Species and sex specific chemical composition in a concealed gland from an internal gland-like tissue of an African frog family

Intraspecific chemical communication in frogs is understudied and the few published cases are limited to externally visible and male-specific breeding glands. Frogs of the family Odontobatrachidae, a West African endemic complex of five morphologically cryptic species, have large, fatty gland-like strands along their lower mandible. We investigated the general anatomy of this strand and analyzed its chemical composition. We found the strand to be present in males and females of all species. The strand varies in markedness, with well-developed strands usually found in reproductively active individuals. Although the strands are situated under the skin, they are connected to particular skin sections, the vocal sac in male frogs and a respective area in females. Gas-chromatography/mass spectrometry and multivariate analysis revealed, that the strands contain sex- and species-specific chemical profiles, which are consistent across geographically distant populations. The profiles varied between reproductive and non-reproductive individuals. Our results indicate that the gland-like strand in the Odontobatrachidae comprise a so far overlooked type of breeding related organ, potentially a gland, that most likely plays a role in the mating and/or breeding behavior of the five Odontobatrachus species. Our results highlight the relevance of multimodal signaling in anurans, and indicate that chemical signaling may not be restricted to sexually dimorphic, apparent skin glands.

Methods
We collected specimens of all five Odontobatrachus species, O. arndti, O. fouta, O. natator, O. smithi and O. ziama. We euthanized, killed and dissected the animals to obtain tissue samples for chemical analysis. Tissue samples where then extracted in dichloromethane and analyzed by gas chromatography/mass spectrometry (GC/MS). To search for sex and species-specific differences in gland composition, we sampled both sexes of each species from at least two locations per species. We also sampled O. arndti under wet and dry season conditions on the same river, to screen for seasonal effects. To check for the effect of geographical distance between populations, we sampled O. arndti males and O. smithi females over a large part of their distribution range. To investigate ontogenetic effects on strand composition, we sampled metamorphic and sub-adult O. ziama frogs and compared them to sexually mature animals. For histological and anatomical analyses, we used voucher specimens from the collection of the Museum für Naturkunde Berlin (ZMB).

蛙类的种内化学通讯（Intraspecific chemical communication）研究仍较为匮乏，已报道的相关案例仅局限于外观可见的雄性特有繁殖腺。齿蛙科（Odontobatrachidae）是西非特有的类群，包含5个形态隐存种，该类群物种的下颌下缘分布有大型脂肪样腺索。本研究对该腺索的大体解剖结构进行了考察，并对其化学成分展开分析。结果显示，所有物种的雌雄个体均具有该腺索；其显著程度存在差异，发育良好的腺索通常见于繁殖活跃个体。尽管腺索位于皮下，但它们与特定皮肤区域相连：雄性个体的声囊以及雌性个体的对应区域。气相色谱-质谱联用法（Gas-chromatography/mass spectrometry，GC/MS）结合多变量分析结果表明，该腺索含有性别和物种特异性的化学特征谱，且该特征谱在地理远缘种群中保持稳定；特征谱在繁殖与非繁殖个体间存在显著差异。本研究结果提示，齿蛙科的腺索属于此前被忽视的繁殖相关器官类型，其极有可能为腺体，大概率在5个齿蛙属物种的交配和/或繁殖行为中发挥作用。研究结果凸显了无尾两栖类多模态信号通讯的重要性，并表明化学信号通讯未必局限于具有性二态性的外观可见皮肤腺。

方法
我们采集了全部5个齿蛙属物种的标本：O. arndti、O. fouta、O. natator、O. smithi及O. ziama。对动物进行安乐处死并解剖，获取用于化学分析的组织样本。随后将组织样本以二氯甲烷萃取，通过气相色谱-质谱联用法（GC/MS）进行分析。为探究腺索成分的性别和物种特异性差异，我们从每个物种的至少两个采样点采集了雌雄个体样本。我们还在同一条河流的干湿季条件下采集了O. arndti样本，以筛选季节效应的影响。为验证种群间地理距离的效应，我们在其分布范围的大部分区域采集了O. arndti雄性个体与O. smithi雌性个体样本。为探究腺索成分的个体发育效应，我们采集了O. ziama的变态期和亚成体个体，并与性成熟个体进行对比。组织学与解剖学分析所用的凭证标本取自柏林自然历史博物馆（Museum für Naturkunde Berlin，ZMB）的馆藏。