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Croaking Science- Another amphibian reproductive mode: newly-hatched caecilians feed on mother’s milk.

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Written by Roger Downie, Froglife and University of Glasgow

Although we mostly think of amphibians mating in water, depositing their eggs there, and leaving them to develop unaided, the group actually includes a vast diversity of reproductive modes, 74 at the last comprehensive count (Nunes-de-Almeida et al., 2021), many of them including some kind of parental care. Now comes something new: Mailho-Fontana et al.(2024) report that in Siphonops annulatus, a widely-distributed South American caecilian, mothers produce and release a milk to feed their newly-hatched babies.

Caecilian babies around the mother’s vent. Credit: Carlos Jared

The 220+ species in the order Gymnophiona, commonly called caecilians, are acknowledged as the most poorly known amphibian group, a situation related to their tropical distribution and secretive burrowing habits. One feature that is known about the group is that a relatively high proportion of species (about 20%) is viviparous (giving birth to live young), compared to the tiny proportion of anurans and urodeles that reproduce in that way. In viviparous caecilians, the eggs are retained in the oviduct and hatch there, with the young nourished by oviductal secretions, growing substantially during a gestation period that can be as long as 11 months.

Egg-laying caecilians also show parental care, with mothers often looking after the eggs, and at least in some cases, feeding the hatchlings on modified, lipid-rich skin which the young browse on using specialised teeth. This behaviour has previously been reported in S. annulatus by Wilkinson et al. (2008).

Nunes-de-Almeida and colleagues were able to capture and observe for an extended period in captivity a sample of 16 adult female S. annulatus. After hatching, the young (4-13 per clutch) clustered around the coiled-up mother, keeping close to her vent. Periodically, the young touched the vent and emitted high-pitched sounds. These behaviours seemed to elicit the release of a transparent, viscous milk, which the young fed on. After feeding, the young became relatively inactive for a time. The young may compete for access to the milk, since data showed considerable variation in growth rate increase among a batch of young; on average, body mass increased by 130% during the week after hatching. Milk release was at least daily, and up to six times a day. As in Wilkinson et al.’s study, skin feeding also occurred, but less frequently, about once a week. Histological analysis of the female reproductive tract showed that milk is produced by oviductal glands which develop during the reproductive period. These are very similar to the glands which appear during gestation in viviparous species, but have not previously been reported in egg-layers. The lipid content of the milk is mainly composed of long-chain fatty acids, palmitic and stearic; the milk is also rich in proteins and carbohydrates. The period of maternal care, during which they do not feed,  lasts about two months, during which maternal body mass declines by 30%, so this behaviour is very costly to the mothers.

Milk production is a diagnostic feature of mammalian reproduction, but it does occur occasionally elsewhere in the animal kingdom: in pigeons and flamingoes among the birds, for example, where the crop produces a nutritive secretion. It will be fascinating to discover if S. annulatus is an isolated case among the caecilians. Frost (2024) lists four species in the genus Siphonops, with five genera and 28 species in the family Siphonopidae, at least some of which are known to show skin-feeding. In my view, it would be surprising if this combination of hatchling behaviour and maternal physiology  is restricted to a single species.

 

Acknowledgement I thank Malcolm Kennedy for drawing my attention to this fascinating discovery.

 

References

Frost (2024). Amphibian Species of the World. Accessed 15/3/24.

Nunes-de-Almeida et al. (2024). Milk provisioning in oviparous caecilian amphibians. Science 383, 1092-1095.

Wilkinson et al. (2008). One hundred million years of skin feeding? Extended parental care in a neotropical caecilian. Biology Letters 4, 358-361.

Wilkinson et al. (2013). A new species of skin-feeding caecilian, and the first report of the reproductive mode in Microcaecilia. PLOS ONE 8, e57756.

 

Reproduction without sex: some mother reptiles can do it on their own.

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Roger Downie, Froglife and University of Glasgow

Recently, media outlets summarised the findings of a paper published in Biology Letters (Booth et al., 2023) that reported the first known case of ‘virgin birth’ in a crocodile. Here, I describe the case and put it into the context of other known examples of reproduction without sex in reptiles.

An 18 year old American crocodile (Crocodylus acutus) had been in captivity, in isolation from other crocodiles, in a Costa Rican reptile park since the age of two. To the keepers’ surprise, it produced a clutch of 18 eggs. These were incubated artificially for 3 months, but failed to hatch. They were then opened up: only one contained recognizable contents, a well-developed baby crocodile, sadly dead. Its gonads showed it to be a female, and genetic analysis demonstrated that it was the offspring only of its mother, with no male input.

