Male Cope's gray treefrogs (Hyla chrysoscelis) in amplexus have elevated and correlated steroid hormones compared to solitary males.

Gonadal steroid hormones are typically elevated during the breeding season-a finding known as an associated reproductive pattern. Though less studied, there is also evidence, in both sexes, for elevated adrenal/interrenal steroids, including acute elevations on the day of mating. I investigated gonadal and interrenal steroids in wild male Cope's gray treefrogs at breeding aggregations. I collected blood from males found in amplexus with female mates (amplexed males) and males sampled at the same time and location that were actively advertising vocally and without a mate (solo males). Concentrations of plasma corticosterone, testosterone, and 17ß-estradiol (CORT, T and E2, respectively) were validated and measured. These two categories of males differed in four ways: (1) amplexed males exhibited significantly elevated concentrations of all three steroids compared to solo males (CORT: +347 %; T: +60 %; and E2: +43 %); (2) these hormone profiles alone accurately predicted male mating category with ca. 83 % accuracy using a discriminant function analysis; (3) amplexed males exhibited significant between-hormone correlations (T and E2 were positively correlated and CORT and E2 were negatively correlated) whereas no correlations were found in solo males; (4) amplexed males showed a negative correlation with CORT concentration and the time of night, whereas no such pattern was present in solo males. These findings suggest an acute and strong coactivation of the interrenal and gonadal axes that could drive phenotypic integration during this fitness-determining moment. I discuss these findings and suggest experiments to determine causation, including the role of motor behavior driving endocrine states and the role of female selection on endocrine profiles.

Natural variation in social conditions affects male mate choosiness in the amphipod Gammarus roeselii.

The extent of male mate choosiness is driven by a trade-off between various environmental factors associated with the costs of mate acquisition, quality assessment and opportunity costs. Our knowledge about natural variation in male mate choosiness across different populations of the same species, however, remains limited. In this study, we compared male mate choosiness across 10 natural populations of the freshwater amphipod Gammarus roeselii (Gervais 1835), a species with overall high male mating investments, and evaluated the relative influence of population density and sex ratio (both affecting mate availability) on male mate choosiness. We investigated amplexus establishment after separating mating pairs and presenting focal males with a novel, size-matched female from the same population. Our analysis revealed considerable effects of sex ratio and (to a lesser extent) population density on time until amplexus establishment (choosiness). Male amphipods are able to perceive variable social conditions (e.g., sex ratio) and modify their mating strategy accordingly: We found choosiness to be reduced in increasingly male-biased populations, whereas selectivity increases when sex ratio becomes female biased. With this, our study expands our limited knowledge on natural variations in male mate choosiness and illustrates the importance of sex ratio (i.e., level of competition) for male mating decisions in natural environments. Accounting for variation in sex ratios, therefore, allows envisioning a distinctive variation of choosiness in natural populations and highlights the importance of considering social background information in future behavioral studies.

Reproductive biology of the emerald frog Hylorina sylvatica (Anura: Batrachylidae) in northwest Patagonia, Argentina / Biología reproductiva de la rana esmeralda Hylorina sylvatica (Anura: Batrachylidae) en el noroeste de la Patagonia Argentina

Abstract Hylorina sylvatica (Anura: Batrachyidae), or the emerald frog, is a pond-breeding anuran endemic to the austral temperate forests of Chile and Argentina. It is considered a vulnerable species in Argentina, where it has a narrow distribution; records and biological information relating to the frog in this area are scarce. In this study, conducted in 2016, the reproductive parameters of the emerald frog were investigated in detail in a ­semi-temporary wetland of Northwestern Patagonia, Argentina. During the spring, the wetland was visited weekly in search of calling males, adults and individuals in amplexus. When amplexus was observed, eggs and larvae were collected from among the aquatic vegetation using dip-nets. Egg size and larval stage at hatching were registered. Calling males were registered during the second week of October, and the first couples were observed on 26 October. Three pairs of emerald frogs were captured and placed in enclosures within the wetlands in the afternoon. The males were smaller than the females. After 24 h the number of eggs laid in each enclosure was counted, and the males and females were released. The eggs were kept in each enclosure until hatching. Hatching occurred after 10-14 days in the enclosures. This information contributes to our knowledge of the biology of the emerald frog in Patagonia, Argentina, and is the only information currently available on this species in Nahuel Huapi National Park, where the species breeds in various aquatic environments, from semi-temporary wet meadows to large permanent ponds.

