Corresponding author: Laura Sandberger-Loua ( firstname.lastname@example.org )
Academic editor: Peter Bartsch
© 2017 Laura Sandberger-Loua, Hendrik Müller, Mark-Oliver Rödel.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Sandberger-Loua L, Müller H, Rödel M-O (2017) A review of the reproductive biology of the only known matrotrophic viviparous anuran, the West African Nimba toad, Nimbaphrynoides occidentalis. Zoosystematics and Evolution 93(1): 105-133. https://doi.org/10.3897/zse.93.10489
Amphibians, and anurans in particular, show the highest diversity of reproductive modes among tetrapods. Nevertheless, viviparity is scarce in anurans and its occurrence is even more often assumed rather than confirmed. Probably the best studied viviparous amphibian is the Nimba toad, Nimbaphrynoides occidentalis. During more than 40 years of research, the Nimba toad’s reproductive morphology, endocrine activity of the ovary as well as the pituitary gland, and to some extent the ecological impact (seasonality, humidity, food availability) on reproduction was examined. Due to the Nimba toad’s unique reproductive mode, summaries are usually included in reviews discussing amphibian reproduction and articles on reproductive biology often discuss the exceptional reproductive system of Nimba toads. However, to our knowledge a detailed synthesis, summarising all the different original studies on the toad’s reproduction, is so far missing. In this paper we review and summarise all available initial publications, which often have been published in French and/or are difficult to access. A short overview is given of the climatic and environmental conditions experienced by Nimba toads and the key findings supporting a “true” viviparous reproduction with matrotrophy (maternal provision of nutrition during the gestation) and pueriparity (birth of juveniles). Then foetal development (morphological, gonad and pituitary development), and the female (ovary, oviduct, pituitary and their endocrine interactions) and the male reproductive system (testes and pituitary) are reviewed. Finally, the reproductive cycle and its link to the Nimba mountains’ seasonality and ecological/ conservation implications are discussed.
Amphibia, development, evolution, ovary, oviduct, pueriparity, seasonality, testes, uterus
Viviparity, in the sense of retaining developing eggs or foetuses within the female genital tract, evolved numerous times in vertebrates and within tetrapods especially in squamates (
Viviparous reproductive modes may differ in two fundamental traits: i) at which developmental stage offspring are born and ii) in the way foetuses receive the nutritional energy for their development. During amphibian development the adult anuran body plan is only achieved after metamorphosis is completed (
Foetal development and growth needs energy. This energy can be provided by the mother via yolk rich (lecithotrophic) eggs, or by unfertilised eggs and smaller siblings (oophagy and embryophagy, sometimes called adelphophagy), or the mother continuously nourishes the foetus during gestation (matrotrophy). A good example for lecithotrophy is direct development: large, yolk rich eggs are deposited at humid locations, outside of water, and the entire development takes place within the egg, exclusively powered by yolk (
This unique matrotrophic (and pueriparous) anuran is a small (snout vent length (SVL) 15–27 mm,
The Nimba mountains. Left: elevation map of the Nimba mountains, with an inset map showing the position of the Nimba mountains within West Africa. Right: a large part of the Nimba mountains showing the steep slopes, the high altitude grasslands, the forests in the lowlands and the ravines.
The Nimba toad was described as Nectophrynoides occidentalis Angel, 1943, due to its similarity to the then two known East African Nectophrynoides Noble, 1926 species, N. viviparous (Tornier, 1905) and N. tornieri (Roux, 1906). For the same reason the new species was assumed to be pueriparous and lecitotrophic (
Nimba toads are endemic to the Nimba mountains, which are a south-west, north-east oriented mountain chain in Liberia, Ivory Coast and Guinea (see Figure
The climate of the area is characterised by first rains in March/ April, a rainy season from May to October and a dry season from November to February/ March. Mean yearly temperature is 25°C in the lowlands (550 m asl) and 19°C at high altitudes (
Rainy and dry season at Nimba. The rainy season (top) is characterised by persistent fog and rain, whereas the dry season (middle) is characterised by little rain, high temperature fluctuations and dry season fires. After dry season fires the grasses sprout very fast (bottom, © Nèma Soua Loua).