The technical term for virgin birth, where a female produces offspring without input from a male is ‘parthenogenesis’, and it can be of two general kinds: a) obligate, where it is the only kind of reproduction occurring in that species; b) facultative, where both sexual and non-sexual reproduction can occur.

How common is parthenogenesis? Obligate parthenogernesis is known in about 80 unisexual species of vertebrates, most of them lizards (Neaves and Baumann, 2011). The best-known examples are the whiptail lizards of Mexico and the southern states of USA, with 12 out of over 40 described species of Aspidoscelis (formerly Cnemidophorus) unisexual; and the rock lizards of the Caucasus mountains, with 7 out of 30 Darevskia species unisexual (Spangenburg et al., 2020). The unisexual lizards are nearly all hybrid clones resulting from matings between related species. The unisexual species have either three (triploid) or four (tetraploid) sets of chromosomes which can be identified in terms of the parental species contributing to the cross. It is believed that the unisexual species overcome any disadvantage from the lack of sexual reproduction through the hybrid vigour derived for their extra sets of genes. In Aspidoscelis, Crews et al. (1986) found that the females of unisexual species show female-female courtship behaviour and pseudo-copulation which enhances ovulation.

Obligate parthenogenesis is relatively easy to detect from the complete lack of males in populations of these unisexual species. Facultative parthenogenesis (FP) is more problematic, partly because many species have long-lived sperm which females can retain in their reproductive tracts after mating. The best evidence therefore comes from captive females kept in the absence of males, as in the crocodile case above. It is the first reported example of FP in any crocodilian, and also the first case from a species with temperature-dependent sex determination. In crocodiles, eggs incubated at low and high temperatures develop as females, with males appearing from incubation at intermediate temperatures: the Costa Rican crocodile’s eggs were incubated at 29-300 C, in the female-determining range. FP has been reported from two captive Komodo dragons (Varanus komodoensis) in Chester and London zoos respectively (Watts et al., 2006). The authors note that Komodo dragons kept for the captive breeding of this endangered species are usually housed as single females, with males brought in when the females are in good condition. Their discovery of eggs laid by unmated females (25 eggs, 8 viable in one case; 4 eggs, one viable in the other) may get in the way of the breeding programme. Komodo dragons have ZZ/ZW sex chromosomes, rather than the XX/XY pattern we are more familiar with in mammals. In ZZ/ZW, males are ZZ and females ZW (in XX/XY, males are XY and females XX). Parthenogenetic development from a female Komodo dragon produces ZZ or WW individuals, with ZZ male, and WW probably non-viable. Booth et al. (2012) published the first report of FP in wild snakes: they collected 22 pregnant copperheads (Agkistrodon contortrix) and 37 cottonmouths (A. piscivorus) and examined the litters for signs of FP (few if any male offspring; high proportion developmental failures). Two litters were selected for molecular analysis, to compare the offspring and maternal genomes, and both indicated that they were the products of only the maternal genomes. The authors note that in both cases, one theory for the occurrence of FP did not fit the facts; it has been suggested that females may use FP to overcome a shortage of males in a population, but in neither case was this true. Although unisexuality with obligate parthenogenesis has some advantages over sexual reproduction, it is not clear why FP should occur as an occasional variant on normal sexual reproduction. The finding of FP in a crocodile means that the only reptile taxon where parthenogenesis is unknown is the Chelonia.

The means by which parthenogenesis works as a reproductive mode can be complex. For example, the entry of the sperm into the ripe egg is usually the trigger for development to begin, so how is development triggered in the absence of fertilization? And what happens to the normal chromosomal reduction divisions of meiosis, when no sperm arrives to restore the diploid number? The references to this article will lead you to some of the answers.

You might also ask: does parthenogenesis also occur in amphibians? So far, it has not been reported, but there are unisexual species of amphibians, though they do not reproduce by parthenogenesis: a story for another day.

References

Booth et al. (2012). Facultative parthenogenesis discovered in wild vertebrates. Biology Letters 8, 983-5.

Booth et al. (2023). Discovery of facultative parthenogenesis in a new world crocodile. Biology Letters 19, 20230129.

Crews et al. (1986). Behavioural facilitation of reproduction in sexual and unisexual whiptail lizards. PNAS 83, 9547-9550.

Neaves and Baumann (2011). Unisexual reproduction among vertebrates. Trends in Genetics 27, 81-88.

Spangenburg et al. (2020). Cytogenetic mechanisms of unisexuality in rock lizards. Scientific Reports 10, 8697.

Watts et al. (2006). Parthenogenesis in Komodo dragons. Nature 444, 1021-2.

