Postnatal ontogeny of the cochlea and flight ... - Wiley Online Library

Journal of

Anatomy

J. Anat. (2015) 226, pp301--308

doi: 10.1111/joa.12284

Postnatal ontogeny of the cochlea and flight ability in Jamaican fruit bats (Phyllostomidae) with implications for the evolution of echolocation Richard T. Carter1 and Rick A. Adams2 1

Ohio University, Chillicothe, OH, USA University of Northern Colorado, Greeley, CO, USA

2

Abstract Recent evidence has shown that the developmental emergence of echolocation calls in young bats follow an independent developmental pathway from other vocalizations and that adult-like echolocation call structure significantly precedes flight ability. These data in combination with new insights into the echolocation ability of some shrews suggest that the evolution of echolocation in bats may involve inheritance of a primitive sonar system that was modified to its current state, rather than the ad hoc evolution of echolocation in the earliest bats. Because the cochlea is crucial in the sensation of echoes returning from sonar pulses, we tracked changes in cochlear morphology during development that included the basilar membrane (BM) and secondary spiral lamina (SSL) along the length of the cochlea in relation to stages of flight ability in young bats. Our data show that the morphological prerequisite for sonar sensitivity of the cochlea significantly precedes the onset of flight in young bats and, in fact, development of this prerequisite is complete before parturition. In addition, there were no discernible changes in cochlear morphology with stages of flight development, demonstrating temporal asymmetry between the development of morphology associated with echo-pulse return sensitivity and volancy. These data further corroborate and support the hypothesis that adaptations for sonar and echolocation evolved before flight in mammals. Key words: Artibeus jamaicensis; bats; cochlea; evolution; ontogeny.

Introduction High-frequency hearing most likely evolved in mammals during the late Cretaceous in the ancestor to marsupials and placentals (Meng & Fox, 1995). Both Metatherians and Eutherians possess cochlear ducts that are elongated and coiled in structure, thereby increasing the frequency range and sensitivity to ultrasound. Cochlear features common to all mammals include a basilar membrane (BM) that is narrow in the basal turn and widens towards the apex, and a tectoral membrane (TM) that increases in cross-sectional € ssl, 2011). Furthermore, area from base to apex (Vater & Ko using CT scans of the inner ear, it has been shown that the common ancestor to all bats was likely capable of such ultrasonic hearing (Davies et al. 2013). However, the ability to hear high-frequency sounds in contemporary mammals is often not accompanied by the Correspondence Richard T. Carter, Ohio University, 101 University Dr., Chillicothe, OH 45601, USA. E: [email protected] Accepted for publication 12 January 2015 Article published online 31 March 2015 © 2015 Anatomical Society

ability to emit comparable vocalizations. Indeed, ultrasonic emissions are often associated with some of the most derived mammals, those in the orders Cetacea and Chiroptera. Both groups evolved specialized echolocation systems independently but show convergent evolution in some character states associated with the inner ear (Ketten, € ssl, 2011) such as a particularly nar1992a,b; Vater & Ko rowed and thickened BM that boosts stiffness, thereby increasing sensitivity of the BM to ultrasound and an ossified secondary spiral lamina (SSL) that provides a lateral anchoring structure for the thickened BM. Further, the basal turn of the cochlea encompasses a relatively larger cross-sectional area in cetaceans and echolocating bats (Solnsteva, 2010a,b). Both of these adaptations increase sensitivity to higher frequencies (Habersetzer & Storch, 1992). Neither nonecholocating flying foxes or echolocating pteropodids from the genus Rousettus (which make use of tongue clicks rather than laryngeally produced ultrasound) exhibit such enlargement of the cochlear basal turn. The evolutionary relationship between echolocation and flight in bats remains controversial as there are no known fossils that show definitive transitions between