The areas outside the World Heritage Site are almost exclusively anthropogenically altered landscapes (
The very pronounced differences between the rainy and the dry season has strong effects on the toad’s activity patterns (
Nimba toad females. Left: a young female towards the end of the rainy season. Right: a large gestating female in June, shortly before parturition.
Nimba toad females have a life expectancy of three to four, rarely five years; males reach even only up to three years (
In summary: Nimba toads are endemic to the high altitude grasslands above 1,200 m asl of the Nimba mountains in West Africa. Their sexual cycle and their activity are strongly linked to the local seasonality. They spend the dry season underground (4–7, on average 6 months) and they are active only during the rainy season. The active time covers the last three months (range 2–5 months) of the gestation and about three months between gestations. All Nimba toad males and 30–70% of females become mature within three months, the remaining females within 15 months. The female life-time reproductive output is very low with a maximum of 20–32 offspring.
Generally females are larger than males (females: SVL: 15–27 mm,
Female and male cloaca of Nimba toads and a pair in amplexus. The female cloaca (top left) is close to the urostyle, whereas the male cloaca (top right) is ventrally oriented. During mating it swells and encloses the female cloaca. During the amplexus lumbalis the female is constantly horizontally swaying (bottom).
Mating occurs without a copulatory organ (
Due to the long duration of mating, it was assumed that Nimba toads may ovulate during this time (
In summary: mating occurs without specialised copulatory organs and sperm transfer is achieved through direct contact of the morphologically differently positioned male and female cloacae. Mating position is an amplexus lumbalis, being exceptional within Bufonidae. No receptaculum was found, nor any other accumulation of sperm, nor indication of ovulation induced by mating. Mating takes several hours and polyandry occurs.
Nimba toad eggs are very yolk poor and with a diаmeter of 500–600 µm (on average 540 µm,
Matrotrophy is characterized by the transport of (nutritional) energy from the mother to the foetus. It is challenging to prove this process, nevertheless matrotrophy was very early (
Foetal labial papillae. Labial papillae are forming during stage Ia (left) and are well developed at stage Ib (right). See foetal development for more information on developmental stages. Redrawn after
Another possibility to transfer nutrients from the mother to the foetuses is the ovulation of unfertilised eggs, or intra-oviductal cannibalism of other foetuses (
In summary: newborn Nimba toads are 15 times larger and > 200 times heavier than the egg. It was shown that marked amino acids injected into mothers are detectable within 30 hours in the foetal digestive system and liver. This supports the hypothesis that foetuses take up their nutrition through the very early developed digestive system and nutrition is provided by oviductal epithelial mucous cells. Additionally, matrotrophy is supported as foetal size at birth is linked to environmental conditions during the last third of the gestation period, during which females are active and most of the foetal growth occurs.
Parturition may take several hours to over two days, depending on the number of offspring (
“Birthing posture” in Nimba toads. A: gestating Nimba toad; the grey shading indicates the size and position of the enlarged distal parts of the oviduct (uterus), B: shows the “birthing posture”, in which females build a double W with their legs and increase pressure on their uteri. Compare the position of legs on the photograph on the right (C) of a female giving birth, showing likewise the double W, of the legs. A and B are redrawn after
Foetal digestive system. Exceptional for an anuran foetus is the straight and differentiated gut, with an oesophagus, the transparent and the dark intestines (stomach) and a rectum, as well as the large liver lobes. Additionally, shown are small lungs (as well small in adults), the heart, kidney and gonads. Redrawn after
There is indication that first all juveniles from one and only thereafter from the other oviduct are born (
In summary: Nimba toad females have not enough muscle power in their oviducts to induce labour. Hence, they induce birth through a unique “birthing posture”. If foetuses die within the oviduct or during parturition, they may not be evacuated, hinting at necessary juvenile activity during parturition.
