RESULTS

Social Calls in Myotis yumanensis

From recordings obtained from outside The Benbow Inn and Smith River roosts, a total of 69 social calls were collected and isolated from a total of 42 bat passes. An average of 1.88 social calls was found in selected recordings, with as many as 7 individual social calls recorded in one bat pass.

Two distinct types of social calls were isolated and identified. Type 1 calls

(Figure 4A) consist of a single syllable, with simple descending frequency modulation

(FM) ending with a small upward FM sweep, with a wide bandwidth (traveling through a broad frequency range). The average duration of Type 1 calls was 11.03 milliseconds

(ms), with an average Fc of about 32 kHz. Fmax averaged about 60 kHz and Fmin averaged about 26 kHz. Type 2 calls (Figure 4B) consisted descending FM sweep, followed by a low slope upward sweep and ending on a second downward FM sweep.

Call duration of Type 2 calls averaged about 10.70 ms, with an average Fc of about 49.8 kHz. The Fmax for Type 2 calls averaged about 58 kHz and Fmin averaged about 40 kHz. Both types of calls were also highly variable, with frequency ranges averaging around 30kHz (Table 1). Only call duration, Fstart and Fmax did not different significantly between call types (P ≤ 0.05) (Table 1).

Figure 2. Sonograms of isolated Yuma myotis social calls. (A) Type 1 social call, (B) Type 2 social call. The call frequency (in kilohertz) is given on the y-axes and the time (in milliseconds) is given on the x-axes. Recordings were done with a Pettersson D500x bat detector with extendable microphone and visualized with the program Sonobat 3.0 (Arcata, CA).

Table 1. Social call parameters of Yuma myotis from northern California, showing Type 1 (n = 19) and Type 2 (n = 50), values of t-statistic and corresponding levels of significance P. For each parameter the value for Type 2 calls is given below that for Type 1 calls. Q1 and Q3 are the lower and upper quartiles. Parameter Mean ± SD Range Median Q1 Q3 t P Duration (ms) 11.03 ± 3.38 6.14 - 17.27 11.42 7.80 13.70 0.36 0.72 10.71 ± 3.03 5.22 - 19.33 10.77 8.78 12.46 Fc (kHz) 31.84 ± 7.67 22.30 - 55.49 29.13 27.31 33.53 -9.07 < 0.001 49.81 ± 5.69 38.95 - 68.09 51.13 44.93 52.92 Fstart (kHz) 60.35 ± 8.68 42.55 - 72.76 60.55 53.23 66.77 1.214 0.23 57.15 ± 12.26 38.69 - 98.65 53.41 50.83 58.14 Fmax (kHz) 60.36 ± 8.45 42.55 - 72.76 60.56 53.23 66.77 0.821 0.416 58.23 ± 11.69 42.34 - 98.65 54.98 51.79 58.71 Fmin (kHz) 26.37 ± 4.97 18.66 - 36.36 26.31 21.70 29.06 -9.71 < 0.001 39.74 ± 5.10 29.98 - 51.79 40.27 34.65 43.86 Fend (kHz) 29.66 ± 7.81 18.65 - 42.18 29.82 21.69 35.64 -7.11 < 0.001 43.65 ± 5.72 33.83 - 58.26 42.83 40.21 46.34 FmaxE (kHz) 39.84 ± 6.82 32.61 - 59.15 37.11 34.64 43.43 -2.38 0.024 44.03 ± 5.01 32.88 - 53.65 44.72 40.20 47.88 Bandwidth 33.98 ± 8.89 17.51 - 44.76 33.77 28.55 42.12 5.93 < 0.001 18.48 ± 11.00 5.55 - 57.31 15.35 10.59 21.48

Field Playback Experiment

One replicate was excluded from each year due to equipment malfunction, resulting

in a total of 9 playback replicates. During 30 observation nights in 2013, I recorded 3,175

bat approaches to 6 replicates in total. During 15 observation nights in 2014, I recorded a

total of 692 bat approaches to 3 replicates. Although I was unable to discern bat species

on video, Yuma myotis and Mexican free-tailed bats consisted of 80 – 90% of the bat activity in each area and it was assumed that most observed bat approaches consisted of these species.