Resumen Hylorina sylvatica (Anura: Batrachyidae) o rana esmeralda, es un anuro que se reproduce en estanques, endémico de los bosques templados australes de Chile y Argentina. Actualmente es considerada una especie vulnerable en Argentina. La distribución es estrecha en Argentina con pocos registros de la especie y muy limitada información sobre la biología de esta especie en la región. En este estudio realizado durante 2016, investigué en detalle los parámetros reproductivos de la rana esmeralda en un humedal semitemporal del Noroeste de la Patagonia Argentina. Durante la primavera de 2016, visité el humedal semanalmente en busca de coros, adultos y amplexos. También después de que se observaron amplexos, recolecté huevos y larvas entre la vegetación acuática utilizando un muestreo con redes de mano. Se registró el tamaño del huevo y el estado larvario al eclosionar. Los coros de machos se registraron durante la tercera semana de octubre y los primeros amplexos se observaron el 26 de octubre. Tres parejas de rana esmeralda fueron capturadas y colocadas en clausuras dentro de los humedales durante la tarde. Los machos eran más pequeños que las hembras. Después de 24 h conté el número de huevos puestos en cada recinto y luego se liberaron machos y hembras. Los huevos se mantuvieron en cada clausura hasta que eclosionaron. La eclosión se produjo entre 10-14 días en las clausuras. Esta información contribuye al conocimiento de la biología de la rana esmeralda que habita la Patagonia Argentina y es la única información disponible al momento de esta especie en el Parque Nacional Nahuel Huapi, donde la especie se reproduce en diferentes ambientes acuáticos, desde humedales semitemporales hasta lagos y lagunas permanentes.

Love bites: male frogs (Plectrohyla, Hylidae) use teeth scratching to deliver sodefrin precursor-like factors to females during amplexus.

BACKGROUND: Efficient transfer of chemical signals is important for successful mating in many animal species. Multiple evolutionary lineages of animals evolved direct sex pheromone transmission during traumatic mating-the wounding of the partner with specialized devices-which helps to avoid signal loss to the environment. Although such direct transmission modes of so-called allohormone pheromones are well-documented in invertebrates, they are considered rare in vertebrates. Males of several species of the frog genus Plectrohyla (Hylidae, Anura) have elongated teeth and develop swollen lips during the breeding season. Here we investigated the possibility that these structures are used to scratch the females' skin and apply allohormone pheromones during traumatic mating in several Plectrohyla species. RESULTS: Our behavioural observations revealed that males press their upper jaw onto the females' dorsum during amplexus, leaving small skin scratches with their teeth. Histological examinations of the males' lips identified specialized mucus glands, resembling known amphibian pheromone glands. Whole-transcriptome sequencing of these breeding glands showed high expression of sodefrin precursor-like factor (SPF) proteins, which are known to have a pheromone function in multiple amphibian species. CONCLUSIONS: Our study suggests SPF delivery via traumatic mating in several anuran species: the males have specialized breeding glands in the lips for production and secretion and use their elongated teeth as wounding devices for application. We hypothesize that these SPF proteins end up in the females' circulatory system, where understanding their exact function will require further molecular, physiological and behavioural testing.

Diagnosing Eulimnadia and Paralimnadia (Branchiopoda: Spinicaudata: Limnadiidae).

Eulimnadia and Paralimnadia are both strongly supported, monophyletic limnadiid lineages based on molecular studies. However, defining the two taxa morphologically relies on the presence/absence of a subcercopodal spiniform projection; otherwise there is considerable overlap and confusion in morphological characters between the two taxa. The most discriminatory of these characters are examined here and applied to Australasian species. As a result, five Eulimnadia species are transferred to Paralimnadia. These characters are then applied to world Eulimnadia species and other limnadiid genera which share key features with Eulimnadia.

May the (selective) force be with you: Spatial sorting and natural selection exert opposing forces on limb length in an invasive amphibian.

Spatial sorting on invasion fronts drives the evolution of dispersive phenotypes, and in doing so can push phenotypes in the opposite direction to natural selection. The invasion of cane toads (Rhinella marina) through tropical Australia has accelerated over recent decades because of the accumulation of dispersal-enhancing traits at the invasion front, driven by spatial sorting. One such trait is the length of the forelimbs: invasion-front toads have longer arms (relative to body length) in comparison with populations 10-20 years after invasion. Such a shift likely has fitness consequences: an increase of forearm length would decrease the strength with which a male could cling to a female during amplexus and so render such a male less competitive in competition for mates, compared to short-armed conspecifics. Our laboratory trials of attachment strength confirmed that males with relatively longer arms were easier to displace, and competition trials show higher duration of amplexus for males with shorter arms. Together with the sharp cline in limb length observed behind the invasion front, these results imply an opposition of selective forces: spatial sorting optimizes dispersal, but as this force wanes behind the invasion front, we see the primacy of natural selection reassert itself.

