Environ Biol Fish (2011) 91:251259 DOI 10.1007/s10641-011-9772-8 Evoked potential audiogram of the lined seahorse, Hippocampus erectus (Perry), in terms of sound pressure and particle acceleration Paul A Anderson & David A Mann Received: 12 March 2010 / Accepted: February 2011 / Published online: 13 May 2011 # Springer Science+Business Media B.V 2011 Abstract The hearing sensitivity of the lined seahorse, Hippocampus erectus (Perry), was determined for both sound pressure and particle acceleration using the auditory evoked potential (AEP) technique Hippocampus erectus demonstrates hearing sensitivity typical of historically characterized hearing generalist fishes, with best sensitivities below 600 Hz and maximum sensitivities of 105.01.5 dB SPL (re: Pa) and 3.46ì 103 7.64ì104 m s2 at 200 Hz The shapes of the audiograms for each modality are similar, suggesting relative similarity in sensitivity between modalities for a given frequency In light of the importance of broadband sound in the acoustic landscape of this fishs environment, and broadband conspecific sound production that may be used in intraspecific acoustic commuP A Anderson (*) IFAS/SFRC Program in Fisheries and Aquatic Sciences, University of Florida, 7922 NW 71st Street, Gainesville, FL 32653, USA e-mail: panderson@flaquarium.org D A Mann College of Marine Science, University of South Florida, 140 7th Avenue South, St Petersburg, FL 33701, USA e-mail: dmann@marine.usf.edu Present Address: P A Anderson Center for Conservation, The Florida Aquarium, 701 Channelside Drive, Tampa, FL 33602, USA nication, audition to broadband sounds was also estimated Maximum broadband sensitivity at 200 Hz is estimated to be 92.01.5 dB SPL (re: Pa) and 7.73ì104 1.71ì104 m s2 Keywords Evoked potential Audiogram Hippocampus erectus Seahorse Acoustic Particle acceleration Introduction The auditory evoked potential (AEP) technique to measure hearing ability is widely practiced among human clinicians (e.g., Davis 1976; Picton et al 1981; Schroeder and Kramer 1989) and has been expanded to test hearing ability of representatives from many vertebrate taxa (Corwin et al 1982), including fishes (Kenyon et al 1998) It is a non-invasive far-field recording of synchronous neural activity in the eighth nerve and brainstem auditory nuclei elicited by acoustic stimuli (Jacobson 1985) Our objective was to characterize the hearing ability of the lined seahorse (Hippocampus erectus Perry) in a comprehensive manner by measuring both the pressure and particle acceleration of the acoustic stimuli in hearing tests These components of sound contribute in different ways in the near-field and far-field of sound sources A vibrating sound source produces two physical changes in the surrounding environment: particle motion and pressure wave propagation of the 252 surrounding medium (Dusenbery 1992) The near-field of a sound source is dominated by local hydrodynamic flow, established by the displacement of water molecules (particle motion) adjacent to the sound source (Bass and Clark 2003) In the far-field, acoustic pressure is proportional to particle velocity (Medwin and Clay 1998) It is generally well-accepted that fishes historically characterized as hearing specialists (Popper et al 2003; but see Popper and Fay 2011) respond to both particle motion and pressure, but are more sensitive to pressure particularly in the far-field and at frequencies above 70 Hz (Fay et al 1982) Fishes historically characterized as hearing generalists, that have no specialized connections between the swim bladder and inner ear, have yielded equivocal data concerning the relative importance of pressure sensitivity to sound detection and processing (Cahn et al 1969; Sand and Enger 1973; Chapman and Johnstone 1974; Fay and Popper 1975; Jerkứ et al 1989; Yan et al 2000; Lovell et al 2005; Horodysky et al 2008; Wysocki et al 2009) The summation of this literature suggests that both acoustic modalities may be detected and processed by fishes classically characterized as hearing generalists, though the relative contributions of each may vary with respect to distance, frequency, and sound pressure level Because fishes historically characterized as generalists may in fact be processing both particle motion and pressure components of sound, this has led Popper and Fay (2011) to discourage the dichotomy of characterizing fishes as either hearing specialists or generalists, but instead to consider the relative importance of sound pressure in hearing among fishes along a continuum of species Thus, both modalities of particle acceleration and sound pressure are reported here Materials