Breeding biology and advertisement call of the horned leaf-frog, Proceratophrys appendiculata (Amphibia: Anura: Odontophrynidae)

We describe the breeding biology and the advertisement call of the horned leaf-frog, Proceratophrys appendiculata (Günther, 1873) in the Parque Nacional da Serra dos Órgãos, municipality of Teresópolis. The reproductive period of P. appendiculata is short and is associated with the end of the winter and the beginning of the spring, when males call night and day under large rocks in sandy bottom rock streams. The amplexus is axillary and one female laid about 656 viscous eggs. The advertisement call of P. appendiculata is unique among congeners. The call lasts approximately two seconds, with about 85 pulses/call at a rate of 45 pulses/s and frequency around 0.620 kHz. It is one of the longest calls and lowest in dominant frequency. Moreover, the call of P. appendiculata is characterized by the greatest number of pulses so far registered, reaching 129 pulses in a single call.

Breeding activity in most species of anurans begins with the emission of an advertisement call (GERHARDT 1994), which plays several roles in anuran reproductive behavior, for instance: attract females to breeding sites, defend territories against other males, and stimulate female ovulation, among others (WELLS 1977).
The advertisement call is species-specific, thus presenting an important mechanism of prezygotic reproductive isolation (RYAN & RAND 1993, FUNK et al. 2009).Because of this property, the advertisement call and all behaviors associated with it are important tools in taxonomic (PADIAL et al. 2009, PADIAL & DE LA RIVA 2009, HEPP & CARVALHO-E-SILVA 2011) and phylogenetic studies (COCROFT & RYAN 1995, ROBILLARD et al. 2006, GOICOECHEA et al. 2010).
Two main phenetic groups are recognized within Proceratophrys according to the presence or absence of palpebral appendages (CRUZ & NAPOLI 2010).Among the species that possess a long and single palpebral appendage are the species complexes of Proceratophrys appendiculata and P. boiei (IZECKSOHN et al. 1998, PRADO & POMBAL 2008, CRUZ & NAPOLI 2010).
Proceratophrys appendiculata (Günther, 1873) is an endemic species of the southeastern Atlantic Forest, occurring amidst the leaf-litter (DIAS et al. 2013a).These are cryptic animals (SAZIMA 1978) found scattered on the forest floor.Their tadpoles develop in small streams inside the forest (DIAS & CARVALHO-E-SILVA 2012).The breeding activity of the species is poorly studied and its advertisement call has not been described.

Breeding biology and advertisement call of the horned leaf-frog,
Proceratophrys appendiculata (Amphibia: Anura: Odontophrynidae) ABSTRACT.We describe the breeding biology and the advertisement call of the horned leaf-frog, Proceratophrys appendiculata (Günther, 1873) in the Parque Nacional da Serra dos Órgãos, municipality of Teresópolis.The reproductive period of P. appendiculata is short and is associated with the end of the winter and the beginning of the spring, when males call night and day under large rocks in sandy bottom rock streams.The amplexus is axillary and one female laid about 656 viscous eggs.The advertisement call of P. appendiculata is unique among congeners.The call lasts approximately two seconds, with about 85 pulses/call at a rate of 45 pulses/s and frequency around 0.620 kHz.It is one of the longest calls and lowest in dominant frequency.Moreover, the call of P. appendiculata is characterized by the greatest number of pulses so far registered, reaching 129 pulses in a single call.KEY WORDS.Atlantic Forest; calling activity; reproductive behavior.Breeding biology and advertisement call of the horned leaf-frog ZOOLOGIA 30 (4): 388-396, August, 2013