Box location was matched for each of the 9 experimental boxes, with no significant difference in the minimal nightly temperatures between the nights per each treatment

(Friedman test: Q = 2.84, degrees of freedom (df) = 4, P = 0.58, N = 9). Other potential

environmental variables such as maximum daily temperature (Friedman test: Q = 6.29,

degrees of freedom (df) = 4, P = 0.17, N = 9), maximum nightly humidity (Friedman test:

Q = 2.69, degrees of freedom (df) = 4, P = 0.61, N = 9) and percent moon illumination

(Friedman test: Q = 2.56, degrees of freedom (df) = 4, P = 0.63, N = 9) also had no effect

on the number of bat approaches per box. Comparison between all five observation nights

showed that treatment order had a significant influence on number of approaching bats

each night (Friedman test: Q = 28.88, degrees of freedom (df) = 4, P < 0.001, N = 9).

Excluding control nights, which were always ordered first and last, there was no effect on playback type order (Friedman test: Q = 0.22, df = 2, P = 0.89, N = 9). Thus, bats did not become more familiar with a certain box over the playback nights, and environmental conditions were unlikely to have influenced my results.

The total number of bat approaches differed significantly between observation nights

(Friedman test: Q = 14.98, df = 4, P = 0.004, N = 9), with more bats observed

approaching during broadcast of social calls than during pre-playback control (Control A)

(Figure 5, Wilcoxon matched pairs test: W = 1.0, P = 0.007, N = 9). There were no significant differences between total bat approaches during either control nights and

TABR echolocation or MYYU echolocation calls. The number of bats observed approaching during playback nights differed significantly between MYYU social calls and either TABR echolocation or MYYU echolocation calls (MYYU social vs. TABR echolocation: Wilcoxon matched pairs test: W = 45.0, P = 0.004, N = 9; MYYU social vs.

MYYU echolocation: Wilcoxon matched pairs test: W = 44.0, P = 0.01, N = 9). The number of bats observed approaching the box also differed significantly between silent control nights (Control A vs. Control B: Wilcoxon matched pairs test: W = 6.0, P = 0.05,

N = 9).

When I analyzed the different types of approaches separately, the corresponding

Friedman tests were all significant (Figure 6, P values ranged from 0.02 to 0.008). Post- hoc tests showed that bats circled and approached the experimental bat boxes significantly more often during nights with playbacks of conspecific social calls than during other playback nights (Wilcoxon matched pairs test, Table 2). While observations of bats circling did not differ significantly between echolocation playback types (TABR and MYYU), the observations of bats circling and approaching differed significantly

28 between both pre- and post-playback controls and MYYU social calls (Wilcoxon matched pairs test, Table 2). Additionally, significantly more bats were seen approaching the experimental boxes during post-playback control nights (Control B) than prior to playback (Wilcoxon matched pairs test W = 6.0, P = 0.03, N = 9). While Friedman tests indicate significant difference between bats landing on experimental boxes between treatment nights (Q = 13.71, df = 4, P = 0.008, N = 9), post-hoc tests did not indicate any significant differences between pair-wise treatments, likely due to low counts. However, bats were only observed landing or entering the experimental boxes when there was an acoustic cue (playback) present at the box, with most bats landing during Yuma myotis call playback (social call and echolocation calls).