Aquatic tadpoles of other anurans generally have a tail, with more or less pronounced fins, first external and later internal gills, a coiled gut, a spiracle, and mouth parts (i.e. horny beaks, labial teeth (keratodonts) and labial papillae,
Embryonic development. Shown are the eight stages as found in the literature. Redrawn after
Stage 0 is the earliest described stage; it has a duration of less than two months. At stage 0 foetuses have a tail, some yolk is still visible, no eyes, no pigmentation and no cloacal opening. During this stage the intestines are forming. Foetus size varies between 1.0–2.7 mm (body: 0.6–1.4 mm, tail: 0.4–1.3 mm,
Stage Ia has a duration of less than two months. Some yolk is still visible in early stage Ia foetuses but completely resorbed in older specimens. During this stage the head develops fast, the eyes appear and become pigmented, the mouth, first visible as a depression, develops into a slit and connects to the oesophagus, and papillae appear on the lower and upper lip (
Stage Ib has the longest duration (about two months). It is characterised by the appearance of the hind limb buds and their separation into thigh and lower leg. Front limbs develop under the transparent opercular skin fold. Pigment cells start to appear on the flanks. The digestive system and large liver-lobes are well developed (
This stage has a duration of about one month and its beginning coincides with the emergence of females from their underground aestivation sites. It is characterised by the further differentiation of the hind limbs into thigh, lower leg and foot. The limbs are still short, and at the end of this stage the toes appear but are not separated. Foetuses of this stage have a large liver and the cloacal tail piece (sensu
This stage has a duration of less than one month. It is characterised by the elongation of the limb and to a lesser extent by their differentiation. The metatarsal tubercle appears, toes become separated and the front limbs continue developing beneath the opercular skin fold and start distending the thin membrane. The eyes grow quickly and the pupil appears. The mouth is still surrounded by papillae (
This stage has a duration of a few weeks. It is characterised by the start of the tail resorption, the rupture of the opercular skin fold and the breaking through of the front limbs. Front and hind limbs are coloured dorsally with the species-specific stripes. The head elongates, nares are visible, the pupils are further developed and the labial papillae start to decrease in size (
This stage has a duration of a few weeks. It is characterised by the resorption of the tail and the labial papillae. The head becomes more elongated; the pupils are fully developed. Foetuses already show the characteristic juvenile colouration (
This stage has a duration of just a few days. At this stage foetal development is completed, the juvenile toads are a bit stockier and have slightly longer extremities and proportionately larger eyes compared to adults. Labial papillae are absent, the nares moved lateral. SVL ranges between 6–10 mm, hind legs ca. 9 mm, front legs, 5.6 mm and on the feet all tubercles are visible (
In summary: Nimba toad foetuses are characterised by the absence of a coiled gut, internal and external gills, spiracle, horny bill and labial teeth, and the presence of a gut similar to those of adults, large livers, a large head with large eyes, a mouth with papillae and the early development of the reproductive, locomotor, digestive, and respiratory systems. The foetal development is linked to the different seasons experienced by the adults. During the first 5–6 months, the time mothers spend underground during the dry season, development is slow (stages 0-IIa,
Within this section the morphology and temporary changes of the oviduct, ovary and the pituitary gland are described and their possible interactions are discussed. The most complete description of the female reproductive system is given in the doctoral thesis of
The oviduct is rather simple, it consists of two parallel strings (Figure
Tube or oviduct sensu stricto. The tube consists of an inner epithelium, a connective tissue, a thin muscle layer and is surrounded by a thin envelop (
Within a reproductive cycle the tube changes little. It is hypertrophic and active only before and during ovulation (diameter between 350–450 µm,
Uterus. As a modification of the tubular oviduct sensu stricto, the uterus also consists of an epithelium, connective tissue, a thin muscle layer and an envelope (
The proliferation phase starts in July/ August when the new epithelium is built from the connective tissue (
The secretion phase lasts the entire gestation period and is characterised by the secretion of mucoproteins by all mucous cells. Nevertheless, the importance of various factors changes over the duration of the gestation. During the first part of the gestation, mainly within the first part of dormancy, the muscle layer increases, the vascularisation of the connective tissue increases and mitoses can be observed (
The apoptosis phase is the shortest and lasts only for about 12 days (
Common tube. The common tube consists of an epithelium, connective tissue, a muscle layer and an envelope. The muscle layer is thicker than in the oviduct sensu stricto. It follows the cyclic changes of the uterus, with the only exception that the apoptosis phase and the glycoprotein secretion are missing. The transition from the secretion to the proliferation phase is achieved as ciliated cells appear (
In summary: the oviduct is separated into the tube, the uterus (distal end of the oviduct) and the common tube. The tube is only active before ovulation and hence, changes the least of the three parts. The uterus supports the foetuses during gestation, undergoes the largest size changes, and rebuilds its epithelium after every gestation. The common tube follows a similar development as the uterus, but is missing the apoptosis phase, in which the epithelium is completely exchanged.