MATERIAL AND METHODS
The present study was conducted in the Parque Nacional da Serra dos Órgãos (PARNASO), municipality of Teresópolis, state of Rio de Janeiro, Brazil, from September 2004 to July 2012, excepting the years of 2005 and 2007.We visited five breeding sites monthly for at least three nights and three days.During the breeding period, we stayed in the field for as long as the calling activity was taking place.Many other observations were made sporadically in different dates and sites of the study area.We considered males calling, amplexed couples, egg clutches and tadpoles in initial development stage as indicators of breeding activity (GOSNER 1960, stages 25-26).
Vocalizations were recorded with a Marantz PMD-670 and Tascam DR-07 digital recorders, at sample rate of 44.100 kHz and sample size of 16 bits and microphone Sennheiser ME-67.The recordings were made in October, 2011 and September, 2006 both with temperature of about 17.5°C.Sound analyses and graphs were made with Raven Pro ver.1.3 from the Cornell Laboratory of Ornithology (Bioacoustics Research Program).The following parameters were measured: call duration; number of pulses per call; pulse rate; pulse periods (measured from the beginning of one pulse to the beginning of the following one, thus encompassing the pulse duration and interpulse interval); dominant frequency; and fundamental frequency.Numerical call parameters are given as a range, followed by the Mean ± SD, Mode and N within parenthesis.
The temporal parameters were measured directly from the waveform and frequency parameters were measured directly from the audiospectrogram (with window function Hann, amplitude logarithmic, window size 512 samples and overlap 99%).We also counted the number of harmonically related frequencies observed in the audiospectrogram and power spectrum (see HEPP et al. 2012).Technical terms and definitions adopted follow LITTLEJOHN (2001) and HEPP & CARVALHO-E-SILVA (2011).
Individuals recorded were collected, fixed in 10% formalin, and preserved in 70% ethyl alcohol.They are housed in the Amphibian collection of the Laboratório de Biossistemática de Anfíbios of the Universidade Federal do Estado do Rio de Janeiro (UNIRIO 2542(UNIRIO -2543, 4471-4473) , 4471-4473) and in the Amphibian collection of the Departamento de Zoologia of the Universidade Federal do Rio de Janeiro (ZUFRJ 8970-8971).The permit for collecting zoological material provided by Instituto Chico Mendes (ICMBio) and Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais e Renováveis (IBAMA) receives the number12164-2.

Breeding Biology
The reproductive period of P. appendiculata was short and associated with the end of winter and spring, from July to November, although the males called only one or two months per year (Table I).During the breeding season, males called continuously at night and during the day in large choruses inside the forest, along shallow rocky streams with sandy bottoms (Fig. 1).Calling activity began a few days after the first heavy rainfall within the period above mentioned.Males of P. appendiculata were only observed calling under large rocks or in small burrows formed by vegetation and tree branches, always with their bodies in contact with the water.
Males called at an approximately equal distance from one another, which varied from one to two meters.A wave like chorus was observed along the streams, where one male called followed by the most adjacent male and so on down and up the river.No satellite males were observed, and it seems that calling activity was also related to territorial defense.
In a plot of 5 x 5 m, about eight to twelve males were observed calling actively.This number decreased each day, and about three to four days after the first record of calling activ-ity, only two or three males were still calling.The frequency of call emission decreased drastically and males called in duets closer than previously, only about 40 cm apart.After this period, the calling activity ceased completely.The end of the calling period was not associated with any abiotic parameter, such as temperature, rainfall or any other.
Three days after the calling activity had stopped, we observed hatched capsules strongly adhered to submerged rocks and aquatic vegetation.We also saw several newly hatched tadpoles (Gosner stage 25 or less) close to these capsules.
In October 2004, one single male was observed calling in a very small stream.It was in contact with the running water.Adhered to its body and to the sandy bottom and to rocks were several eggs (Fig. 2).They were collected and raised in the laboratory (Fig. 3).Four days later, tadpoles at Gosner stage 23 hatched.
In September 2006, during calling activity, an amplexed coupled was found in contact with shallow water after removing a large rock.The male was strongly clasped to female's armpits (Fig. 4).The couple was collected and later the female spontaneously oviposited in a plastic box.
In August 2008, a solitary male was heard calling from under a large rock.About five minutes later, a female was intercepted moving through the water in the direction of the male'.Both were captured and maintained in a plastic bag with a small amount of water and vegetation.In the laboratory, they spontaneously engaged in amplexus (Fig. 5), although no eggs were laid.
In 2010, two breeding periods were recorded, one in July and another in November, although not in all breeding sites.The July activity took place only at one of the sites, and the November one in all other sites except the first one.