Table 2. Wilcoxon matched pairs test for pairwise comparisons of the numbers of bat approaches combined and separately for each type of approach between five treatments, the W- test statistic and corresponding P value. Treatment Total Passing Circling Direct Landing Approaches Approach W P W P W P W P W P Control A vs. 1.0 0.007 4.0 0.02 0.0 0.009 0.0 0.01 0.0 0.10 Social Control A vs. 7.0 0.14 12.0 0.25 7.5 0.08 4.5 0.24 0.0 0.34 TABR Control A vs. 9.0 0.12 11.0 0.20 2.5 0.03 0.0 0.03 0.0 0.18 Echo Control A vs. 6.0 0.05 8.0 0.09 22.5 1.00 0.0 0.02 0.0 NA Control B Social vs. 45.0 0.003 36 0.01 26.5 0.04 29.0 0.14 10.0 0.10 TABR Social vs. 44.0 0.01 44 0.01 23.0 0.15 31.5 0.06 6.0 0.17 Echo Social vs. 38.0 0.07 35.5 0.13 27.0 0.03 32.0 0.05 10.0 0.10 Control B TABR vs. 11.0 0.36 22.0 1.0 8.0 0.67 4.0 0.40 0.0 0.17 Echo TABR vs. 21.0 0.91 21.0 0.90 24 0.10 24..0 0.10 3.0 0.34 Control B Echo vs. 23.0 1.00 8.0 0.09 0.0 0.003 0.0 0.003 6.0 0.18 Control B

When Myotis call types were combined (total number of bat approaches/2), the total number of bats observed approaching experimental boxes differed significantly between

treatment nights (Figure 7, Friedman Test: Q = 9.61, df = 3, P = 0.02, N = 9). More bats

approached experimental boxes during Yuma myotis (MYYU) acoustic cues than during

Control A (Wilcoxon matched pairs test: W = 1.0, P = 0.007, N = 9) or TABR echolocation calls (Wilcoxon matched pairs test: W = 44, P = 0.007, N = 9). When I analyzed the different types of approaches separately, the corresponding Friedman test remained significant (P values between 0.04 and 0.007). With MYYU treatments combined, significantly more bats circled and approached the experimental boxes during

MYYU playback compared to Control A (Table 3). Also, significantly more bats approached experimental boxes during Control B than during Control A nights

(Wilcoxon matched pairs test: W = 0.0, P = 0.02, N = 9).

Table 3. Wilcoxon matched pairs test for pairwise comparisons of the numbers of bat approaches combined and separately for each type of approach between four treatments (MYYU social calls and echolocation calls combined into one treatment), the W- test statistic and corresponding P value. Treatment Total Passing Circling Direct Landing Approaches Approach W P W P W P W P W P Control A vs. 1.0 0.007 8.0 0.09 0.0 0.008 0.0 0.01 0.0 0.10 MYYU Control A vs. 7.0 0.14 12.0 0.25 7.5 0.08 4.5 0.24 0.0 0.34 TABR Control A vs. 6.0 0.05 8.0 0.09 22.5 1.0 0.0 0.02 0.0 NA Control B MYYU vs. 44.0 0.007 36.0 0.01 30.0 0.10 21.0 0.27 10.0 0.10 TABR MYYU vs. 35.0 0.16 29 0.49 40.0 0.03 30.5 0.09 10.0 0.10 Control B TABR vs. 21.0 0.91 21 0.90 24.0 0.10 13.5 1.0 3.0 0.34 Control B

Individual Playback Experiment

A total of 29 Yuma myotis were captured between July 23 and September 9, 2014, resulting in 29 individual playback trials. Seven trials were conducted for each treatment type, except for Yuma myotis echolocation calls, for which there were 8 trials conducted.

Six of the individuals captured were males and 23 were females. Twenty two of these were adults and 7 were juveniles. No lactating bats were captured. Most females were either post-lactating or not reproductive.

There was no effect of playback treatment on the proportion of time bats spent performing Positive, Neutral or Negative categories during the 30 seconds of playback.

Sex significantly influenced the amount of time bats spent performing different behaviors, with males spending significantly greater proportions of time performing neutral behaviors (Figure 8). When comparing between all 6 behaviors during the 30 seconds of playback, there was still no effect of treatment on proportions of behaviors.

Males spent significantly more time flying large circles around the flight tent than females, regardless of treatment type (MANOVA: F = 7.53, df = 1, P = 0.01). Juvenile bats also spent significantly more time flying large circles around the tent compared to adult bats (Figure 9, MANOVA: F = 4.41, df = 1, P = 0.04).