An occasional record of the amplexus in epigean Niphargus (Amphipoda: Niphargidae) from the Russian Western Caucasus.

The precopulatory mate guarding (amplexus) was observed in epigean Niphargus cf. magnus Birstein, 1940 collected near Tuapse in the Western Caucasus of Russia. The records of amplexing representatives of the subterranean genus Niphargus Schiödte, 1849 (Crustacea: Amphipoda: Niphargidae) are extremely rare, and there are no publications describing the amplexus in this amphipod genus. The presented report shows that Niphargus have a chemically stimulated short-time amplexus immediately after the molt of the female, similar to the representatives of relative epigean and subterranean gammaridean amphipods. It is relatively short in time, possibly, because of adaptation to inhabit narrows spaces in underground habitats.

Population density and structure drive differential investment in pre- and postmating sexual traits in frogs.

Sexual selection theory predicts a trade-off between premating (ornaments and armaments) and postmating (testes and ejaculates) sexual traits, assuming that growing and maintaining these traits is costly and that total reproductive investments are limited. The number of males in competition, the reproductive gains from investing in premating sexual traits, and the level of sperm competition are all predicted to influence how males allocate their finite resources to these traits. Yet, empirical examination of these predictions is currently scarce. Here, we studied relative expenditure on pre- and postmating sexual traits among frog species varying in their population density, operational sex ratio, and the number of competing males for each clutch of eggs. We found that the intensifying struggle to monopolize fertilizations as more and more males clasp the same female to fertilize her eggs shifts male reproductive investment toward sperm production and away from male weaponry. This shift, which is mediated by population density and the associated level of male-male competition, likely also explains the trade-off between pre- and postmating sexual traits in our much broader sample of anuran species. Our results highlight the power of such a multilevel approach in resolving the evolution of traits and allocation trade-offs.

Sperm competition and the evolution of precopulatory weapons: Testis size and amplexus position, but not arm strength, affect fertilization success in a chorusing frog.

Trade-offs between pre- and postcopulatory traits influence their evolution, and male expenditure on such traits is predicted to depend on the number of competitors, the benefits from investing in weapons, and the risk and intensity of sperm competition. Males of the chorusing frog Crinia georgiana use their arms as weapons in contest competition. Previously, we showed that increased numbers of rivals elevated the risk and intensity of sperm competition due to multimale amplexus, and caused a reversal in the direction of precopulatory selection on arm girth. Here, we focused on the factors affecting postcopulatory fertilization success during group spawning, using paternity data from natural choruses. Competitive fertilization success depended on the time spent amplexed and amplexus position. Relative testes size but not arm girth, contributed to fertilization success, but the effect of testes size depended on amplexus position. Our findings offer within species empirical support for recent sperm competition models that incorporate precopulatory male-male competition, and show why an understanding of the evolution of animal weapons requires a consideration of both pre- and postcopulatory episodes of sexual selection.

A unique mating strategy without physical contact during fertilization in Bombay Night Frogs (Nyctibatrachus humayuni) with the description of a new form of amplexus and female call.

Anurans show the highest diversity in reproductive modes of all vertebrate taxa, with a variety of associated breeding behaviours. One striking feature of anuran reproduction is amplexus. During this process, in which the male clasps the female, both individuals' cloacae are juxtaposed to ensure successful external fertilization. Several types of amplexus have evolved with the diversification of anurans, and secondary loss of amplexus has been reported in a few distantly related taxa. Within Nyctibatrachus, a genus endemic to the Western Ghats of India, normal axillary amplexus, a complete loss of amplexus, and intermediate forms of amplexus have all been suggested to occur, but many species remain unstudied. Here, we describe the reproductive behaviour of N. humayuni, including a new type of amplexus. The dorsal straddle, here defined as a loose form of contact in which the male sits on the dorsum of the female prior to oviposition but without clasping her, is previously unreported for anurans. When compared to known amplexus types, it most closely resembles the form of amplexus observed in Mantellinae. Furthermore, we prove that, opposed to the situation in most anurans, male semen release happens before egg deposition. We hypothesize that the male ejaculates on the female's dorsum and that sperm subsequently runs from her back and hind legs before fertilizing the eggs. A second feature characterizing anuran breeding is the advertisement call, mostly produced solely by males. Despite recent descriptions of several new Nyctibatrachus species, few studies have explored their vocal repertoire. We describe both the male advertisement call and a female call for N. humayuni. The presence of a female call has not been reported within Nyctibatrachidae, and has been reported in less than 0.5% of anuran species. Altogether, our results highlight a striking diversity and several unique aspects of Nyctibatrachus breeding behaviour.