Advertisement Call
Eighty-two calls from three (UNIRIO 4471-73) specimens were analyzed (Figs 6 and 7).The advertisement call was composed of a sequence of 51 to 129 pulses (x = 85.5 ± 18.7, Mo = 70.0,n = 79) repeated quasi-periodically.All pulses have a shorter attack than decay, probably as a consequence of the natural reverberant environment of the calling sites (rock caves).The amplitude of the call usually increases and decreases abruptly, although, on occasion, this rise and fall were gradual.The middle or the last quarter of the call is the part with most amplitude (Fig. 8).The difference between the amplitude of the pulses throughout the call is variable.Some calls (63.4%) have pulses with approximately similar amplitudes while others (36.6%) have middle pulses with clearly more amplitude, resulting in a prominent middle amplitude peak (Fig. 9).The calls without a prominent peak vary from a low pulse rate with few pulses to a high pulse rate with high number of pulses (Fig. 13).The calls with a prominent peak have a high pulse rate with high number of pulses (Figs 9 and 11).Call duration ranges from 1.322 s to 2.416 s (x = 1.907 ± 0.223, n = 79).The call has a harmonic structure with up to four visible harmonics, though only the first two harmonics are highlighted.The dominant frequency corresponds to the fundamental one and ranges from 0.562 to 0.656 kHz (x = 0.621 ± 0.046, Mo = 0.656, n = 79) (Fig. 8).The pulse period varies from 0.012 to 0.119 s (x = 0.021 ± 0.008, Mo = 0.015, n = 944).The pulse periods decrease slightly and gradually until to the middle of the call where they begin to increase slightly and gradually until the end in calls with prominent amplitude peak.In calls without a prominent peak, they only increase slightly and gradually all the way until the end (Fig. 11).Eventually (19.5%), the first or second period is the longest one, mainly (93%) in calls with a prominent peak (Fig. 8).The pulse rate ranges from 30.0 to 65.4 pulses per second (x = 45.3 ± 10.4, n = 79) and varies inversely to the pulse periods throughout the call, being higher in the middle or beginning of the call (Fig. 11).
In September 2006, sixty sections with calls partially overlapped, emitted by two or three specimens in a chorus, were recorded (Fig. 12).In this social context, an isolated pulse (introductory pulse) precedes the overlapping calls and the final pulse periods are long and their pulses have high amplitudes.Moreover, on many occasions, a long pulse with modulated and low amplitude was emitted immediately after the end of the overlapping calls (Figs 12 and 13).