Figure 8. Proportion of time all bats spent performing three types of behaviors during the 30 seconds of playback compared between sexes, regardless of treatment type.

Figure 9. Proportion of time spent performing each of 6 different behaviors during the 30 seconds of playback, compared between juveniles (n=7) and adults (n=22), regardless of treatment type.

When comparing proportions of three behavior categories (Positive, Neutral and

Negative) during the five minute post-playback observation period, treatment was

borderline significant overall. Bats spent a greater proportion of their time performing

neutral behaviors during control treatments than during other treatment types

(MANOVA: F = 3.54, df = 3, P = 0.02). This effect remained significant when age and sex were controlled for in the model.

However, the proportion of time bats spent doing each of the six specific behaviors

(Near Circling, Near Landing, Large Circling, Side Landing, Far Circling or Far Landing) differed significantly between treatment types in the five minute period following playback (Figure 10, MANOVA: F = 1.85, df = 3, P = 0.04, N = 29). Specifically, bats spent significantly more time circling near the playback speaker following playback of social calls (Figure 10, MANOVA: F = 4.95, df = 3, P = 0.007) and more time flying large circles around the flight tent following control (Figure 10, MANOVA: F = 6.8, df =

3, P = 0.001). Accounting for sex and age of bats did not affect the significance of these behaviors, though males spent a greater proportion of time landing on the sides of the flight tent than females (MANOVA: F = 4.74, df = 3, P = 0.039). Interactions between the type of treatment and sex did not have significant effects on the time bats spent doing each behavior. However, the interaction between treatment type and age was significant

(MANOVA: F = 2.8, df = 2, P = 0.01). Adults and juveniles were not equally represented across all treatments, with only one juvenile tested for control and social call treatment types.

DISCUSSION

Social Calls in Myotis yumanensis

Few studies have investigated the specifics of social calls emitted by North American

Myotis species, with most studies having been conducted over 30 years ago on a limited

number of species (Fenton 1977, Barclay et al. 1979). The social calls of Yuma myotis I

describe were all emitted immediately adjacent to known roosting sites, prior to the

presence of pups and assumed to be emitted by adult bats.

Pfalzer and Kusch (2003) compiled a variety of bat social calls from 16 European bat

species, including seven species of Myotis. Structure and associated behavior of these

calls was variable and highly diverse, though many recorded calls from Myotis species

occurred at maternity and mating roosts. Noisy aggressive calls were common within

large bat colonies and can frequently be heard outside of roosts. These types of calls are

produced as an alternative to physical aggression, and have been associated with jostling

between individual little brown bats while roosting (Barclay et al. 1979). These calls

were commonly recorded outside Yuma myotis roost during this study, but often failed to

produce clean recordings that could be used for analysis.

Distress calls are also prevalent at roost sites, in response to some specific irritant.

These types of calls are also common during interference, such as human handling or

human presence. Distress calls have been known to attract conspecifics, and may function

as a way to deter or mob predators, such as in some pipistrelle species (Russ et al 1998).

In other species, such as Rafinesque’s big-eared bats (Corynorhinus rafinesquii), these short, repeated trills instead act as a spacing mechanism and deter other conspecifics

(Loeb and Britzke 2010). Social calls can be used as agonistic food defense calls, particularly at foraging sites. Pipistrelle bat species in Europe have been shown to use social calls for food patch defense (Barlow and Jones 1997, Russo and Jones 1999) and big brown bats (Eptesicus fuscus) that use social calls to “claim” food resources while foraging were more successful at prey capture (Wright et al. 2014). Social calls obtained in this study were not associated with obvious signs of foraging activity such as feeding buzzes (shortened echolocation pulses emitted immediately before an attack on an airborne insect) and are unlikely to be used in this context.

Many of these calls can be detected both inside and outside of roost locations. In this study, social calls were recorded immediately outside of known roost entrances/exits.