302 Postnatal ontogeny of the bat cochlea, R. T. Carter and R. A. Adams

nonvolant and volant mammals, with molecular evidence providing no clear consensus either. It has been suggested that the oldest known full-bodied fossil bat specimens (Onychonycteris finneyi) are from an extinct species of flying microbat that did not echolocate, supporting the flight first theory (Simmons et al. 2008, 2010). However, this was refuted when, based on the articulation of the stylohyal and tympanic bones, it was shown that this species might have echolocated and therefore does not represent a transitional form (Veselka et al. 2010). Not only has molecular analysis of extant bat species provided no conclusive evolutionary links between echolocation and flight in bats but there is also disagreement regarding how many times echolocation has evolved in bats. One hypothesis is that echolocation evolved independently in Vespertilioniformes (Yangochiroptera) and Pteropodiformes (Yinpterochiroptera) (Teeling et al. 2000; Eick et al. 2005; Jones & Teeling, 2006). Another is that, based on substantial sequence overlap in the genes that regulate synthesis of Prestin (a motor protein responsible for cochlear sensitivity), laryngeal echolocation evolved once in extant bats (Li et al. 2008, 2010; Jones, 2009; Parker et al. 2013). This striking overlap in Prestin gene sequence has also been attributed to molecular convergence and, like morphology, can provide misleading signals regarding evolutionary history. In previous papers that classified bats into the same developmental stages (flutter, flap, flight, adult), using the same drop tests as the present paper, we showed that the ontogeny of flight and of laryngeal emission of echolocation are disjunct. Indeed, 1-day-olds emitted adult-like sonar calls approximately 36 days before the onset of flight. We went on to hypothesize that the evolution of sonar ability occurred before the evolution of powered flight in bats and that the protobat inherited a primitive sonar system that was later co-opted into the highly derived sonar system observed today (Carter & Adams, 2014; Carter et al. 2014). Although the relative developmental and functional timing among compartmentalized systems may be altered evolutionarily, in many cases they are not and thus ontogeny can act as a surrogate for understanding ancestor–descendant transitions (see Wagner, 2014 for review). In this paper we further corroborate this theory by showing that the morphological prerequisite for cochlear sensitivity to high-frequency sounds develops prepartum and is already available for use at birth in bats with no significant changes during flight development. These data align with the fact that the ability of mammals to hear high-frequency sounds evolved very early in mammalian history and in and of itself is decoupled from the ability to generate ultrasound by any given species (Meng & Fox, 1995). We feel these data raise interesting new questions regarding the evolution of echolocation in bats that are not under consideration in current discussions of bat evolution.

Methods Sample population A captive breeding colony of Artibeus jamaicensis (approximately 60 individuals) was housed at the University of Northern Colorado in a 200-m2 room that allowed for free-flight and proper development of flight ability in young bats. Bats were fed a prepared mixed fruit diet (apples, bananas, cantaloupe, mangoes, and watermelon) daily with a twice weekly blended supplement of Harlan Global, 25% protein primate diet mixed with corn syrup for added sugar, powdered milk for extra calcium, and dry Jell-O gelatin for flavor (Shaw, 2011). We also hung larger pieces of fruit from the walls, which helped alleviate competition at the two feeding trays and also provided some environmental enrichment. We used a reversed 12-h light/dark light cycle to ensure that the bats were active during daylight hours. Room temperature was maintained at about 23 °C and relative humidity between 50 and 65%. Females typically gave birth to one pup twice a year with each cohort of newborns ranging between 15 and 20 individuals. Pups are precocial, born with some fur and eyes open, much like other phyllostomids (Gould, 1975).

Flight ability tests Flight ability data were gathered using the drop test method, which began on the first day of parturition and involved a free fall from a 1-m-high perch onto a soft, padded surface in a flight chamber (2 9 3 m) (Moss et al. 1997). We categorized flight ability of each individual into one of four categories: flutter – individuals fell straight to the pad with no horizontal displacement but with wing movement; flap – individuals fell to the pad with horizontal displacement (within 200 cm of the perch) but without sustained flight across the flight chamber; flight – individuals achieved powered flight across the chamber; adult – individuals achieved powered flight across the chamber and had an age of ≥ 104 days, which is the average age at which the epiphyseal gap of the 4th metacarpal closes in A. jamaicensis and the animal has completed skeletal growth (Ortega & Castro-Allano, 2001).