Sperm competition and the evolution of precopulatory weapons: Increasing male density promotes sperm competition and reduces selection on arm strength in a chorusing frog.

Sperm competition theory assumes a trade-off between precopulatory traits that increase mating success and postcopulatory traits that increase fertilization success. Predictions for how sperm competition might affect male expenditure on these traits depend on the number of competing males, the advantage gained from expenditure on weapons, and the level of sperm competition. However, empirical tests of sperm competition theory rarely examine precopulatory male expenditure. We investigated how variation in male density affects precopulatory sexual selection on male weaponry and the level of sperm competition in the chorusing frog Crinia georgiana, where males use their arms as weapons in male-male combat. We measured body size and arm girth of 439 males, and recorded their mating success in the field. We found density-dependent selection acting on arm girth. Arm girth was positively associated with mating success, but only at low population densities. Increased male density was associated with higher risk and intensity of sperm competition arising from multimale amplexus, and a reversal in the direction of selection on arm girth. Opposing patterns of pre- and postcopulatory selection may account for the negative covariation between arm girth and testes across populations of this species.

Desarrollo embrionario y larva del sapo Incilius aucoinae (Bufonidae) en Golfito, Costa Rica / Embryonic development and larva of the toad Incilius aucoinae (Bufonidae) in Golfito, Costa Rica

Embryonic development and larva of the toad Incilius aucoinae (Bufonidae) in Golfito, Costa Rica. The larva and developmental stages of Incilius aucoinae are described using specimens from Río Cañaza, Golfito, Costa Rica. We collected the individuals in January 1997, and January and February 1998. The total sample was 696 individuals, 317 eggs in 1-12 stages, 176 eggs with embryos in stages 15-16, and 204 tadpoles in stages 21-43. The average number of eggs per nest was 8 940 (n = 4). These eggs were laid in strings, attached to the river strata. Eggs in stages 1-12 had an average diameter of 1.75 (s = 0.38) mm and eggs with embryos in stages 15 -16 measured 1.33 (0.34) mm wide and 3.06 (1.46) mm long. The description of the tadpole based on UCR 19982 is as follows: dorsal, ventral surfaces, and tail are brown with dark brown spots. Length of tail 9.43 mm, body length 6.18 mm (total length 15.62). Translucent light brown small fins, dorsal fin with scattered spots, ventral fin without dark pigments. Dorsal-ventral flattened oval body with anteroventral snout. A few individuals had varied colors, from black (3.4%) to uniform (0.5-1.5%). Rev. Biol. Trop. 57 (Suppl. 1): 291-299. Epub 2009 November 30.

Se describe la larva y los estadios del desarrollo del huevo y larva de Incilius aucoinae utilizando especímenes del río Cañaza, Golfito, Costa Rica, recolectados en enero de 1997 y enero-febrero de 1998. La muestra fue de 696 individuos, 317 huevos en estadios 1-12, 176 huevos con embriones en estadios 15-16, y 204 renacuajos en estadios 21-43. El promedio del número de huevos por nidada fue de 8 940 (n = 4), los cuales fueron colocados en una hilera, sujetos al sustrato del río. Los huevos en estadios 1-12 tienen un promedio de diámetro de 1.75 (s = 0.38) mm y los huevos con embriones en estadios 15-16 miden 1.33 (0.34) de ancho y 3.06 (1.46) mm de largo. La descripción del renacuajo basada en el espécimen UCR 19982, es como sigue: el dorso, el vientre y la cola son de color café y punteado de café oscuro; la cola mide 9.43 mm y el cuerpo 6.18, para un tamaño total de 15.62; las aletas son pequeñas café claro translúcidas, la dorsal con puntos dispersos y la ventral sin ningún tipo de pigmento oscuro. También es el único renacuajo ovoide con una tendencia a ser aplastado dorso-ventralmente, y con la boca antero-ventral. Dentro de la variación observada, algunos individuos son de color negro (3.4 %) y pocos son uniformes (0.5- 1.5 %).