Thus, it is likely that recorded social calls were emitted just prior to flight or while in flight as bats approached the roost entrance. Social calls are important in tandem flights of bats, where pups learn to locate roost sites or foraging areas by following their mother

(Wilkinson 1992a, Kerth and van Schaik 2012). Calls in this study were recorded prior to the presence of any volant juveniles, but tandem flights have also been observed as

“following behavior” in adult bats (Racey and Swift 1985, Wilkinson 1992a), increasing individual foraging success and allowing coordination between individuals that commonly roost together (Wilkinson and Boughman 1998).

Social calls can also play a significant role in male advertisement and mate attraction.

Several bat species use complicated songs to attract mates, often broadcasting or displaying at central roost locations or mating roosts (Behr and von Helverson 2004,

Bohn et al. 2009a). Social calls in this study were obtained just prior to parturition at

maternity roosts, with most callers likely to be pregnant females. Additionally, many

Myotis species like Yuma myotis mate primarily in the fall and winter (Dalquest 1947, P.

Cryan, pers. comm., 2014) and it is unlikely that any mate attraction calls or behaviors

would be observed in the summer.

Social calls at roost sites can help facilitate information transfer about potential

foraging or roosting resources (Ratcliffe and ter Hofstede 2005, Chaverri et al. 2010).

Pallid bats (Antrozous pallidus) emit directive social calls while exiting roosts, which act both as an advertisement for a suitable crevice and as part of a “rallying flight” to coordinate between flying group members (Arnold and Wilkinson 2011). Tent-making

bats that roost in highly ephemeral rolled up leaves produce social calls associated with roost-mate recruitment (Gillam et al. 2013) and have developed call-and response systems to locate familiar individuals at roost sites (Chaverri et al. 2012). It is unknown if the social calls I recorded were produced by bats entering or exiting the roost, but it is plausible that both Type 1 and Type 2 social calls may be used as contact or recruitment calls between individuals within and around the roost or to coordinate bats as they enter

and exit the roost. It would be highly rewarding to investigate the specific context of

these calls through synced acoustic and video recording and determine timing and

identity of calling individuals using marked bats.

Regardless of specific behavioral contexts, it is increasingly important to develop

a better understanding of social call structure and build a more complete vocal repertoire

for common North American bat species. Non-invasive and cost effective, acoustic

surveys are increasingly used to detect bat presence and activity and make management

and conservation decisions. Many species can be confidently identified from their

echolocation calls, though there can be significant variation due to habitat structure,

geographical area, sex, age and conspecifics (Russo and Jones 2002, Gillam and

McCracken 2007). Additionally, many species have similar echolocation call structures

due to phylogenetic relatedness or adaptive convergence (Jones and Holderied 2007).

Social calls have the potential to aid bat species identification, particularly in areas with problematic cryptic species (Georgiakakis and Russo 2012, Russo and Papadatou 2014), but require more comprehensive knowledge of the variation and structure of these social calls.

There have been published efforts to describe social calls among a diversity of

European species (Pfalzer and Kusch 2003, Middleton et al. 2014), but there is a dearth of knowledge of social calls of many common North American bat species, particularly of Myotis species. Echolocation structure between Myotis species can be difficult to determine in all cases. For example, approximately 5-15% of Indiana bat (Myotis sodalis) calls will resemble little brown bat calls, depending on the recording context (Szewczak et al 2011b). This can have significant consequences for habitat protection, since Indiana bats are listed as endangered under the Endangered Species Act and little brown bats are not. Knowledge of and observation of species-specific social calls could increase species identification accuracy in these confounding situations. This study presents one of the first descriptions of social calls in a common Myotis species at a roost site, though there is still much to learn about the diversity of Myotis social calls.

Field Playback Experiment

The aim of this study was to investigate whether wild free-ranging bats are attracted to social calls of conspecifics, specifically as a potential method for attracting bats to artificial roost sites during exclusions or relocations. Additionally, I attempted to investigate whether Yuma myotis can distinguish between conspecific social and echolocation calls and calls of a sympatric, heterospecific bat species (Mexican free- tailed bat), though ultimately I was unable to identify species on video recordings.