Tissue and data collection Cochleae were collected from individuals from the captive colony at different stages of development. All procedures used in this study were approved by the University’s IACUC (protocol #1108B-RA-14). Following assessment of flight ability, all individuals were euthanized with an overdose of isoflurane followed by cervical dislocation. Both cochleae were immediately exposed and doused with neutral buffered formalin (NBF) to begin the fixation process in situ, following which both cochleae were carefully removed and placed in separate vials of NBF. We collected 42 cochleae from 30 bats distributed across the various flight stages in the following numbers: flutter – 12 cochleae from nine individuals; flap – nine cochleae from seven individuals; flight – 11 cochleae from seven individuals; and adult – 10 cochleae from seven individuals (Table 1). Histological preparation and sectioning was carried out by Colorado Histoprep (Fort Collins, CO, USA). Cochleae were run through 10% NBF followed by 70, 85, and 100% ethanol and then 100% xylene and three stages of paraffin, all for 45 min each. One midsagittal section (5 lm thick) was taken per cochlea, sections were parallel to the modiolus at planes A, B or C, which were © 2015 Anatomical Society

Postnatal ontogeny of the bat cochlea, R. T. Carter and R. A. Adams 303

Table 1 Number of cochleae sampled for basilar membrane, and SSL morphometrics across each developmental stage and the cochlea region (1–12) found in each section orientation. Developmental flight stage

Section orientation (from oval window)

Cochlea region

No. of cochleae

Flutter

0° 60° 120° 0° 60° 120° 0° 60° 120° 0° 60° 120°

1, 2, 3, 1, 2, 3, 1, 2, 3, 1, 2, 3,

4 5 3 3 3 3 4 4 3 4 3 3

Flap

Flight

Adult

4, 5, 6, 4, 5, 6, 4, 5, 6, 4, 5, 6,

7, 8, 9, 7, 8, 9, 7, 8, 9, 7, 8, 9,

10 11 12 10 11 12 10, 11 12 10 11 12

radially 60° apart (using the oval window as a reference landmark) and stained with hematoxylin and eosin (H&E; Bruns, 1976) (Fig. 1). Section A was taken through the oval window (0°), section B 60° from the oval window, and section C 120° from the oval window. This sectioning technique provided a series of cross-sections through the organ of Corti at relatively evenly spaced intervals along the entire cochlea for all flight stages. Cochlear regions were assigned as follows: the first half turns from sections A, B, and C (0°, 60°, and 120°) were assigned as regions 1, 2, and 3, respectively, the second half turns from these same sections were assigned as regions 4, 5, and 6, respectively, and so forth for all cochlear regions. This technique provided 12 regions (1–12) in a base to apex direction with each section (0°, 60°, and 120°), providing four regions (Table 1). Cochleae from the same individual were always sectioned in different planes to ensure independence of the data. Digital photographs of each cochlea section were taken at 409 and each organ of Corti cross-section at 4009 with an Olympus CX41 microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) with an Insight 2 Spot Image Sample (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) camera with associated SPOT (version 4.0.4) software. A 100-lm bar was digitally added to each photograph for calibration

and morphometrics were then made with Sigma Scan (SPSS, Inc., Chicago, IL, USA). The following morphometrics were used to quantify any differences between the cochleae of different flight stages: cochlea height, basal and apical turn diameter, BM thickness and width (pars tecta and pars pectinata), and SSL length. A stiffness coefficient of the BM was also calculated by finding the quotient of the thickness and width of the pars pectinata (Bruns, 1976; Vater & € ssl, 2011). Cochlear height, basal and apical turn diameter data Ko were measured from sections made through the oval window from four cochleae from four different individuals for each developmental stage except the flap stage, from which three cochleae from three different individuals were used (Fig. 2). Comparisons of measurements of cochlear morphology between developmental stages were made using one-way ANOVA with a = 0.05 (Number Cruncher Statistical Systems, NCSS, LLC, Kaysville, UT, USA).

