De Hyla : pars I.

Thesis (doctoral)--Universitas Litterarum Viadrina.

Interindividual Differences in Leg Muscle Mass and Pyruvate Kinase Activity Correlate with Interindividual Differences in Jumping Performance of Hyla multilineata

Frog jumping is an excellent model system for examining the structural basis of interindividual variation in burst locomotor performance. Some possible factors that affect jump performance, such as total body size, hindlimb length, muscle mass, and muscle mechanical and biochemical properties, were analysed at the interindividual (intraspecies) level in the tree frog Hyla multilineata. The aim of this study was to determine which of these physiological and anatomical variables both vary between individuals and are correlated with interindividual variation in jump performance. The model produced via stepwise linear regression analysis of absolute data suggested that 62% of the interindividual variation in maximum jump distance could be explained by a combination of interindividual variation in absolute plantaris muscle mass, total hindlimb muscle mass ( excluding plantaris muscle), and pyruvate kinase activity. When body length effects were removed, multiple regression indicated that the same independent variables explained 43% of the residual interindividual variation in jump distance. This suggests that individuals with relatively large jumping muscles and high pyruvate kinase activity for their body size achieved comparatively large maximal jump distances for their body size.

Ecological correlates of successful colonization in the life history of the Cuban treefrog, {\it Osteopilus septentrionalis\/} (Anura: Hylidae)

Ten correlates of successful colonization were tested and met in the life history of the Cuban treefrog in Florida and the Caribbean. Like many successful colonizing species of animals, the Cuban treefrog was highly fecund; reproduction was possible at a small body size in males (27.0 mm) and females (45.0 mm), and large females could lay large clutches and eggs throughout the year. Generation times were short in this species thereby accelerating the colonization process. Tadpoles and post-metamorphic individuals could exploit a wide range of physical conditions with respect to weather conditions and structure of the habitat. The Cuban treefrog occupied the terrestrial-arboreal niche which was only marginally exploited by other species in Florida. Habitat preference of the Cuban treefrog was for mesophytic forests and disturbed areas, and both habitats were found in native and introduced ranges. The ability to coexist with man further enabled the Cuban treefrog to expand its geographic range. A broad diet enabled the Cuban treefrog to exploit a wide range of prey species and sizes thereby alleviating an important constraint to colonization success. The Cuban treefrog was gregarious and vagile, thereby accelerating the process of dispersal which is crucial to the colonization process. Thus, many features in its life history enabled the Cuban treefrog to rapidly disperse and colonize, often in high population densities, many kinds of sites in its native and introduced range. Conformity to these correlates by the Cuban treefrog ultimately provides predictive power regarding the future colonization of this tropical frog. ^

Creation of temporary ponds for amphibians in northern and central Europe

More than 4000 ponds have been created or restored in Denmark since 1985 as part of a large-scale pond-digging programme to protect endangered amphibians in particular and pond flora and fauna in general. Most ponds are created on private land with public financing. The programme was triggered by, among other factors, a drastic decline in amphibian populations in Denmark between 1940 and 1980. However, in recent years there has been an increased awareness in Denmark that temporary ponds are important for the conservation of some of the most rare amphibian species, such as fire-bellied toad Bombina bombina, natterjack toad Bufo calamita and green toad Bufo viridis. Other rare species such as moor frog Rana arvalis and European tree frog Hyla arborea also benefit from temporary ponds. The last 15 years of work on the conservation of endangered species and their habitats has resulted in a last-minute rescue and a subsequent growth in the size of most Danish populations of fire-bellied toad and green toad; some populations of the relatively more common natterjack toad have also increased. The creation of temporary ponds plays an important role in the success of these three species. The creation of ponds to help restore viable populations of the most rare amphibians has not been easy. To study the conditions that may need to be created, Danish herpetologists searched for areas with temporary ponds that had good water quality, natural hydrological conditions and a management regime influenced by traditional agricultural methods. The paper gives an overview of pond creation and restoration projects in Denmark and Poland and their significance for amphibian diversity.