and methods Animal accession, holding, and husbandry procedures Lined seahorses (H erectus) were collected as bycatch from shrimp trawl nets and donated by local fishermen Upon accession, animals were quarantined for month prior to transfer to a sound-dampened holding system Clear round acetate tags (approx cm diameter) were marked with alphanumeric codes, on monofilament line collars, and tied around the necks of Environ Biol Fish (2011) 91:251259 seahorses (tagging methods modified from Vincent and Sadler 1995) Animals were fed frozen mysids (Piscine Energetics, Kelowna, BC, Canada) in the mornings and live Artemia sp (Sea Critters, Key Largo, FL, USA) enriched with Roti-Rich (Florida Aqua Farms, Inc., Dade City, FL, USA) in the afternoons Tanks were siphoned clean of debris twice daily and system water changes of 10% were performed weekly Water quality parameters remained within the following ranges during holding: Temperature, 2527C; salinity, 28.531.5; ammonianitrogen, mg L1; nitritenitrogen, mg L1; nitratenitrogen, 2.822.7 mg L1 Eleven animals were transferred to a soundproofed tank with an established biological filter to 11 days prior to testing Soundproofing was accomplished by resting the frame of the tank on a sturdy lab bench with sections of bearing felt, installing a subsurface drain that transferred water to a sump resting on the floor where filtration occurred, and using a quiet, 15 W water pump with a flexible return pipe that returned water to the tank below the water surface A loop was suspended in the flexible return line; this attenuated vibration and sound travelling through the return water and pipe walls (A Noxon, Acoustic Sciences Corp., Eugene, OR, USA, pers comm., Fig 1) The ambient noise profiles of both the holding system and the soundproofed tank were measured with an HTI-96-min hydrophone (High Tech Instruments, Inc., Gulfport, MS, USA, sensitivity = 164.1 dB re: VPa1, bandwidth = 230,000 Hz), for sound pressure level (SPL) measurements The ambient noise profile of the sound-proofed tank was also measured with an Acoustech geophone probe (Acoustech Corp., State College, PA, USA, sensitivity = 212 Vm1 s1, bandwidth = 1001,000 Hz) for measurements of particle motion Both instruments, when in use, were connected to the line-in port of a laptop computer running CoolEdit (Syntrillium Software Corp., Phoenix, AZ, USA) Hydrophone recordings were collected from the middle and bottom of tanks Geophone recordings were collected from the bottom of tanks in the center, along three orthogonal axes, because particle motion is a vector quantity (as opposed to pressure, which is a scalar quantity) Resulting sound files were calibrated according to manufacturer instructions and postprocessed with SpectraPlus (Pioneer Hill Software, Poulsbo, WA, USA) Analysis settings used in Spec- Environ Biol Fish (2011) 91:251259 253 Fig Soundproofed laboratory tank Subsurface Return Subsurface Drain Rag Animal Holding Tank Bearing Felt Lab Bench Clamp Flexible Tubing Soundproofing Loop Sponge Sump Pump Stand traPlus are summarized in Table The Acoustech geophone probe measures particle velocity To convert to acceleration, Fast-Fourier Transforms (FFTs) processed by SpectraPlus were exported into a spreadsheet program and particle velocity values converted to particle acceleration using the following formula: a ẳ v2pf 1ị Table SpectraPLUS analysis settings Ambient noise analysis AEP stimulus analysis Sampling rate (Hz) 44100 48828 Sampling format 16-bit 16-bit Standard Hz weighting Flat (none) Flat (none) Decimation ratio 11 12 Frequency limit 2004.545 Hz, Low-pass filter enabled 2034.500 Hz, Low-pass filter enabled FFT size 4096 4096 Spectral line resolution 0.979 Hz 0.993 Hz Smoothing window Hanning Hanning Averaging settings Infinite, linear, Disable peak hold 1, linear, Enable peak hold FFT overlap 0% 99% Time resolution 1021.68 ms 10.07 ms Input signal overload Enable overload detection Enable overload detection Exclude overloaded data from processor Exclude overloaded data from processor 254 Environ Biol Fish (2011) 91:251259 where a = acceleration (m s2), v = velocity (m s1), = pi (~3.14), and f = frequency (Hz) (Casper and Mann 2006) The magnitude of particle acceleration was calculated by vector averaging according to the following equation: p a ẳ x2 ỵ y2 ỵ z 2ị SigGen and BioSig software Sounds were generated by an RP 2.1 Enhanced Real-Time processor, fed through a PA5 programmable attenuator to control sound level, and amplified by a Hafler Trans.Ana P1000 110 W professional power amplifier before being sent to the UW30 speaker, where sound was emitted where