Results Cochleae from all developmental stages had 2.5 full turns and cochlea height was not significantly different among stages (flutter, 2632.9 lm, SD = 185.7; flap, 2640.7 lm, SD = 30.3; flight, 2665.9 lm, SD = 101.8; adult, 2680.1 lm, SD = 174.0) (F3,11 = 0.09, P = 0.96). Basal turn diameter was also not significantly different among stages (flutter, 3311.6 lm, SD = 133.1; flap, 3321.3 lm, SD = 47.2; flight, 3526 lm, SD = 98.7; adult, 3509.5 lm, SD = 159.2) (F3,11 = 3.46, P = 0.054). In addition, apical turn diameter was not significantly different among stages (flutter, 2352.8 lm, SD = 131.9; flap, 2339.2 lm, SD = 308.7; flight, 2288.3 lm, SD = 177.7; adult, 2367.7 lm, SD = 161.1) (F3,11 = 0.13, P = 0.94).

BM and SSL morphology For all flight stages the BM is radially segmented into the pars tecta and pars pectinata (Fig. 3) and was narrower and thicker at the base of the cochlea, thinning and widening in an apical direction. There was an obvious SSL present in the first 1.25 turns of the cochleae for all flight stages (cochlear regions 1–6), all SSLs began to diminish in the second half of the apical turn (cochlear regions 7–9), and all SSLs were greatly reduced in the last three cochlear regions (10–12) (Fig. 4). When the width and thickness of the BM (pars tecta and pars pectinata) at the same cochlear regions were compared among flight stages we found no significant difference (Tables 2–5). There was also no significant difference between the SSL from the same cochlear region among flight stages. Finally, there was no significant difference between the stiffness coefficients of the BM from the same cochlear regions among flight stages.

Discussion Fig. 1 Diagram of a cochlea showing orientation of planes sectioned around the modiolus (from Bruns, 1976 with permission). Planes A, B, and C represent planes cut at 60° intervals around the axis of the modiolus (M). A 60° wedge has been removed to illustrate the orientation of the intact BM. © 2015 Anatomical Society

The gross morphology of the cochlea of adult A. jamaicensis was previously described as approximately 2.2 mm high (as measured from midmodiolar sections) with a BM that follows the same general mammalian pattern of increasing


A

B

Fig. 2 Cross-section through the modiolus of cochleae from individuals from the flutter (A) and adult (B) flight stages at 409 (H&E stain). Dashed lines represent cochlea height (red), basal turn diameter (blue), and apical turn diameter (green). Primary spiral lamina (PSL) and secondary spiral lamina (SSL) are indicated with arrows. The first half turn is indicated with T1, the second with T2, third with T3, and fourth with T4.

A

B

Fig. 3 Cross-section through the first half turn (T1) of cochleae from individuals from the flutter (A) and adult (B) flight stages at 4009 (H&E stain). The dashed boxes surround the pars tecta (red) and pars pectinata (blue) of the BM. PSL, SSL, and TM are indicated by arrows.

Fig. 4 Average SSL length (lm) for all regions (base to apex) of all developmental stages. Flutter is represented by the black bars, flap by the gray bars, flight by the parallel striped bars, and adult by the cross-hatched bars. The error bars represent standard deviation.

width and decreasing thickness from base to apex (Pye, 1967). Our data agree with those of Pye (1967) in that we found adult bats to have an average cochlea height of 2.6 mm (2680.1 lm, SD = 174.0) and an increase in width and a decrease in thickness of the basilar membrane from base to apex. Our data also showed that nonvolant neonates possess cochleae that were not significantly different from adult cochlea in height, width (basal and apical turns) or morphometrics associated with high frequency specialization. Although the microchiropteran skull has undergone much of its development prior to parturition, it is still only approximately 80% of the expected adult size (Pedersen, 1993, 1995, 1998, 2000). Thus, the similarity in size of newborn and adult cochlea was surprising. However, this pattern of adult-sized cochlea housed in the newborn skull has also been found in many rhinolophids and some hipposiderids (Vater, 2000). Additionally, the width and thickness of the BM has also been found to be no different between © 2015 Anatomical Society


Table 2 Average width (lm) of the pars tecta at all cochlear regions (1–12, base to apex) for all flight stages. Values in parentheses represent standard deviation.