Bats approached artificial roost boxes significantly more often during playback of

Yuma myotis social calls compared to other playback treatments (Figure 5). This indicated that bats are more attracted by social calls than echolocation calls, and suggests that social calls can be used to get more bats to investigate potential roosts. Of the two social calls isolated from roost recordings, only Type 2 was used for playback trials. Type

2 calls were the only calls of high enough quality to use for playback when this study started and so were used as playback files. Though Type 1 and Type 2 calls were recorded in the same general context (outside of a roost entrance), it is possible that only using Type 2 calls biased these results. Further study can investigate whether broadcast of

Type 1 calls would elicit the same responses. Social calls used in this study were recorded specifically at a roost site, so the presence of such a call at a potential roost could indicate a quality roost locations.

As intentionally emitted signals, bats may use social calls as a way to actively guide conspecifics to potential roost sites and maintain group cohesion between individuals.

Searching for roosts can be energetically demanding and bats are more efficient at

44 finding roosts when social acoustic cues are present (Ruczyński and Barton 2012). Long- term social relationships maintained by some bat species despite high fission-fusion dynamics are likely partially facilitated through social communication at common resources (Kerth et al. 2011), and allow bats to benefit from group living including predator detection, information transfer (Kerth and Reckardt 2003) and social thermoregulation (Willis and Brigham 2007). Social calls at a roost can provide a mechanism to reunite individuals after separate foraging and can contain information about the calling individual, allowing familiar recognition (Arnold and Wilkinson 2011).

Attracting familiar conspecifics to roost sites allows bats living in maternity roosts, such as those in this study, to benefit from cooperative behaviors such as allo-nursing of young

(Wilkinson 1992b) and pup-guarding (Bohn et al. 2009b).

Playback of Yuma myotis echolocation calls did not result in significantly more bat approaches than either pre- or post-playback control nights (Table 2). While primarily used for foraging and orientation, echolocation calls can be used as a source of social information by eavesdropping bats (Fenton 2003). Echolocation calls can provide information about the presence of bats at a given foraging or roost site (Dechmann et al.

2009, Gillam 2007). Under lab conditions, Ruczyński et al. (2007) demonstrated that noctule bats (Nyctalus noctula) used echolocation calls of conspecifics played back from an artificial tree cavity to find the roost.

This discrepancy in behavior responses to Yuma myotis social calls and echolocation calls could be a function of the physics of the call rather than the social or behavioral context. Minimum frequency of Yuma myotis echolocation calls generally only reach as

low as 44 kHz (Szewczak et al. 2011a). High frequency sounds attenuate quickly in air,

traveling limited distances (Siemers and Kerth 2006). In contrast, the minimum

frequency of Yuma myotis social calls averaged about 38 kHz. Depending on

environmental factors such as temperature, humidity and air pressure, a 50 kHz call can

range from 122 to about 30 decibels (dB) up to 35 meters away, with a low threshold for

detection averaging around 30 dB (Arnett et al. 2013). Frequencies around 30 to 40 kHz

generally don’t drop below 30 dB before reaching 45 to 60 meters away (Arnett et al.

2013), increasing detection distance by almost 50%. It is possible that lower frequency

social calls attracted more eavesdropping bats due to increased audible range.

Interestingly, bats previously demonstrated to use echolocation calls to convey social

information generally have mid to low frequency echolocation calls, such as big brown

bats, noctule bat and African large-eared free-tailed bats (Otomops martiensseni) (Kazial

et al 2001, Ruczyński et al. 2007, Fenton et al. 2004).

The number of bats approaching during Mexican free-tailed bat echolocation call

playback did not differ significantly from either control nights or Yuma myotis

echolocation playback nights. Mexican free-tailed bats have been demonstrated to eavesdrop on echolocation calls containing feeding buzzes of conspecifics but not on calls without feeding buzzes (Gillam 2007). Based on acoustic data collected from field

sites, Mexican free-tailed bats were abundant and it could be expected that at least some individuals would respond to acoustic cues of conspecifics. However, Mexican free-tailed

bats have also been shown to emit sounds that jam the sonar of conspecifics while

foraging, causing conspecifics to miss their prey (Corcoran and Conner 2014). Thus, it is

46 possible that free-tailed bats will avoid playback of conspecific echolocation calls as a spacing mechanism and to decrease interspecific food competition.