Herpetofaunal Inventories of the National Parks of South Florida and the Caribbean: Volume II. Virgin Islands National Park

Amphibian declines and extinctions have been documented around the world, often in protected natural areas. Concern for this alarming trend has focused attention on the need to document all species of amphibians that occur within U.S. National Parks and to search for any signs that amphibians may be declining. This study, an inventory of amphibian species in Virgin Islands National Park, was conducted from 2001 to 2003. The goals of the project were to create a georeferenced inventory of amphibian species, use new analytical techniques to estimate proportion of sites occupied by each species, look for any signs of amphibian decline (missing species, disease, die-offs, etc.), and to establish a protocol that could be used for future monitoring efforts. Several sampling methods were used to accomplish these goals. Visual encounter surveys and anuran vocalization surveys were conducted in all habitats throughout the park to estimate the proportion of sites or proportion of area occupied (PAO) by amphibian species in each habitat. Line transect methods were used to estimate density of some amphibian species and double observer analysis was used to refine counts based on detection probabilities. Opportunistic collections were used to augment the visual encounter methods for rare species. Data were collected during four sampling periods and every major trail system throughout the park was surveyed. All of the amphibian species believed to occur on St. John were detected during these surveys. One species not previously reported, the Cuban treefrog (Osteopilus septentrionalis), was also added to the species list. That species and two others (Eleutherodactylus coqui and Eleutherodactylus lentus) bring the total number of introduced amphibians on St. John to three. We detected most of the reptile species thought to occur on St. John, but our methods were less suitable for reptiles compared to amphibians. No amphibian species appear to be in decline at this time. We found no evidence of disease or of malformations. Our surveys provide a snapshot picture of the status of the amphibian species, so continued monitoring would be necessary to determine long-term trends, but several potential threats to amphibians were identified. Invasive species, especially the Cuban treefrog, have the potential to decrease populations of native amphibians. Introduced mammalian predators are also a potential threat, especially to the reptiles of St. John, and mammalian grazers might have indirect effects on amphibians and reptiles through habitat modification. Finally, loss of habitat to development outside the park boundary could harm some important populations of amphibians and reptiles on the island.

Herpetofaunal Inventories of the National Parks of South Florida and the Caribbean: Volume III. Big Cypress National Preserve

Amphibian declines and extinctions have been documented around the world, often in protected natural areas. Concern for this trend has prompted the U.S. Geological Survey and the National Park Service to document all species of amphibians that occur within U.S. National Parks and to search for any signs that amphibians may be declining. This study, an inventory of amphibian species in Big Cypress National Preserve, was conducted from 2002 to 2003. The goals of the project were to create a georeferenced inventory of amphibian species, use new analytical techniques to estimate proportion of sites occupied by each species, look for any signs of amphibian decline (missing species, disease, die-offs, and so forth.), and to establish a protocol that could be used for future monitoring efforts. Several sampling methods were used to accomplish these goals. Visual encounter surveys and anuran vocalization surveys were conducted in all habitats throughout the park to estimate the proportion of sites or proportion of area occupied (PAO) by each amphibian species in each habitat. Opportunistic collections, as well as limited drift fence data, were used to augment the visual encounter methods for highly aquatic or cryptic species. A total of 545 visits to 104 sites were conducted for standard sampling alone, and 2,358 individual amphibians and 374 reptiles were encountered. Data analysis was conducted in program PRESENCE to provide PAO estimates for each of the anuran species. All of the amphibian species historically found in Big Cypress National Preserve were detected during this project. At least one individual of each of the four salamander species was captured during sampling. Each of the anuran species in the preserve was adequately sampled using standard herpetological sampling methods, and PAO estimates were produced for each species of anuran by habitat. This information serves as an indicator of habitat associations of the species and relative abundance of sites occupied, but it will also be useful as a comparative baseline for future monitoring efforts. In addition to sampling for amphibians, all encounters with reptiles were documented. The sampling methods used for detecting amphibians are also appropriate for many reptile species. These reptile locations are included in this report, but the number of reptile observations was not sufficient to estimate PAO for reptile species. We encountered 35 of the 46 species of reptiles believed to be present in Big Cypress National Preserve during this study, and evidence exists of the presence of four other reptile species in the Preserve. This study found no evidence of amphibian decline in Big Cypress National Preserve. Although no evidence of decline was observed, several threats to amphibians were identified. Introduced species, especially the Cuban treefrog (Osteopilus septentrionalis), are predators and competitors with several native frog species. The recreational use of off-road vehicles has the potential to affect some amphibian populations, and a study on those potential impacts is currently underway. Also, interference by humans with the natural hydrologic cycle of south Florida has the potential to alter the amphibian community. Continued monitoring of the amphibian species in Big Cypress National Preserve is recommended. The methods used in this study were adequate to produce reliable estimates of the proportion of sites occupied by most anuran species, and are a cost-effective means of determining the status of their populations.