Table 4 Average width (lm) of the pars pectinata at all cochlear regions (1–12, base to apex) for all flight stages. Values in parentheses represent standard deviation.

Developmental stage and average pars tecta length (lm) Cochlear region 1 2 3 4 5 6 7 8 9 10 11 12

Flutter 29.2 34.6 28.5 46.8 44.7 43.1 49.3 54.1 51.9 55.8 52.7 55.6

(5.0) (5.3) (5.3) (0.9) (5.2) (2.6) (3.9) (6.2) (2.5) (11.0) (7.4) (1.1)

Flap 30.03 31.9 35.6 39.9 42.2 39.9 49.9 53.0 52.2 56.4 56.1 56.3

(3.3) (4.1) (3.2) (6.8) (2.1) (2.2) (0.8) (6.2) (1.7) (6.1) (10.6) (1.5)

Flight 28.5 29.4 33.9 42.0 45.6 41.9 49.3 51.1 53.3 53.7 52.2 56.0

(2.3) (4.4) (7.1) (4.9) (5.9) (2.6) (4.2) (8.8) (5.3) (3.4) (6.1) (4.4)

Adult 27.1 32.2 28.7 44.2 44.5 43.3 52.7 52.8 60.2 58.3 57.8 60.3

(13.9) (4.6) (5.3) (6.5) (9.9) (2.3) (5.9) (1.3) (10.8) (3.3) (2.9) (1.5)

Table 3 Average thickness (lm) of the pars tecta at all cochlear regions (1–12, base to apex) for all flight stages. Values in parentheses represent standard deviation.

Developmental stages and average pars pectinata length (lm) Cochlear region 1 2 3 4 5 6 7 8 9 10 11 12

Flutter 62.8 63.7 61.9 75.2 79.1 69.0 82.4 82.1 78.4 77.4 80.6 78.8

1 2 3 4 5 6 7 8 9 10 11 12

Flutter 11.0 8.8 9.7 7.1 6.5 7.7 4.1 4.6 5.9 2.6 3.1 4.7

(2.3) (1.4) (3.4) (1.4) (2.0) (3.7) (1.6) (1.6) (2.0) (1.1) (0.7) (3.4)

Flap 9.2 8.1 8.3 7.0 6.9 7.3 4.3 4.6 4.2 4.4 3.7 3.7

(1.5) (1.4) (2.9) (1.6) (4.1) (1.0) (1.6) (2.1) (1.5) (0.9) (0.6) (2.0)

Flight 11.6 9.6 9.6 8.8 8.2 9.3 8.3 4.0 5.4 4.8 2.4 3.7

(2.2) (1.0) (2.8) (1.8) (2.1) (5.9) (3.30 (1.3) (2.7) (2.3) (0.5) (0.6)

Adult 9.5 10.3 9.4 8.8 7.1 8.4 7.1 4.4 6.3 4.6 5.0 4.0

(2.3) (1.2) (3.2) (1.1) (0.9) (3.0) (2.1) (0.9) (1.1) (1.5) (3.4) (2.3)

newborns and adults in gerbils and horseshoe bats, indicating that this pattern may be primitive in mammals and possibly other vertebrates (Harris & Dallos, 1984; Vater, 1998). Along with our previously published evidence on the ontogeny of echolocation and laryngeal development (Carter & Adams, 2014; Carter et al. 2014) in bats, we feel that the present study corroborates a strong case for the echolocation-first evolutionary scenario in bats (Fenton, 1984; Fenton et al. 1995; Arita & Fenton, 1997). Specifically, that a cochlea sensitive to high frequencies is a primitive trait in early and contemporary mammals (Metatherians and Eutherians) (Meng & Fox, 1995) and was a preadaptation to the eventual evolution of high-frequency sonar emission. © 2015 Anatomical Society