a = magnitude of acceleration (m s2), and x, y, and z refer to acceleration (m s2) in each of three orthogonal axes Sound generation, calibration, and AEP acquisition Experimental setup Acoustic stimuli were calibrated with the HTI-96-min hydrophone for pressure measurements of tones and the Acoustech geophone in three orthogonal axes for particle motion, all connected to the RP 2.1 processor During calibration, the hydrophone or geophone was positioned in the experimental setup in place of the fish at the level of the animals head, and the stimulus presentation protocol as described for AEP acquisition was executed, except without phase alternation Signals were captured and averaged by BioSig Resulting time domain averaged signals were exported as ASCII formatted files, imported in SpectraPlus, and 4,096 point FFTs run (SpectraPlus settings in Table 1) to generate power spectra, from which peak amplitude measurements were taken Particle acceleration was calculated per Eqs and Calibration runs were conducted daily The testing chamber consisted of a steel tube (1.22 m high, 20.32 cm diameter, 0.95 cm thickness), closed at the bottom with a square steel plate (60.96ì60.96 cm), and oriented vertically Four 51700 Series anti-vibration floor mounts (Tech Products Corp., Miamisburg, OH, USA) were placed under each corner of the base of the tank The tube was filled with saltwater of approximately 26C up to a height of 1.12 m A laboratory stand was supported on an adjacent vibration-isolated table and scaffolding descended into the tube for animal suspension A UW30 speaker (University Sound, Oklahoma City, OK, USA) was placed at the bottom of the tube in the centre This setup was enclosed inside an audiology booth For testing, individual fish were secured in a harness constructed from Nitex mesh, fastened with clamps to scaffolding 2.5 cm below the water surface The harness restricted movement while allowing normal respiration Sub dermal stainless steel needle electrodes (Rochester Electro-Medical, Inc., Tampa, FL, USA) were used to record the AEP signal An electrode was inserted about mm into the head, over the medulla region Reference and ground electrodes were placed directly in the water in close proximity to the fish Evoked potentials recorded by the electrode were fed through an HS4 fiber optic head stage (Tucker-Davis Technologies [TDT], Alachua, FL, USA) and into an RP 2.1 processor (TDT), routed into the computer and averaged by BioSig software (TDT) All eleven seahorses were tested for AEPs to tone stimuli (described below); also, a dead goldfish (Carassius auratus) was run to generate control AEP signals Sound stimuli and AEP waveform recordings were produced with a TDT AEP workstation running Calibration AEP acquisition Stimuli consisted of 60 ms pulsed tones gated with a Hanning window The phase of the tone was alternated between presentations to minimize electrical artifacts from the recordings During each trial, nine different frequencies were presented: 100, 200, 300, 400, 600, 800, 1,000, 1,500, and 2,000 Hz Amplitudes at each frequency were presented within a range of approximately 74 to 148 dB re: Pa and 8.60ì106 to 0.67 ms2, beginning at amplitudes below threshold and increasing in dB steps until a threshold was visually detected in the digital signal output (see Data analysis) Post-hoc trials were run at amplitudes that were dB below visual threshold to improve the resolution of threshold determination Up to 2,000 signal presentations (or until detection was visually confirmed) were averaged to measure the evoked response at each level of each frequency Environ Biol Fish (2011) 91:251259 255 Data analysis Evoked potential traces were transformed with the Hanning window function and converted to power spectra with a 2048-point FFT in BioSig Evoked potentials are visualized as peaks that occur at twice the frequency of the presented stimulus (Fig 2) This is a well-established phenomenon in evoked potentials of fishes to pure tones in the frequency domain (Egner and Mann 2005) Visualized peaks were considered true evoked potentials if they were at least dB above the average of all peaks occurring within a window of 50 Hz above the presented stimulus frequency AEP thresholds were defined as the lowest amplitude at which a true evoked potential, according to these criteria, was visualized AEP waveforms of live seahorses were checked against AEP waveforms of dead goldfish to ensure that the identified peak was not a stimulus or electrical artifact Audition of broadband noise was estimated using calculations proposed by Yost (2000) and employed by Egner and Mann (2005) for hearing in damselfish Specifically, the tonal audiogram was adjusted by an estimated critical bandwidth that is assumed to be 10% of the center frequency Because sound pressure is expressed on a logarithmic scale (dB), this estimated adjustment is: tb ẳ ts 10 log0:1f ị 3ị Fig Auditory evoked potentials (AEPs) to a 400 Hz tone pip depicted in the time domain (left) and in the frequency domain (right) a Control AEP waveform (dead goldfish) b,c,d AEP waveforms of H erectus at progressively lower amplitudes d where tb = broadband threshold, ts = spectrum-level threshold (both in dB re: Pa), and f = frequency (Hz) A similar estimate of the audition of broadband noise in terms of acceleration was calculated by first converting acceleration thresholds to velocity (because in an ideal far-field situation, velocity is proportional to pressure, Urick 1975), and using Eq with dBvelocity substituted for pressure The resulting velocity was then converted to acceleration using Eq Results Ambient noise The long-term holding tanks in which animals were housed prior to transfer to the AEP laboratory demonstrated an average total RMS power (within the to 998 Hz frequency range) of 117.40.9 dB SPL (re: Pa) at the middle of the water column and 128.8 1.4 dB SPL (re: Pa) at the bottom The soundproofed AEP laboratory tank demonstrated an average total RMS power of 115.80.5 dB SPL (re: Pa) at the middle of the water column and 120.50.2 dB SPL (re: Pa) at the bottom In terms of particle acceleration, the soundproofed AEP laboratory tank demonstrated a vector-averaged total RMS power of 4.58ì103 m s2, comprised of total RMS powers of 2.38ì103 on the represents amplitude at threshold Asterisks denote AEPs that occur at twice the frequency of the presented stimulus (in this case, 800 Hz) 256 Environ Biol Fish (2011) 91:251259 horizontal-length (x) axis, 1.82ì103 on the horizontaldepth (z) axis, and 3.47ì103 on the vertical (y) axis AEP Average hearing thresholds from 11 H erectus are reported in Table for both sound pressure and particle acceleration For sound pressure, this species most sensitive hearing range is below 400 Hz, with minimum thresholds at 200 Hz (tonal threshold, 105.01.5 dB SPL re: Pa; estimated broadband threshold, 92.0 1.5 dB) Above 600 Hz, tonal hearing thresholds increase to levels above most environmentally relevant noise (Wenz 1962; Urick 1975) For particle acceleration, this species most sensitive hearing range is below 800 Hz, with minimum thresholds of 3.46ì103 7.64ì104 m s2 (tonal) and 7.73ì104 1.71ì104 m s2 (estimated broadband) at 200 Hz (Fig 3) Discussion Hippocampus erectus demonstrates sound pressure sensitivity that falls within the range of sensitivities documented for other fishes historically characterized as hearing generalists (e.g., Kenyon et al 1998; Yan 2001; Scholik and Yan 2002; Lugli et al 2003; Egner and Mann 2005; Lovell et al 2005; Casper and Mann 2006; Fig 4a) While this species hearing sensitivity was tested at frequencies up to 2,000 Hz (for sound pressure), tonal thresholds at frequencies above 600 Hz begin to rise into a range of high SPLs that animals are not likely to encounter in the natural Table Hearing thresholds of H erectus (mean SE) Tonal thresholds were adjusted by an estimated critical bandwidth that is assumed to be 10% of the center frequency to estimate broadband thresholds, per Yost (2000) Frequency (Hz) environment (Wenz 1962; Urick 1975) Like other generalists, H erectus also does not appear to have any bony or gaseous vesicular connections between the swimbladder and the inner ear (pers obs.) In light of these observations, it is especially pertinent to report this animals audiogram in terms of particle acceleration, as direct detection of particle motion in the inner ear is thought to be the predominant mode of sound reception in fishes historically characterized as hearing generalists (Fay and Popper 1975; Popper and Fay 1993) Likewise, particle acceleration thresholds for the lined seahorse fall into a range of sensitivities documented in other generalist fishes and elasmobranches (Casper and Mann 2006; Wysocki et al 2009; Fig 4b) There is remarkable similarity between the shapes of the audiograms for sound pressure and particle acceleration Despite the fundamental differences between the pressure and particle motion component of sound, and the fundamental differences in the way each modality is processed by fishes, fishes historically characterized as specialists (e.g., Ictalurus punctatus, Fay and Popper 1975; some sciaenids, Horodysky et al 2008) and generalists (e.g., Ginglystoma cirratum, Casper and Mann 