The ovaries are situated in the posterior third of the body (
Follicles consist of a theca layer and granulosa cells (
After ovulation, follicles decrease slightly in size (280–320 µm diameter,
Hence, within the ovary two phases can be observed, a follicular phase, which is characterised by follicle growth, and a luteal phase, during which the corpora lutea are present (
The luteal phase starts with the gestation. Corpora lutea decrease in size during the active life of females after emergence, and are rapidly disappearing after parturition (
In summary: Nimba toads have small ovaries, which contain only few follicles (< 60), of which a small proportion (4–20) reaches maturity every year. After ovulation follicles develop into corpora lutea, which are present during the entire gestation period. Within follicles androgens (oestrogen and testosterone) are produced within the theca cells, whereas the granulosa cells produce progesterone. During the luteal phase, exclusively progesterone is produced by the granulosa cells. Progesterone levels are highest during the dry season, when foetal and follicle development is slow or absent.
The pituitary gland is comparatively small (
The glycoprotein type 1 cells are widely distributed within the pituitary gland (
Protein type 1 cells are large, widely distributed cells within the pituitary gland (
Within one sexual cycle this means that at the beginning of the gestation glycoprotein and protein type 1 cells are present and protein type 2 cells are appearing. Glycoprotein type 2 cells are less abundant but easily and brightly stainable (
In summary: five different cell types can be distinguished within the pituitary gland, three glycoprotein cell types and two protein cell types. The glycoprotein type 1 cells are visible and active during the toad’s active life, follicle development and vitellogenesis. This indicates a connection between the glycoprotein type 1 cells and the ovarian follicular phase, which makes them gonadotropic cells. The protein type 1 cells are well visible and active during most of the time, with a slight decrease in activity for some time after emergence. They had been shown to secret prolactin.