62.3 64.4 62.5 76.5 72.0 74.4 78.7 80.5 78.4 84.0 76.4 78.6

(9.3) (5.2) (1.4) (3.5) (3.0) (4.1) (3.0) (0.9) (2.6) (11.1) (7.7) (4.3)

Flight 62.9 60.7 63.9 73.0 75.5 74.5 79.8 80.5 82.4 76.3 71.0 78.9

(4.0) (3.8) (6.2) (3.1) (4.0) (0.7) (4.3) (4.7) (9.10 (9.9) (8.4) (7.9)

Adult 71.3 66.5 59.9 76.0 81.1 72.9 80.3 82.9 81.3 75.7 86.2 79.5

(11.4) (1.3) (4.7) (5.1) (7.0) (4.3) (3.1) (2.8) (1.8) (4.1) (8.0) (8.2)

Table 5 Average thickness (lm) of the pars pectinata at all cochlear regions (1–12, base to apex) for all flight stages. Values in parentheses represent standard deviation.

Developmental stages and average pars tecta height (lm) Cochlear region

(2.9) (5.5) (2.6) (1.0) (6.0) (1.0) (1.6) (3.20 (3.9) (6.8) (8.6) (4.0)

Flap

Developmental stages and average pars pectinata height (lm) Cochlear region 1 2 3 4 5 6 7 8 9 10 11 12

Flutter 9.0 10.0 8.2 8.2 8.8 8.5 5.3 4.9 4.8 3.9 3.2 4.1

(1.0) (1.7) (1.0) (1.9) (2.5) (1.8) (0.5) (1.4) (2.1) (1.0) (1.0) (1.3)

Flap 9.5 8.3 7.5 9.5 7.1 6.0 6.4 5.3 4.3 4.6 3.8 3.2

(0.5) (0.6) (2.1) (0.8) (1.0) (0.9) (1.2) (1.5) (0.7) (0.9) (1.2) (1.0)

Flight 9.6 9.4 10.3 9.4 8.3 7.8 8.1 6.3 6.2 5.4 3.8 4.8

(2.0) (1.4) (1.9) (2.8) (0.8) (2.9) (3.7) (1.2) (3.6) (2.0) (0.9) (5.2)

Adult 10.9 9.2 9.7 9.3 8.6 9.7 7.8 5.8 7.7 3.9 4.6 4.8

(1.5) (1.3) (1.2) (1.2) (1.5) (1.2) (1.9) (1.1) (0.9) (0.3) (0.5) (0.8)

Table 6 Averages and standard deviations for sonar call duration, high frequency, low frequency, and sweep rate (sonogram slope) for A. jamaicensis across all developmental stages (from Carter & Adams, 2014 with permission).

Flight stage

Call duration (ms)

High frequency (kHz)

Low frequency (kHz)

Sweep rate (kHz ms 1)

Flop Flutter Flap Flight Adult

1.8 1.6 1.1 1.0 1.1

70.9 72.6 74.6 79.1 74.6

52.1 52.8 54.9 58.1 51.8

13.8 14.7 18.7 21.2 22.1

(0.8) (0.8) (0.2) (0.2) (0.1)

(3.8) (5.1) (6.4) (3.7) (3.8)

(3.4) (4.2) (6.8) (5.1) (4.1)

(5.6) (4.5) (4.8) (3.0) (3.8)