Eavesdropping on acoustic cues can be a mechanism for social learning within and between different species, particularly when involved species share a common niche

(roosting, foraging, etc). Current research has focused on the use of eavesdropping on echolocation calls between sympatric bat species that share a similar diet (Gillam 2007,

Ubernickel et al. 2013, Clarin et al. 2014). Bats will discriminate and respond to heterospecific echolocation calls, though the strength of the response depends on the amount of niche overlap (Li et al. 2014). Species that share a greater ecological similarity in foraging receive a greater benefit from eavesdropping on the foraging calls of heterospecifics, such as between little brown bats and big brown bats (Barclay 1982).

However, the species in this study demonstrate little diet overlap. Mexican free-tailed bats forage high above the ground compared to Yuma myotis, feeding primarily on moths and beetles (McCracken et al. 2008). Yuma myotis forage low over water sources, where they feed on aquatic emerging insects (Brigham et al. 1992). These species can share roosting resources such as a bat box, but may seek different temperature regimes or other environmental factors that result in natural segregation within a structure. Additionally, the response to echolocation calls of either species was not significant compared to silent controls. It could be that echolocation calls at roost sites are not a reliable signal to indicate quality roosting habitat, even in species that share that resource.

Although results were not statistically significant due to low total numbers of bats entering boxes, it is notable that bats were only observed entering roost boxes during

playback nights. It is unknown if these were different individuals entering the boxes or

the same individuals each time. There was also evidence of a latent effect of playback at

the roost sites, with more bats seen approaching the experimental boxes during post-

playback nights than prior to playback. In this study, it is unclear if the same or different

individuals were approaching the bat boxes during observation nights. Individual bats

may use the presence of social calls to initially learn about new roosts. Individual

Bechstein’s bats (Myotis bechsteinii) sometimes did not occupy roosts until several

weeks after they were first inspected (Kerth and Reckardt 2003). Bats have been

demonstrated to have strong spatial memory, even after long periods of time (Rydell

1990, Jensen et al. 2005). If it was not the same individuals returning to the experimental

bat boxes following playback, there is the possibility of some information transfer

between individuals (Kerth and Reckardt 2003). Most Yuma myotis in the area roosted within the same colony and could be sharing information about these new potential

roosts.

It should be noted that bats are not the only animals that might eavesdrop on acoustic

cues. The presence of lower frequency, conspicuous social calls could also result in the

attraction of natural bat predators. At three experimental boxes in 2013, owls were

observed perching and flying around the box only during playback nights. Interestingly,

owl presence seemed to have no obvious effect on bat activity, with bats still entering

video field of view while an owl was present. Owls and other raptors have been observed

predating on bats, with avian predation accounting for about 11% of the annual mortality

in British bats (Speakman 1991). Bat bones have also been found in spotted owl and barn

48 owl pellets (Duncan and Sidner 1990, Kowalski and Lesiriski 1990, Vargas et al. 2002).

Owls rely heavily on sound while hunting and it is possible they may be able to eavesdrop on social calls or associated background noise of social call playback recordings. The effect of predators on bat behavior is still largely unknown (Lima and

O’Keefe 2013) and this observation provides anecdotal evidence of a predator perceiving bats via auditory cues, indicating a potential interaction between prey acoustic vocalizations and predator detection, though further research would be needed.

Individual Playback Experiment

Overall, there was little difference in bat behaviors in different playback situations.

However, age and sex were demonstrated to have some effect on the proportion of time bats spent doing six defined behaviors in a flight tent situation.