Within the female reproductive cycle three time periods lead to changes within the reproductive system: i) before toads become dormant ovulation and mating occur and gestation starts. Within the oviduct this leads to the cease of activity within the tube, the start of the secretion phase within the uterus and within the ovary to the start of the luteal phase, in the pituitary glycoprotein type 1 cells become scarcer and protein type 2 cells appear; ii) With emergence at the beginning of the rainy season, the uterus starts to increase considerably in size as foetal growth and development takes up speed, within the ovaries the corpora lutea start decreasing in size, the follicles slowly start development and within the pituitary the glycoprotein type 1 cells start to appear, protein type 1 and 2 cells become less abundant; iii) With the birth of juveniles in June the tube increases developmental speed, the uterus collapses and rebuilds a new epithelium, within the ovary the corpora lutea disappear and follicle growth intensifies, within the pituitary the glycoprotein type 1 cells show very high activity, the protein type 1 cells increase their activity as well and protein type 2 cells are absent. Hence, the question arises, whether these changes in the different organs are coincidences or one change is triggering the change in another organ. To answer this question several experiments were carried out. If not stated otherwise studies are described in
Ovary and foetus development. During the dry season corpora lutea are present, large and active, and it was shown that they produce progesterone. Foetal development is slow during dormancy (
If females are separated from males during the mating season, ovulation occurs and a “pseudo-gestation” develops (
Ovary and oviduct. The tube’s endocrine cycle is synchronised with the follicular phase of the ovary. Follicles and tube start slowly developing after emergence and are most active before and during ovulation, and are not active during dormancy. In females ovariectomised after parturition the tube does not show any activity towards the end of the rainy season, usually the time of highest activity. In females ovariectomised at about the time of ovulation, the tube maintains high activity for the next 6 months and activity is terminated only after two years. This indicates that the ovary is controlling tube activity (
Another connection between ovary and tube became apparent by an observation of
Experiments on female ovary, oviduct and uterus removal. Shown are the positions of eggs in the uteri after unilateral ovariectomy (A), after unilateral ovariectomy and the connection between oviduct and uteri bound at the side with the still present ovary (B), unilateral ovariectomy and the oviduct and uterus at the side of the still present ovary removed (C) and the unilateral removal of the oviduct and the uterus without ovariectomy (D). The red cross indicates the ovary removed, the red lines indicate positions where either oviduct (B) or the uterus (C and D) were bound. The two-headed arrow with the two red lines indicates that eggs apparently do not wander from one uterus to the other passing the common tube. Redrawn after
The uterus does not follow the same cycle as the follicles and the tube. The largest change of the uterus is during gestation. Nevertheless, during the follicular phase after parturition in ovariectomised females the injection of oestrogen initiated the development of the mucous layer and some blood vessels, the injection of testosterone the development of the connective tissue and some blood vessels, and the injection of progesterone the development of the muscle layer and an epithelium without ciliated cells. Only if oestrogen and three weeks later progesterone were injected together, did the uterus develop as in non-ovariectomised females (
The removal of ovaries at the beginning of gestation leads to abortion 3–4 months after the operation in 50% of gestating females. Whether ovariectomy leads to abortion depends on the female’s age. In all females 3–4 months old (hence, born the same year) and within 50% of females 15–16 months old, ovariectomy resulted in abortion. In all older females the gestation continued. As females may either get mature within 3–4 months, or one year later, females 15–16 months of age may be gestating for the first or the second time. Françoise
After emergence, ovariectomy of gestating females never results in abortion, irrespective of the female’s age. This indicates that at this stage of the gestation the ovary has no longer an important effect on gestation (
Hysterectomy (the removal of uteri) at the beginning of the gestation leads to atresia of small follicles and the corpora lutea disappear faster. After ovulation in non-hysterectomised females only nearly mature follicles are destroyed by atresia. Hence, ovary and uterus are influencing each other. To examine whether this connection is linked to the uterus, or to the developing foetuses, foetuses were removed by caesarean section (
Ovary and pituitary gland. After ovariectomy at the beginning of gestation the glycoprotein type 1 cells are contracted and de-granulated. The protein type 2 cells, which are normally appearing with the corpora lutea, are absent, but protein type 1 cells are very abundant. Ovariectomy after emergence does not lead to large changes within the pituitary gland. Ovariectomy one month after parturition leads to the degranulation of the glycoprotein type 1 cells and they develop into “castration cells”. Hence, the absence of the ovary has the largest effect on the pituitary at the beginning of the gestation and after parturition during vitellogenesis (
Examining the effect of the pituitary on the ovary is much more problematic, as females die within the next 15–25 days after the flattening (destruction) of the pituitary, indicating that the pituitary has other vital functions in Nimba toads (
Summary of interactions. The ovary seems to have the largest effect on the other reproductive organs from June to emergence at the beginning of the rainy season. Nevertheless, the ovary has different functions between parturition and ovulation (vitellogenesis) and during the first six months of the gestation females rest underground (dormancy). During vitellogenesis follicles grow and develop quickly and they synthesise oestrogen, testosterone and progesterone. Ovariectomy at the beginning of this period hinders the development of the oviduct – tube and uterus. This non-development can be circumvented with the injection of oestrogen and progesterone, indicating, that these hormones are vital for oviduct development during vitellogenesis. As well within the pituitary gland, glycoprotein type 1 cells (gonadotropic cells) develop into “castration cells” if the ovaries are removed at the beginning of vitellogenesis. During dormancy the corpora lutea are large, numerous and active and synthesise progesterone. Progesterone slows foetal development during dormancy and if experimentally applied as well after emergence. Ovariectomy of first-gestating females leads to abortion, which was as well linked to lower progesterone levels in first-gestating, than in older females. Within the pituitary protein type 2 cells appear with the development of corpora lutea. In neutered females they are absent during the whole dry season. Hence, most of the year the ovary influences the development of the pituitary gland, the tube and the uterus and during dormancy as well foetus development.