三种两栖类动物皮肤和脑中活性多肽结构、功能与多样性分析

两栖动物是最原始的陆生脊椎动物，分布比较广泛。无尾目两栖动物现有3000 多种，它们皮肤裸露、光滑，为适应广泛的栖息地和生态条件，已进化出各种有效的皮肤防御系统。抗菌肽（Antimicrobial peptides，AMPs）作为两栖类先天防御系统的重要组成部分，在皮肤分泌液中含量异常丰富。我们以来源于云南省普洱市景东县的铃蟾科微蹼铃蟾（Bombina microdeladigitora）和楚雄州双柏县雨蛙科华西雨蛙（Hyla annectans）为实验材料，对其皮肤分泌液中抗菌肽的分子多样性并对其结构和功能进行研究。微蹼铃蟾皮肤抗菌肽多样性非常丰富，我们从单一个体中克隆得到了64 条编码不同抗菌肽的cDNA 序列，其中有两条序列只编码Maximin 一种抗菌肽，其余 62 条均编码Maximin 和Maximin H 两类抗菌肽。这64 条cDNA 序列共编码44 种Maximins 和30 种Maximin Hs，其中有32 种Maximins 和20 种Maximin Hs 为新鉴定的抗菌肽，其余和铃蟾属其它种中发现的抗菌肽具有相同的序列。除了皮肤外，两栖动物的脑也是抗菌肽的丰富资源库。我们分别从微蹼铃蟾和大蹼铃蟾（B. maxima）脑中得到了大量新的抗菌肽cDNA 序列。其中从微蹼铃蟾脑中克隆到21 条新的cDNA序列，共编码16 种Maximins 和10 种Maximin Hs，其中7 种Maximins 和4 种Maximin Hs 为新鉴定的抗菌肽。从大蹼铃蟾脑中克隆到39 条新的cDNA 序列，编码27 种Maximins 和20 种Maximin Hs，其中16 种 Maximins 和12 种Maximin Hs 为新鉴定的抗菌肽。在以上新鉴定的抗菌肽中，Maximins 均为阳离子抗菌肽，Maximin Hs 中除以前鉴定的Maximin H5 外，尚有十余种阴离子抗菌肽。抗菌肽碱基转换/颠换（R=s/v）分析表明，RMaximin<1 而RMaximin H>1，说明这两种抗菌肽碱基转换和颠换发生的几率并不相同，Maximin 间差异主要由碱基颠换引起，而Maximin H 则主要由碱基转换引起。种间进化分析表明，大蹼铃蟾和微蹼铃蟾的遗传距离较近，而它们与欧洲花铃蟾（B. variegata）的遗传距离均较远。种内各部分遗传距离差异较大。与信号肽和酸性间隔肽相比，成熟肽的遗传距离明显增大，其中Maximin 的进化速度比Maximin H 更快。抗菌肽Maximin 和Maximin H 种内、种间均存在正选择（ω>1），而信号肽和酸性间隔肽在分化过程中没有正选择（ω<1），说明Maximin 和Maximin H 经受着达尔文正选择驱动的快速进化，是抗菌肽多样性产生的根本原因。这与抗菌肽参与最终的生物防御功能，从而增加物种对环境的适应是一致的。