This is supported by morphological evidence of the inner ear that shows that high-frequency hearing was likely present in the ancestor to all bats (Davies et al. 2013). The ontogeny of hearing in phyllostomids has been assessed using audiogram data. For example, Carollia perspicillata (Phyllostomidae) have two peaks of increased auditory sensitivity that appear during the first week (Sterbing, 2002), with the most sensitive peak at approximately 20 kHz and the second peak between 40 and 60 kHz. As C. perspicillata develops, the first peak (~ 20 kHz) becomes more sensitive and the second (40–60 kHz) expands to higher frequencies and becomes more sensitive. Adult A. jamaicensis exhibit a similar two-peaked audiogram, which seems typical of phyllostomid bats (Heffner et al. 2003). Although audiogram data from newborns are lacking for A. jamaicensis, the cochlear pattern we found of highly similar BM and SSL morphology across flight stages indicates that neonates of this species exhibit the morphology necessary for a second high-frequency sensitivity peak in hearing. Morphological prerequisites for high-frequency sensitivity and emission of short duration ultrasonic frequency modulated (FM) calls in newborns (Table 6) (Carter et al. 2014) suggest that aspects of echolocation begin developing prior to and independently of the flight system. Indeed, it has also been found that during the first week of postnatal life, parts of the auditory cortex of Pteronotus parnellii contain functional circuits that calculate distance € ssl based on temporal separation of pulse and echo (Ko et al. 2012). Furthermore, during the flap stage there are concurrent changes in the larynx that allow for a faster emission rate of the already developed high-frequency FM call (Carter & Adams, 2014). Thus, if interpreted in an evolutionary perspective, it appears that bats co-opted the primitive sonar capacity of a common ancestor into a more complex echolocation system in coordination with improved flight ability. This is supported by the realization that sonar emission, and even echolocation, has evolved in the shrew genera Blarina, Crocidura, and Sorex (Gould et al. 1964; Buchler, 1976; Tomasi, 1979; Forsman & Malquist, 1988; Siemers et al. 2009), suggesting that the temporal developmental sequence that we observed is also a probable evolutionary sequence. Thus, the eventual evolution of flight in the protobat would have driven a concurrent sophistication of the already preexisting echolocation system (Fenton, 1984). This concurrent sophistication would have involved adaptations in cochlear micro-anatomy, the nervous system, Prestin expression, and the ear ossicles, which we feel can be assessed using ontogenetic data. The majority of work carried out on bat cochlea morphology, ontogeny, and function has been done on high duty cycle-emitting species which exhibit sharp tuning to specific frequencies and make use of Doppler shift (Suga et al. 1974, 1976). The high duty cycle-emitting rhinolophids and hipposiderids (Pteropodiformes) are thought to have evolved echolocation independently of the Vespertilioniformes and

be more closely related to the largely nonecholocating pteropodids (Teeling et al. 2000; Eick et al. 2005; Jones & Teeling, 2006). This is, however, at odds with cranial morphology and development, neuroacoustic systems, flight € ller, musculature, and reproductive data (Pedersen & Mu 2013). Interestingly, one species (P. parnellii) within the Vespertilioniformes utilizes high duty cycle echolocation and also exhibits sharp tuning to specific frequencies. In both cases (Pteropodiformes and Vespertilioniformes) sharp tuning is a result of evolution of an acoustic fovea, which has characteristic BM, TM, and SSL morphology (Suga & Jen, € ssl, 1994, 1997; Ko € ssl & Rus1977; Henson & Henson, 1991; Ko € sell, 1995; Kossl & Vater, 1996). Not surprisingly, we found no acoustic fovea in any of the cochlea we sampled, likely because A. jamaicensis, like other phyllostomids, is a low duty cycle emitter. Furthermore, with the exception of P. parnellii, all Vespertilioniformes species exhibit low duty cycle echolocation, indicating that this form of echolocation and the corresponding BM lacking sharp tuning, are most likely primitive traits of this group. Interestingly, there appears to have been a decrease in the rate of morphological evolution of the BM in multiple ancestral branches of Vespertilioniformes and an increase in the rate in Pteropodiformes (Davies et al. 2013). We feel that further studies in the ontogeny of integrated systems in bats will give further insights into the evolution of bats and perhaps help fill the wide gaps in knowledge about one of the most successful groups of mammals on Earth today.

Acknowledgements The authors would like to thank Patrick Burns for advice regarding microscopy and Jason Shaw for advice regarding the analyses of the photographs with SIGMA SCAN. We also thank Scott Pedersen and one other anonymous reviewer for comments on earlier versions of the manuscript. We would like to thank the University of Northern Colorado School of Biological Sciences and Graduate Student Association, and the University of Northern Colorado 2009 Provost Award for funding this research.

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