Between emergence and parturition foetuses develop quickly, stretching the uterus and restricting the other organs within the female. This development is only stopped by the destruction of the pituitary gland, which is lethal to females. Development can be slowed down by the injection of progesterone. On the other hand, the absence of foetuses in pseudo-gestating females leads to faster development of the ovary (decrease of corpora lutea and development of follicles), but has no effect on the pituitary gland. If foetuses are removed through caesarean section after emergence, with the apoptosis phase the uterus starts a new cycle, earlier than in normally gestating females. These results are in accordance with the hypothesis that the presence and development of foetuses are important for normal development of ovaries, and through them on the oviduct and the pituitary gland. On the other hand, change of the females from the inactive, low nutrition life underground to an active high nutrition life above ground and external factors might be important too.
The reproductive system of Nimba toad males received less attention than that of females. Respective research focused more on the environmental and cyclic dependencies of the reproductive cycle than on morphology and physiology of the reproductive system. Primary publications are by M. Zuber-Vogeli (
Nimba toad males lose at 13 mm SVL their juvenile colouration (
In summary: males have been studies less than females; are smaller, darker and at least during the mating season more active than females. Due to their more active life-style they may suffer from higher predation pressure and possibly as well higher energy demands.
Nimba toad testes are small (< 2 mm) ovoid masses (
Most bufonids have a Bidder’s organ, a part of the testes that contains oocytes, and which is separated from the testes by a separate envelope. During Nimba toad foetal development testes usually contain 1 or 2 (rarely 5 or 6) oocytes per male (
The spermatozoa show no apparent modification compared to those of other bufonids. The only differences are that the perforatorium ends slightly posterior to the acrosome vesicle, the distal centriole is penetrated throughout its length by the central singlets of the axoneme and no mitochondrial collar is present, but mitochondria are located around the anterior axoneme (
In summary: Nimba toad testes are ovoid masses, which are white during the rainy and dark during the dry season. During embryonic development the testes may contain a few oocytes, which disappear before birth and are never surrounded by an envelope. The seminiferous tubes are always numerous, but vary their size during the reproductive cycle. Most gonad cells are grouped around Sertoli cells and within each group development is synchronised. The spermatozoon is a typical bufonid spermatozoon with little modification.