功能研究发现，有些微蹼铃蟾Maximins 抗菌肽是多功能分子，不但对革兰氏阴性菌、革兰氏阳性菌和真菌起抗菌作用，而且还具有很强的抗氧化功能。脑中抗菌肽基因的大量表达也预示着抗菌肽可能在神经信号传导中起一定作用。用基因克隆方法我们从微蹼铃蟾皮肤得到大量缓激肽前体序列，由1-4 个拷贝的Bombinakinin 或1-4 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成。这与大蹼铃蟾皮肤中缓激肽前体由1-8 个拷贝的Bombinakinin 或1-8 个拷贝的Bombinakinin 和1 个拷贝的Bombinakinin-GAP 组成有所不同。按同样方法，我们从大蹼铃蟾脑中也得到了三条缓激肽前体序列，其中两条含有6 个 Bombinakinin 拷贝，另一条含2 个Bombinakinin 拷贝。通过比较只含Bombinakinin 和同时含有Bombinakinin 和Bombinakinin-GAP 的前体cDNA 序列后发现， 前者序列中缺失了一段碱基序列TGCGGGTA， 从而导致移码突变， 终止了 Bombinakinin-GAP 的表达。通过生物化学的手段从微蹼铃蟾皮肤分泌液中分离到一种丝氨酸蛋白酶抑制剂BMSI1，与铃蟾属其它种中的胰蛋白酶抑制剂具有很高的相似性。根据已有铃蟾属丝氨酸蛋白酶抑制剂cDNA 序列设计引物，以皮肤cDNA 为模板，扩增丝氨酸蛋白酶抑制剂的基因序列，结果得到两条不同的序列。这两条前体序列与铃蟾属其它两栖动物皮肤中的丝氨酸蛋白酶抑制剂具有高度相似性（>70%），而且它们都含有10 个半胱氨酸残基。BMSI1 对五种丝氨酸蛋白水解发色底物的抑制活性测定表明，BMSI 1 能抑制胰蛋白酶和凝血酶的水解活性，其K(i)分别为0.02 μM 和0.15 μM。通过随机筛选cDNA 文库的方法，我们从大蹼铃蟾脑中得到了一条完整的 Somatostatin（SST）序列，根据该序列，我们在大蹼铃蟾和微蹼铃蟾脑cDNA 文库中筛选到两条变异体序列SST-L(Leu11-SST-14)和SST-R(Arg14-SST-14)。功能研究表明，这两种变异体具有和SST 相似的生物学功能，可抑制肿瘤细胞增殖、抑制细胞因子释放以及具有一定的镇痛作用。从大蹼铃蟾和微蹼铃蟾脑中得到了阿片肽前体POMC 和Proenkephalin 的 cDNA 序列，序列比对发现与东方铃蟾具有较高的同源性。从华西雨蛙皮肤cDNA 中克隆得到两类活性多肽，命名为Annins。其中一类为抗菌肽类似肽，共11 条序列，编码单一的成熟肽序列，其信号肽与雨蛙科信号肽具有很高的同源性，但酸性间隔肽和成熟肽相差较大。其成熟肽由15-17 个氨基酸残基组成，活性分析表明无抗菌和抗氧化作用，但在较高浓度时对部分细菌和多种细胞有促进生长作用，推测可能在使伤口快速愈合方面起重要作用；另一类编码具有2 个拷贝的成熟肽序列，成熟肽由5 个氨基酸残基组成，具有一定的镇痛活性，其镇痛机理可能是拮抗bradykinin 作用。