2006; some sciaenids, Horodysky et al 2008; Gobius cruentatus, Chromis chromis, Sciaena umbra, Wysocki et al 2009) demonstrate similarities between the shape of audiograms for each acoustic modality Tests that have shown dissimilarities (e.g., Hawkins and Johnstone 1978; Kelly and Nelson 1975) may be due to artifactual acoustic discontinuities between sound pressure and particle motion in constrained testing environments Sound pressure (dB re: Pa) Particle acceleration (m s2) Tonal Tonal Broadband Broadband 100 107.52.2 97.52.2 0.0062480.001295 0.0019760.000409 200 105.01.5 92.01.5 0.0034580.000764 0.0007730.000171 300 109.92.5 95.12.5 0.0076160.001695 0.0013910.000310 400 116.11.7 100.11.7 600 117.61.8 99.81.8 0.0118510.003116 0.0015300.000402 800 127.71.0 108.71.0 0.0399840.005986 0.0044700.000669 1,000 132.71.9 112.71.9 0.0815320.029967 0.0081530.002997 1,500 133.21.8 111.41.8 2,000 136.31.9 113.31.9 Environ Biol Fish (2011) 91:251259 b 140 SPL (dB re: àPa) 135 130 125 120 115 110 105 100 95 90 100 Particle acceleration (m s-2) a 257 1000 0.1 0.01 0.001 0.0001 100 10000 Frequency (Hz) 1000 Frequency (Hz) Fig Audiogram of the lined seahorse, H erectus, for a sound pressure, and b particle acceleration () = threshold to tonal stimuli, () = predicted threshold to broadband stimuli Error bars represent mean SE the lined seahorse is exposed are broadband in nature Ambient environmental noise is broadband (Wenz 1962; Urick 1975) Greatest hearing sensitivity among hearing generalist fishes tends to fall in the range of 50 500 Hz, precisely the range at which ambient noise in shallow water tends to be propagated (Bass and Clark 2003), leading Popper and Fay (1999) to postulate that hearing among (generalist) fishes first evolved to evaluate the broadband auditory scene. The role of hearing in intraspecific acoustic communication is also important for soniferous fishes, including the seahorse Fish (1953) and Colson et al (1998) characterized the seahorse click; it is a broadband sound that is emitted by stridulation of the posterior process of the supra- The lack of commercially available accelerometers has hindered ability to evaluate audition to particle motion Audition to true sound stimuli that are comprehensively characterized in terms of sound pressure and particle motion has only recently been reported (e.g., Casper and Mann 2006; Horodysky et al 2008; Wysocki et al 2009) as a result of recent developments in commercially available accelerometers (McConnell 2003; McConnell and Jensen 2006) This work is a contribution to these few, but growing, number of studies In the audiograms presented, audition of broadband sound is estimated (per Yost 2000; Egner and Mann 2005), as most biologically significant sounds to which b 140 Particle acceleration (m s-2) a SPL (dB re: àPa) 130 120 110 100 90 80 70 60 100 1000 Frequency (Hz) Fig Audiograms of representative hearing generalist fishes, measured by the AEP technique a Sound pressure: In this comparative audiogram, the hearing specialist Carassius auratus is also represented for comparison () = Hippocampus erectus (this study), () = Lepomis macrochirus (Scholik and Yan 2002), () = Opsanus tau (Yan 2001), () = Padogobius martensii (Lugli et al 2003), () = Astronotus ocellatus 0.1 0.01 0.001 0.0001 100 1000 Frequency (Hz) (Kenyon et al 1998), () = Carassius auratus (Kenyon et al 1998) b Particle acceleration: () = Hippocampus erectus (this study), () = Ginglymostoma cirratum (Casper and Mann 2006), () = Urobatis jamaicensis (Casper and Mann 2006), () = Sciaena umbra (Wysocki et al 2009), () = Gobius cruentatus (Wysocki et al 2009), () = Chromis chromis (Wysocki et al 2009) 258 occipital against the coronet (Colson et al 1998) It is demonstrated in many behavioral contexts, including feeding (Colson et al 1998), aggression and competition for mates (Vincent 1994), and distress (Fish 1953) Traditional audiograms present audition to tonal stimuli of a range of frequencies, but in light of the relevance of broadband sounds in the natural history of fishes, audition to broadband stimuli should also be measured or estimated Acknowledgments Many thanks to B Casper, M Hill Cook, R Hill, and J Locascio (University of South Florida) for guidance with AEP methodology, A Noxon (Acoustic Sciences Corporation), who shared soundproofing design concepts, R Shrivastav (University of Florida) and J Pattee (Pioneer Hill Software) for guidance with sound analysis, P Perkins (Florida Aquarium) for illustrations provided in Figs and 2, and W.J Lindberg, D Murie, D Parkyn, C St Mary (University of Florida), I Berzins (Florida Aquarium), H Masonjones (University of Tampa), and two anonymous reviewers, who provided constructive criticism for improvement of the manuscript Seahorses were donated by Above the Reef and R Stevens, his crew, and the Twin Rivers Marina Live brine shrimp was donated by Sea Critters, Inc P Anderson was supported by the University of Florida Alumni Fellowship, the Morris Animal Foundation, The Florida Aquarium Center for Conservation, The Spurlino Foundation, and an anonymous donor Animal collection was authorized by the Florida Fish and Wildlife Conservation Commission Special Activities License #05SR-944 and husbandry and experimental protocols were authorized by the University of Florida IACUC Protocol #D-432, by the University of South Florida IACUC Protocol #2118, and by the Florida Aquarium Animal Care and Use Committee References Bass AH, Clark CW (2003) The physical acoustics of underwater sound communication In: Simmons AM, Popper AN, Fay RR (eds) Acoustic communication Springer, New York, pp 1564 Cahn PH, Siler W, Wodinsky J (1969) Acoustico-lateralis system of fishes: tests of pressure and particle-velocity sensitivity in grunts, 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Auditory evoked potentials from the human cochlea and brainstem J Otolaryngol 10(Suppl 9):141 Popper AN, Fay RR (1993) Sound detection and processing by fish: critical review and major research questions Brain Behav Evol 41:1438 Popper AN, Fay RR (1999) The auditory periphery in fishes In: Fay RR, Popper AN (eds) Comparative hearing: fish and amphibians Springer, New York, pp 43100 Popper AN, Fay RR (2011) Rethinking sound detection by fishes Hear Res 273(12):2536 Popper AN, Fay RR, Platt C, Sand O (2003) Sound detection mechanisms and capabilities of teleost fishes In: Collins SP, Marshall NJ (eds) Sensory processing in aquatic environments Springer, New York, pp 338 Environ Biol Fish (2011) 91:251259 Sand O, Enger PS (1973) Evidence for an auditory function of the swimbladder in the cod J Exp Biol 59:405414 Scholik AR, Yan HY (2002) The effects of noise on the auditory sensitivity of the bluegill sunfish, Lepomis macrochirus Comp Biochem Physiol A 133:4352 Schroeder LL, Kramer SJ (1989) The very basics of ABR The Interstate Printers and Publishers, Danville Urick RJ (1975) Principles of underwater sound McGraw-Hill, New York Vincent ACJ (1994) Seahorses exhibit conventional sex roles in mating competition, despite male pregnancy Behaviour 128:135151 Vincent ACJ, Sadler LM (1995) Faithful pair bonds in wild seahorses, Hippocampus whitei Anim Behav 50:15571569 259 Wenz GM (1962) Acoustic ambient noise in the ocean: spectra and sources J Acoust Soc Am 34(12):19361956 Wysocki LE, Codarin A, Ladich F, Picciulin M (2009) Sound pressure and particle acceleration audiograms in three marine fish species from the Adriatic Sea J Acoust Soc Am 126(4):21002107 Yan HY (2001) A non-invasive electrophysiological study on the enhancement of hearing ability in fishes Proc Inst Acoust UK 23(2):1525 Yan HY, Fine ML, Horn NS, Colon WE (2000) Variability in the role of the gasbladder in fish audition J Comp Physiol A 187:371379 Yost WA (2000) Fundamentals of hearing Academic, San Diego 354 However, there have been no studies on the other three species, A megastoma, A obscura and A dieffenbachia, that live in the WSP In addition to molecular genetic analyses, counts of the total number of vertebrae (TV) of anguillid eels have been shown to be a useful morphological character for examining differences among populations (Watanabe et al 2009a) as first realized in the important monograph about the morphological characteristics of the genus Anguilla published by Ege (1939) Recent statistical comparisons of TV including the data of Ege (1939) have shown there is significant variation that reflects the likely population structure of A marmorata (Watanabe et al 2008, 2009a) Statistical differences in TV were also found recently between the two subspecies of A australis (Watanabe et al 2006) These differences in TV in anguillid eels appear to reflect genetic differences, despite the fact that environmental factors are thought to cause differences in these types of characters in some fishes For example, a phenomenon known as Jordans rule (Jordan 1892, but see McDowell 2008) states that TV increases with latitude, which is often regarded as an effect of the varying water temperature (Hubbs 1922; Fowler 1970) at each latitude on the development of the number of vertebrae in their early life history However, because of their unique reproductive ecology, both temperate and tropical species of the genus Anguilla have their spawning areas