Male adaptations to the viviparous reproductive mode are less distinct than in female Nimba toads; however, the male reproductive cycle is likewise tightly linked to the climatic cycle of the environment. Nimba toad males have a discontinuous reproductive cycle, (
The dry season dormancy is characterised by low metabolism (
In April, when males emerge, one month after females, testes are still black and nuptial pads are not yet visible and their fat bodies are very small (
In June nuptial pads start to turn black, testes increase further in size (
During the mating season the nuptial pads are blackest and spiniest (Figure
Male during the mating season, showing pronounced nuptial pads on the thumbs. © Joseph Doumbia
Similarly to females, the male reproductive cycle is linked to three important seasonal points, the mating season and the subsequent dormancy underground (slow or no development), emergence during the next rainy season (beginning of spermatogenesis with the appearance of secondary spermatogonia), and in June/ July when rain becomes permanent spermatogenesis intensifies (appearance of spermatocytes). Despite this strong link individual males may finish their spermatogenesis at different times due to individual differences in developmental speed (
In summary: the male reproductive cycle is interrupted during the dry season, when only divisions of primary spermatogonia are observed. Only after emergence, which is later in males than in females, spermatogenesis starts and is accelerated after June, when rain is more permanent, and results in the presence of spermatozoids during the mating season. During the mating season only primary spermatogonia divide. Spermatogenesis is accelerated through the availability of prey and high humidity. Nevertheless, individual males differ in the quantity and speed of the different gonad cell stages and larger males develop faster and mate earlier than smaller (but nevertheless, sufficiently old) males.
Monique Zuber-Vogeli examined the male pituitary gland and focused on the annual reproductive cycle (
In summary: in the male pituitary, all five cell types were present, but only three of these five showed temporal modification. The glycoprotein type 1 cells are linked in males and females to gonad development and are rare during the dry season and most abundant during spermatogenesis/ vitellogenesis in July/ August and during the mating season. The protein type 1 cells are always the most abundant in males and females and show some, but little variability. The glycoprotein type 3 cells, which are assumed to be the thyrotropic cells, are always rare in both sexes. The glycoprotein type 2 cells and the protein type 2 cells differ in their annual activity between the sexes. Glycoprotein type 2 cells show no variability in females, but in males they are absent during the dry season and increase in number after emergence until they reach their maximum in July/ August and they were linked to nuptial pad development. The largest discrepancy is between female and male protein type 2 cells. In females they are abundant and active during the dry season, whereas in males they are only present in July/ August.
Within anurans, Nimba toads have a highly derived and unique reproductive mode. They retain eggs and foetuses within their oviducts and are pueriparous and matrotrophic. As in other viviparous amphibian species, they have internal fertilisation, a reduction in number of developing eggs, and a prolonged developmental period (
In Nimba toads the ovaries are small and contain small follicles, of which only very few mature within each reproductive cycle. For example, in A. malcolmi and N. tornieri ovaries are considerably larger and contain more follicles at very different developmental stages (
Nimba toad foetuses have no internal, nor external gills, no spiracle, no coiled gut and neither labial teeth nor horny beaks at any time during their development, but they possess labial papillae, a gut similar in structure to that of adults, well developed livers, and their development takes nine months. It was hypothesised that within viviparous amphibians metamorphosis is prolonged (
Based on morphology
The other three unique traits of the Nimba toad reproduction can be regarded as adaptations to an unpredictable environment and time constraints due to the seasonality. First, the developmental break during the dry season coincides with an inactive life with presumably a low energy budget (
Within one year two important periods mainly determine the reproductive cycle of a female: one, at the end of the rainy season with ovulation, mating and the beginning gestation, the second in June with the birth of juveniles. These seasonal periods are important for males as well, as during mating season spermatozoids are present, but disappear afterwards, in June at the time juveniles are born, spermatogenesis is intensified. Nevertheless, one further important period in the season is the emergence at the beginning of the rainy season, which results as well in changes within the reproductive system. Hence, within one year three periods lead to important changes within the Nimba toad reproductive system, one might be triggered by reproductive and/ or environmental clues (late rainy season), one is characterised by environment – the beginning of the rainy season (emergence), and one is characterised by reproduction at least in females - the end of gestation and intensification of spermatogenesis and intensification of rains (June). In Figure
Summary of the temporal development of the foetus and male and female reproductive system. The outer layer gives the months as abbreviations and the seasons by colour (yellow: dry season, blue: rainy season). Toads mate between mid-September and mid-October on average, are going underground mid-October and emerge in mid-March from their dormancy sites. Juveniles are born in mid-June (pictograms). Spermatogenesis shows the development within the male reproductive system. Foetal growth gives the speed of development and growth. The uterus shows three phases: a proliferation phase in which the uterine epithelium develops, a secretion phase during the gestation and an apoptosis phase during which the uterine epithelium is completely exchanged (old one removed and new one built). The ovary has two phases: the follicles phase, characterised by follicle growth and the luteal phase characterised by the presence of corpora lutea. Within the female pituitary three cell types show variation within the annual cycle, the glycoprotein type 1 cells and the protein type 1 and type 2 cells. Within the male pituitary as well three cell types show variation (not shown), glycoprotein type 1 cells and glycoprotein type 2 cells in the same way as those of females, whereas protein type 2 cells are only present in July/ August.