雨蛙皮肤分泌液中两种活性蛋白结构和功能的研究

在本研究中我们首次从雨蛙皮肤分泌液中分离得到了一种神经毒素（命名为Anntoxin）和一种干细胞自我更新支持因子（命名为AnSF）。随后，我们通过构建雨蛙皮肤cDNA 文库，利用特异引物筛选到Anntoxin 和AnSF 的cDNA 编码序列，前者的Gene Bank 登录号为FJ598043，后者还在等待分配登录号。Anntoxin 具有60 个氨基酸，是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，构建Anntoxin 的3D-NMR 溶液结构，证实Anntoxin 不同于有三对二硫键（键组合模式：1-6，2-4，3-5）的Kunitz 类型丝氨酸蛋白酶抑制剂，它只有两对二硫键（组合模式：1-4，2-3）。AnSF 具有123 个氨基酸，在C 端具有和Calmadolin 同源的两个EF 手指结构，能够支持人类胚胎干细胞（hESC）和猴神经干细胞（rNSC）的自我更新。为了进行Anntoxin 的生物活性和结构分析，我们在体外成功表达了 Anntoxin，获得了大量的重组Anntoxin（rAnntoxin）。经过生物活性分析， rAnntoxin 和天然分离到的Anntoxin 生物活性相当，都具有很强的胰蛋白酶抑制剂活性。Anntoxin 是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，和来源于芋螺（Cone Snail）的神经毒素Conkunitzin-S1，黑色眼镜蛇毒液（black cobra, Dendroaspis polylepis polylepis）的树突毒素δ-DaTX 或蛋白酶抑制剂K 分别具有32.8%和36.7%的相同序列，和鱼类（fish）来源的Stonustoxin 也有一定的同源性。利用膜片钳技术分别检测Anntoxin 对大鼠背根神经节（rat DRG）上Na+通道，K+通道，Ca2+通道的作用，结果证明Anntoxin 对河豚毒素敏感（TTX-S）的钠离子通道（Nav）有较强的抑制活性，对 K+通道，Ca2+通道作用不明显。随后我们在非洲爪蟾卵母细胞上表达几种典型和常用于测试对亚型K+通道作用的Kv1.1，Kv1.2，Kv1.3，Kv2.1 和 Kv4.2，Kv4.3，Anntoxin 对这些亚型K+通道上的K+电流都没有明显影响。我们成功构建了Anntoxin 的3D-NMR 溶液结构（NMR 号：PDB ID 2KCR， BMRB ID 16094），证实Anntoxin 具有典型的Kunitz 结构，由反向平行的 β–折叠片和α–螺旋及转角组成梨形结构。利用RT-PCR，WesternBlot 以及 ELISA 技术，发现在皮肤、脑、肝、胃和肠中都能检测Anntoxin mRNA 转录，但只在皮肤、脑、肝和胃中有蛋白表达，表达量分别为29.5、5.39、 4.80 和2.02 微克/克鲜重，可以看出Anntoxin 在皮肤中大量表达，是皮肤分泌液中非常重要的组成部分。因为皮肤是雨蛙接触外界的第一屏障，雨蛙的生存环境中存在很多潜在威胁，比如微生物、吸血昆虫、鸟类、爬行动物、哺乳动物等，所以Anntoxin 有可能是雨蛙适应环境的重要化学武器，于是我们测试了Anntoxin 对甜菜夜蛾幼虫（Laphygma exigua Hubner）、水蛇（Enhydris plumbea）、鹌鹑（Coturnix coturnix）、昆明小鼠（Kunming mice）的急性毒性，其LD50 分别为50，450，2500 和3000 微克/千克体重，说明在华西雨蛙皮肤中大量表达的Anntoxin 对几类潜在天敌确实有较强的杀灭作用。为了检测AnSF 的生物学活性，我们在体外成功表达了AnSF，获得了大量rAnSF。设计三个浓度梯度10、100 和500ng/ml，把AnSF 和hESC 共培养，发现在10~100 ng/ml 浓度时对hESC 的自我更新有支持作用；设计三个浓度梯度10、100 和500ng/ml，把AnSF 和rNSC 共培养，发现在 10ng/ml 时对rNSC 的自我更新有较强的支持作用。在超过500ng/ml 高浓度时，AnSF 对hESC 和rNSC 都有明显的细胞毒性作用，对rNSC 的毒性作用更明显。利用RT-PCR 技术，我们检测了雨蛙的皮肤、肌肉、肝脏、胰脏、胃、肠、心脏和脑，AnSF 只在皮肤中有少量表达。这表明AnSF 可能只参与雨蛙皮肤干细胞库的维持，保持皮肤内环境稳定，因为蛙类的皮肤细胞要负责产生大量活性物质参与先天免疫和抗氧化等重要的生理活动，需要经常更新，而AnSF 的存在可能保证雨蛙皮肤干细胞库容量稳定，不断分化出各种成熟的皮肤细胞来使皮肤能够得到足够和及时的更新，保证其功能的正常行使。所以AnSF 是维持华西雨蛙皮肤内环境稳定的重要物质。

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic ((15)N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (delta(15)N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (+/- 0.07 SE) and 49.6% (+/- 0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian-bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.