in tropical to subtropical regions with similar water temperatures This is the case for species whose spawning areas are known, such as the temperate species A anguilla, A rostrata, and A japonica (Schmidt 1925; McCleave et al 1987; Tsukamoto 1992, 2006) and the tropical species A bicolor bicolor, A borneensis, A celebesensis, and A marmorata (Jespersen 1942; Miller et al 2002; Aoyama et al 2003; Kuroki et al 2009) are all in tropical to subtropical regions with similar water temperatures In addition, both A anguilla and A rostrata spawn in the almost same area in the Sargasso sea (McCleave et al 1987), but their TV are different with very little overlap (A anguilla: range 110119, mean 114.7; A rostrata: range 103111, mean 107.2; Ege 1939) This indicates that variations of TV in anguillid eels typically have a species-specific genetic basis rather than being a reflection of the effects of environmental factors such as water temperature Furthermore, the population structure of A marmorata (Watanabe et al 2008, 2009a) and the recognition of Environ Biol Fish (2011) 91:353360 subspecies in A australis (Watanabe et al 2006) based on morphological differences in TV correspond well with the results of molecular genetic analyses of the population structure of A marmorata (Ishikawa et al 2004; Minegishi et al 2008) and A australis (Shen and Tzeng 2007a, but see Dijkstra and Jellyman 1999) This evidence suggests that differences in TV are well correlated with the amount of evolutionary divergence among populations of anguillid eels The objective of this study was to determine if the variation of TV among different localities shows any indication of separate populations in the three tropical eels, A megastoma, A obscura, and A reinhardtii in the WSP through comparison of TV values of specimens collected throughout the majority of the geographic ranges Each species was tested by combining newly collected data with that of the historical work by Ege (1939) The morphology of these species was examined by Ege (1939), and he proposed that there were different races within A megastoma The present study examines this hypothesis using statistical analyses in comparison to the phylogeography of tropical anguillid eels based on molecular genetic analyses to examine the possible population structure of these species, which need to be tested using population genetic studies and early life history studies of their spawning areas Materials and methods A total of 141 eel specimens of these three species were collected from four locations in the WSP from September 1994 to 18 February 2005 (Fig 1; Table 1) Five specimens (total length: 179677 mm) of A megastoma were collected from Fiji (N=3) and New Caledonia (N=2), 73 specimens (total length: 213 580 mm) of A obscura were collected from Fiji, and 63 specimens (total length: 124450 mm) of A reinhardtii were collected from the Albert River area of Queensland (N=52) in Australia and New Caledonia (N=11) Each specimen was preserved in 10 to 20% formalin after collection All specimens were radiographed by Soft-X (Softex Co., Ltd.) to count their TV Based on Ege (1939), all the specimens were identified as A megastoma, A obscura, and A reinhardtii using characters of color markings, dentition, ratios of the distance between verticals through the anus and origin of the dorsal fin to total length and TV Environ Biol Fish (2011) 91:353360 Fig The geographic distribution of three anguillid species in the western South Pacific and the collection localities of the specimens used in this study (solid circles) and Eges (1939) study (open circles) 355 15N New Guinea A obscura A megastoma Samoa New Caledonia Cook Islands Tahiti Fiji Morea 15S A reinhardtii Queensland 30 New South Wales Lord Howe Island 45 120 135 Eges (1939) TV data included some museum specimens and many collected specimens of A megastoma, A obscura, and A reinhardtii that were from New Guinea (A obscura: N=18), New Caledonia (A megastoma: N=9, A obscura: N=71, A reinhardtii: N=191), Samoa (A megastoma: N=32), Fiji (A megastoma: N= 1, A obscura: N=208), Cook Islands (A obscura: N= 94; Rarotonga and Mitiare islands), Tahiti (A megastoma: N=159, A obscura: N=190), Morea (A megastoma: N=21), Queensland (A reinhardtii: N=30), New South Wales (A reinhardtii: N=143) and Lord Howe Island (A reinhardtii: N=48) (Table 1) Eges (1939) data were combined with our original data for TV from New Caledonia and Fiji for A megastoma, Fiji for A obscura and Queensland and New Caledonia for A reinhardtii (Fig 1; Table 1) The TV data sets of A megastoma and A obscura were compared using the nonparametric Kruskal-Wallis test (P0.05, see Table 1) using SPSS (version 16.0) for Macintosh (SAS Institute Inc., Chicago, Illinois) The four individuals of A megastoma from Fiji could not be analyzed because the number of specimens was too small (Table 1) The TV data sets of A reinhardtii were initially tested with ANOVA using Prism (version 4.0c) for Macintosh (GraphPad Software Inc., San Diego, California), since the data from all 150 165E 180 165W 150 135 localities for this species had normal distributions (Kolmogorov-Smirnov one-sample test, P[...]