Breeding behavior
Few studies have presented detailed information about the reproductive behavior of Proceratophrys species, and the information available generally corresponded to natural history notes in species descriptions (e.g., WEYGOLDT & PEIXOTO 1985, CRUZ & NAPOLI 2010, MARTINS & GIARETTA 2011) with few exceptions (e.g., BOQUIMPANI-FREITAS et al. 2002).
On the other hand, the peculiar behavior of calling from under large rocks is less diffused in the genus, and seems to be shared only with Proceratophrys moheringi (WEYGOLDT & PEIXOTO 1985).As GERHARDT (1994) pointed out, natural selection usually favors morphological characteristics and behaviors that increase the range of effective communication in the context of mate attraction.In the burrowing frogs Eupsophus Fitzinger, 1843, acoustic properties of calling sites may affect signal emission and reception (PENNA & SOLÍS 1999), contributing to the antiphonal and chorus activity.The ongoing antiphonal calling would in turn direct the females toward the breeding areas (PENNA 2004).The rocks may play a similar role in the social interactions of P. appendiculata, working as natural sound amplifiers.
Several behavioral traits may be indicative of mate choice by the female, such as 1) males calling from a fixed site; 2) absence of physical interactions between males; and 3) females moving toward the male (WELLS 1977).The breeding behavior recorded for P. appendiculata match these characteristics.
In addition, the absence of egg clutches and amplexed pairs during peak calling activity and the constant decline in the number of males in the chorus per day may indicate that females are choosing the males who remain in the chorus for longer.Despite the fact that our data support this hypothesis, it still must be tested.
The short reproductive period recorded for P. appendiculata may be a consequence of the calling activity pattern, acting as a  1985).The same was observed for P. tupinamba in Ilha Grande, where the species breeds from one to three months per year (BOQUIMPANI-FREITAS et al. 2002).This pattern was also registered for species of the Proceratophrys bigibbosa group.CALDART et al. (2010) reported that P. avelinoi reproduces from September to November, and males can be heard both at night and during the day.The same period was cited by KWET & FAIVOVICH (2001) for P. avelinoi.These authors also mentioned that P. brauni calls throughout the day from November to February and P. bigibbosa, which calls during the night and sometimes during the day, breeds from September to November.
Female fecundity of P. appendiculata is very similar to that of P. tupinamba (729 to 946 eggs) and P. melanopogon (664 eggs) (BOQUIMPANI-FREITAS et al. 2002, ALMEIDA-GOMES et al. 2007).There are no other records for Proceratophrys species, and more data are necessary to generalize this pattern.The hatching time for P. appendiculata is about three to four days after the eggs are laid.This fact was observed in the field and in the laboratory.
There are many forms of parental care in anurans, of which egg attendance the most common (KLUGE 1981).MAR-TINS et al. (1998) mentioned the facultative parental care in the Hylid frog Hypsiboas faber (Wied-Neuwied, 1821), in which males keep calling near fertilized clutches, thus protecting their territory and, consequently, the eggs from other males.The single male captured in 2004 was calling near laid fertilized eggs and no other male or female was caught in the area.This behavior may be associated with facultative parental care, presenting the first record for the genus, although more observations are necessary.

Advertisement Call
Possibly, the function of the introductory pulse in the chorus (Fig. 12) is to advertise to neighboring males that the main call is about to begin.This pulse could be important to stimulate the partial overlap of the calls.The long pulse recorded after the overlapping calls may be related to a territorial or agonistic context, since it is frequently emitted after intense overlap and/or after high amplitude pulses in the chorus (Fig. 12).

Figures 8 -
Figures 8-10.Advertisement call of Proceratophrys appendiculata, spectrogram and power spectrum with window function Hann, amplitude logarithmic, window size 512 and 256 samples respectively, overlap 99%.(8) Oscillogram (top), spectrogram (middle) and power spectrum (bottom) of one call.Note the first longest period and the first two harmonics with more energy (harmonics indicated by Roman-numbered arrows in power spectrum).(9) Oscillogram (top) and spectrogram (bottom) of sixteen calls with and without prominent amplitude peak (see text).The first ones are flagged with asterisk and show higher pulse rates.(10) Oscillogram (top) and spectrogram (bottom) of three calls without prominent amplitude peak.Note the pulse rate variation, increasing from the first to the last call.

Figures 11 -
Figures 11-13.Advertisement call of Proceratophrys appendiculata, oscillogram(top) and spectrogram (below).Spectrogram with window function Hann, amplitude logarithmic, window size 512 samples, overlap 99%.(11) Two calls with and without prominent amplitude peak respectively.Note that the first call shows the highest pulse rate section in the middle while the last one shows the same section in the in the beginning, decreasing until the end.(12) Overlapping calls of three males.Note the introductory pulse and the territorial final pulse after the loud pulses at the end of the main emission.(13) Territorial pulse in detail.

Table I .
Month of calling activity of Proceratophrys appendiculata per year in the period of 2004 to 2012.Breeding site not visited in the years of 2005 and 2007.