Energy substrate utilization during nightly vocal activity in three species of Scinax (Anura/Hylidae)

Anuran amphibians exhibit different patterns of energy substrate utilization that correlate with the intensity of vocal and locomotor activities. Given the remarkable differences among species in breeding and feeding strategies, and the different ways energy is used in the whole animal, the suggested correlations between calling and locomotor behavior and the level of energy substrates in the muscles responsible for such activities are more complex than previously reported. We explored the relationships between calling and locomotor behavior and energy supply to trunk and hindlimb muscles, respectively, within the ecologically diverse tree-frog genus Scinax. Specifically, we measured the relative amount of carbohydrates and lipids in these two groups of muscles, and in the liver of three species of Scinax that differ in vocal and locomotor performance, and compared our results with those of two other species for which comparable data are available. We also compared the contents of lipids and carbohydrates of conspecific males collected at the beginning and after 4 h of calling activity. The stomach content to potential feeding opportunities across species was also assessed in both groups of males. Scinax hiemalis and S. rizibilis exhibit comparatively low and episodic calling during long periods of activity whereas S. crospedospilus calls at higher rates over shorter periods. Male S. hiemalis had highest levels of trunk muscle glycogen followed by those of S. rizilbilis and S. crospedospilus, respectively. There was no correlation between total lipid content in trunk muscle and calling rate among different species, suggesting that other metabolic aspects may be responsible for the energetic support for vocal activity. The levels of lipids and carbohydrates in trunk and hindlimb muscles and liver of males collected at the beginning and 4 h into the calling period were similar across species, so the extent of energetic reserves does not appear to constrain vocal or locomotor activity. Finally, we found exceptionally high levels of carbohydrates and lipids in the liver of S. rizibilis, a trait perhaps related to a long and demanding breeding period.

Phylogenetic position of Dendropsophus gaucheri (Lescure and Marty 2000) highlights the need for an in-depth investigation of the phylogenetic relationships of Dendropsophus (Anura: Hylidae)

Dendropsophus gaucheri is a recently described species which inhabits open areas of the eastern part of the Guiana Shield and is currently assigned to the D. parviceps species group based on the presence of a subocular cream spot. Herein we investigate its phylogenetic position including material from the type locality and newly documented populations from Suriname and Brazil based on mtDNA sequences. The species, as well as D. riveroi which is assigned to the D. minimus species group, were recovered nested within the D. microcephalus species group which implies the paraphyly of the three Dendropsophus species groups. Such result, along with other evidences, highlights the need for a thorough revision of the genus. The genetic distances among D. gaucheri samples studied are low confirming their conspecificity and suggesting recent connections among populations from open areas currently isolated by rainforest in the lowlands of the Guiana Shield.

Reproductive biology of Scinax fuscomarginatus (Anura, Hylidae) in south-eastern Brazil

The present study describes the reproductive biology of Scinax fuscomarginatus in a remnant of Cerrado in south-eastern Brazil. Observations were made between September 2002 and March 2004 at Estacao Ecologica de Itirapina, State of São Paulo, south-eastern Brazil. Breeding activities occurred in lentic and temporary bodies of water during the rainy season. Scinax fuscomarginatus exhibited a prolonged breeding pattern and a lek mating system. Males were smaller than females and defended individual calling areas through acoustic and physical interactions. Resident males consistently won encounters, but did not differ in size or mass from intruder males. Satellite behaviour was observed, but no female or amplected pair interception was registered. Scinax fuscomarginatus exhibited low operational sex ratios and the general reproductive mode in which eggs are laid in the water and tadpoles are aquatic. Amplexus was axillary and the eggs were deposited at the bottom of temporary ponds. Details on oocytes, egg masses, and eggs are included.

Modeling a spatially restricted distribution in the Neotropics: How the size of calibration area affects the performance of five presence-only methods

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evolutionary history of Scinax treefrogs on land-bridge islands in south-eastern Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gene and species trees of a Neotropical group of treefrogs: Genetic diversification in the Brazilian Atlantic Forest and the origin of a polyploid species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)