... mark profile Profile of mean increment width of ten increments from both sides of the settlement mark (n=16), which is represented by the decrease in width between increments 1 and 0 282 Environ Biol Fish (2011) 91:275286 Fig 4 Bellapiscis medius otolith of a recently settled fish Transverse section of sagitta of a recently settled B medius Dark and wide increments are visible up to the edge of the... significance of the resulting division by reach and the BIO-ENV procedure within the BEST function of PRIMER version 6 software (Clarke and Gorley 2006) to evaluate potential environmental correlates of community structure We performed the BIO-ENV test using a resemblance matrix of normalized environmental variables calculated using Euclidean distance (Clarke and Ainsworth 1993) and tested the significance of. .. Biol Fish (2011) 91:261274 DOI 10.1007/s10641-011-9776-4 Spatial and seasonal patterns in freshwater ichthyofaunal communities of a tropical high island in Fiji Aaron P Jenkins & Stacy D Jupiter Received: 14 July 2010 / Accepted: 16 January 2011 / Published online: 7 April 2011 # Springer Science+Business Media B.V 2011 Abstract We surveyed freshwater ichthyofaunal communities in streams of Vanua Levu,... edge of the otolith The hatch mark is also visible (marked as the first red +) Validation of settlement marks Otoliths of recently settled fishes displayed either no transition area (i.e settlement mark) or a transition area that was at the very edge of the otolith (Fig 4) Otoliths of juvenile fishes contained a clear settlement mark Environ Biol Fish (2011) 91:275286 281 Table 2 Validation of daily... northwest, which receives only 20% of the annual total in the dry months compared with 33% on the windward side (Terry 2005) Freshwater fish surveys We surveyed fish species richness and abundance in rivers and stream of Kubulau District and Macuata Province of Vanua Levu, Fiji, during the dry season of Environ Biol Fish (2011) 91:261274 263 Fig 1 Map of rainfall isohyets of the main Fiji islands and stream... within 50 m reaches of upper, mid and lower sections of river basins in Kubulau and Macuata using the exact methods of Jenkins et al (2010), modified from field protocols of Parham (2005) and Fitzsimons et al (2007) In brief, we used a variety of techniques to collect fauna from the rivers/streams to ensure comprehensive presence/absence assessment These techniques included: electrofishing using either... and settlement marks in the otoliths The formation of daily increments was validated using larvae of known age and tetracycline marking of settled juveniles Settlement mark identity was verified by comparing total increment counts from otoliths of recently settled fishes with PLD counts from post-settlement fishes A similar pattern of three groups of increments across the otolith was found in all specimens... may facilitate greater gene flow between populations of F lapillum than between populations of F nigripenne An alternative explanation for the low levels of gene flow in F nigripenne is that the larvae of this species return to their natal estuary after the pelagic phase There is evidence of larval retention in triplefin species from other parts of the world (Brogan 1994 (California); Sabatộs et al... 91:275286 Brogan MW (1994) Distribution and retention of larval fishes near reefs in the Gulf of California Mar Ecol Prog Ser 115:113 Brothers EB, Williams DA, Sale PF (1983) Length of larval life in 12 families of fishes at one tree lagoon, Great Barrier Reef, Australia Mar Biol 76:319324 Campana SE (1992) Measurement and interpretation of the microstructure of fish otoliths In: Stevenson DK, Campana SE (eds)... coastal waters of northeastern New Zealand Mar Ecol Prog Ser 80:4154 Kingsford MJ, Choat JH (1989) Horizontal distribution patterns of presettlement reef fish: are they influenced by the proximity of reefs? Mar Biol 101:285298 Leis JM, Carson-Ewart BM (1997) Swimming speeds of the late larvae of some coral reef fishes Mar Ecol Prog Ser 159:165174 Leis JM, Hay AC, Howarth GJ (2009) Ontogeny of in situ behaviours