“Environmentally linked” developments start with emergence and end with the beginning of the dormant life underground which coincides with mating (shown in blue in Figure
“Reproductively linked” developments start with mating and the establishment of the gestation and end with the birth of juveniles (shown in yellow on Figure
The uterine apoptosis and proliferation phases and the presence and activity of protein type 1 cells (shown in red in Figure
In Nimba toads the reproductive cycle is tightly linked to the seasons. In most other viviparous amphibians an environmental dependency is assumed, but detailed data rarely exist (
Most amphibian species with derived reproductive modes are threatened (
The strong link between the Nimba toad’s reproductive and life-cycle with the Nimba mountains seasonality and other environmental factors indicates that these conditions might have favoured the evolution or at least the maintenance of viviparity. Hence, ecological studies on N. occidentalis and the other species within the Nimba toad’s clade (Didynamipus, Altiphrynoides, Schismaderma, Nectophrynoides), several of which have derived reproductive modes as well, may give insights into the evolutionary drivers of viviparity in African bufonids. Comparatively little is known about the other species with and without derived reproductive modes within this clade. For example, the reproductive mode of D. sjostedti is only assumed to be direct development (
Viviparity is rare in anurans and the only known matrotrophic anuran is the Nimba toad. In Nimba toads three observations support matrotrophy: first, newborn Nimba toads are 15 times larger and > 200 times heavier than the egg. Second, amino acids injected into the mother were recorded first within the digestive system and liver and later in other areas of the foetuses. Third, foetal size at birth is linked to environmental conditions during the last third of the gestation period, during which females are active and most of the foetal growth occurs. We have identified six characteristics linked to viviparity specific of Nimba toads, which provide strong evidence that viviparity evolved independently in this species. These characteristics comprise three traits which are common in other amphibians, but not usually found in other viviparous species (small and yolk poor eggs, mucoprotein secretion by oviduct/ uterus epithelium, labial papillae of the foetuses), two behavioural or ecologically important traits (“behavioural birthing” and the developmental break during the dry season) and one characteristic which to our knowledge is not known from any other amphibian, but from mammals (complete apoptosis and rebuilding of the uterine epithelium after parturition). Apart from the mucoprotein secretion of the oviduct/ uterus which is found in all amphibians before fertilisation - but in Nimba toads additionally after fertilisation - these traits are more likely present in oviparous species with free-swimming tadpoles than in direct developing and lecitotrophic viviparous species. The other three unique traits can be regarded as adaptations to an unpredictable environment and time constraints due to the environments seasonality. Most reproductive developments can be placed in either of two categories: i) linked to the gestation period, “reproductively linked”, or ii) linked to the active above ground life of toads and hence, “environmentally linked”. Hence, it is likely that the harsh unpredictable environment and scarcity of open water promoted viviparity in Nimba toads, or supported the survival of this unique reproductive mode in these special and isolated conditions. Considering their complex life cycle, in which reproductive and seasonal cycles are tightly linked, understanding and protecting the Nimba toad’s threatened environment is of utmost importance.
For support to receive rare and difficult to access publications we thank Martina Rißberger and Hans-Ulrich Raake from the MfN library. Johannes Penner organised access to the doctoral thesis of Françoise Xavier. Nèma Soua Loua provided one photograph in Figure