Regardless of treatment type, males spent significantly more time behaving in a neutral way, neither attracted nor repelled by playback (Figure 8). While female Yuma myotis roost in large maternity colonies sometimes numbering in the thousands, males tend to roost separately during the summer, in smaller bachelor roosts (Dalquest 1947).

Males and females differ in roosting ecology, especially in the summer. During lactation, females benefit from larger group sizes (Kerth et al. 2001) while males roost in smaller groups and change roosts more frequently (Randall et al. 2014). During this time, there is little benefit for males to respond to social calls emitted at maternity roosts. Additionally, studies have indicated that bats may be able to recognize sex of a caller from echolocation calls (Kazial and Masters 2004, Knornschild et al. 2012), so male bats may respond more favorably to female social calls during other times of the year, such as

49 during fall mating swarms. It is also possible that flight behaviors will not accurately reflect social responses, which may reflect in more subtle movements such as wing displays, head movements or facial expressions.

Surprisingly, juvenile bats also spent most of their time performing neutral behaviors, flying large circles around the flight tent (Figure 9). Social calls are important in facilitating social relationships between mothers and pups in maternity colonies. Female bats can identify individual pup isolation calls (Scherrer and Wilkinson 1993, Bohn et al.

2007) and pups can also respond to specific female isolation calls (Balcombe and

McCracken 1992). Social calls can also be important in tandem flights as young bats learn to fly and locate foraging and roost sites (van Heerdt and Sluiter 1965, Vaughan

1976). For these reasons, I would have predicted juvenile bats would respond strongly to social calls of conspecifics. However, calls used for playbacks were recorded outside of the roost and the social context may have been aimed at other adults and not at juveniles.

Additionally, juvenile bats may be less agile flyers than adults. Studies have shown habitat partitioning in juvenile bats, with juvenile little brown bats more often foraging in less cluttered environments compared to older and larger individuals (Adams 1997).

Placed in a flight tent where their movements are restricted may limit the range of behaviors juvenile bats can perform, and young bats may be more concerned with increasing foraging time than social interactions.

Results from this experiment were limited by demographic sampling bias. Most of the sampled individuals were adult females. Treatments were assigned randomly across all

50 individuals, resulting in a skew of types of bats exposed to different treatment types and it is possible that sample sizes were insufficient to accurately reflect different behaviors.

These results indicate there is some potential for age and sex specific responses to social calls, though further testing needs to be done. Bats were given a minimum of two minutes to acclimate to the experimental set up prior to the start of the playback experiment. Increasing this time may allow bats to be more relaxed and act more naturally under experimental conditions. More detailed information about an individual’s response may have been obtained by presenting each individual with all four different treatment types (on different occasions), instead of just one treatment type. By presenting the same individuals with five different playback treatments, Voigt-Heucke et al. (2010) were able to demonstrate bat recognition of familiar and unfamiliar conspecifics using echolocation calls. Habituation-dishabituation experiments have also been used to determine behavioral responses to acoustic playbacks in bats by testing responses to a new or different acoustic treatment following habituation (Kastein et al. 2013, Li et al.

2014). Permitting and funding limited these options in the present study, but there is still much potential to investigate these responses in a common myotis species.

CONCLUSION

Previous field studies have shown that playbacks of social calls of a European bat

species can attract several bat species at foraging sites (Russ et al. 2004), into mist-nets

(Hill and Greenaway 2005), or to artificial roost boxes (Schöner et al. 2010). This study provides further evidence that social calls be used as management tool to attract bats to new roost locations, especially in cases of habitat loss or roost exclusions, as well as demonstrating this effect in a North American bat species. Additionally, this study has provided the first description of social calls in a North American Myotis species. Yuma

myotis may possibly use these calls as contact calls between individuals or as a spacing

mechanism for bats entering and exiting at roost openings. Understanding bat social calls

can provide insight into roosting, foraging and mating behaviors that can inform

conservation and management decisions. Finally, improving our knowledge of common social calls can also improve non-invasive acoustic surveys by broadening our knowledge

of bat call repertoires, which may improve detection of rare and cryptic species.

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