The astonishing diversity of reproductive modes in amphibians: a new classification

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Written by Roger Downie, Froglife Trustee and University of Glasgow

In the UK, we are accustomed to amphibians breeding in the spring and depositing their eggs in freshwater bodies, usually ponds rather than streams or lakes. Frogs deposit their eggs as a clump of jelly; toads as strings; and newts wrap theirs individually in folded leaves. The embryos hatch as larvae and feed in the water until they are ready to metamorphose into juvenile versions of the adult form. The adults spend no time with their eggs after deposition. So far, so familiar. But, when we look beyond our UK species, we find a wide diversity of reproductive modes. How many, and what are they like?

The term ‘reproductive mode’ (RM) was coined by Breder and Rosen (1966) to help them make sense of reproductive diversity in fish. Later, Salthe and Duellman (1973), in the context of amphibians, defined RM as a set of characters including oviposition site, ovum and clutch characteristics, rate and duration of development, stage and size of hatchlings, and type of parental care, if any. Without using the term RM, Boulenger (1886) had identified 10 amphibian modes. A hundred years later, Duellman and Trueb’s (1986) textbook recognised 29 RMs in anurans, seven in urodeles and two in caecilians. Haddad and Prado (2005) extended this to 39 modes for all amphibians, and there have been a few additions since. However, Nunes-de-Almeida et al. (2021) have now published a new classification, identifying 74 RMs in amphibians, almost a doubling of the 2005 list. How and why?

Their method is to divide the reproductive process into a set of eleven characters where each species can be assigned to one of two (occasionally more) states. The characters are:

  1. Reproduction type: oviparity (egg-laying) or viviparity (eggs not laid: the female gives birth to larvae or juveniles). Viviparity is common in caecilians, but also occurs in a few frogs and salamanders.
  2. Oviposition macrohabitat: eggs are deposited into the environment or they develop in or on the body of either the female or the male parent.
  3. Spawning type: the distinction here is between cases where eggs are immersed in froth, or not. Froth is made from oviduct secretions in two ways: either a foam is generated by beating movements of the adults’ limbs; or bubbles are made by the female’s jumping movements.
  4. Oviposition substrate: either in water, or not in water: on the ground, or in vegetation, or attached to a parent.
  5. Medium surrounding the eggs: the main distinction here is between two kinds of aquatic habitat: lentic (still water, like a pond) or lotic (flowing waters, such as streams). The medium can also be air, as in eggs deposited on the ground, or attached to a parent’s body.
  6. Nest construction: a constructed nest is defined as a place to deposit eggs which the parents have made by digging, or cleaning, or building in some way. ‘Froth’ nests are excluded from this category (I’m not sure this exclusion is fully justified). Constructed nests can be burrows, or depressions, or cleared areas on the forest floor, or leaves folded around the eggs.
  7. Oviposition microhabitat: here, Nunes-de Almeida and colleagues find 15 variables: eggs on the surface of water, at the bottom of a pool, on the ground, on a leaf, on a rock, in a bromeliad tank etc.

The remaining characters distinguish different patterns of development:

  1. Embryonic development: can be indirect, with a larval stage, or direct – lacking a distinct larval form, and progressing directly from embryo to juvenile.
  2. Embryonic nutrition: all amphibians have yolky eggs, and the yolk provides the nutrients needed for embryonic development, but in some cases the mother provides additional nutrients. Where all nutrients derive from the yolk, development is termed lecithotrophic; where the mother provides extra, it is matrotrophic.
  3. Larval and newborn nutrition: when embryos hatch and become free-living, we consider them as larvae. Generally, this marks the stage when they begin to forage for food, although they still have some of the egg-yolk left. However, some species do not feed as larvae, but obtain their nutrition from their large remaining yolk reserves: these are termed endotrophic. Most larvae are exotrophic, obtaining most of their nutrition from external food sources. In a few cases, parents provide this nutrition. For example, so-called trophic eggs, unfertilised eggs deposited by females to feed their hatched larvae. Another example is the feeding of some caecilian young on their mother’s skin secretions.
  4. Place of larval development: mostly this occurs either in a pool (lentic) or a stream (lotic), but there are also cases of larval development on land, or attached to a parent’s body.
Credit: Julia Page

Overall, the authors reviewed RMs in 2171 species on which they could find adequate information: this is 26 % of all amphibians (8393 species, November 2021). Anurans showed 71 of the 74 RMs; urodeles 16 and caecilians seven. Most species showed a single RM, but some fitted up to four of the modes.

Nunes-de-Almeida and colleagues have made a valiant effort to classify the rich diversity of amphibian RMs, but it is not without some problematic aspects. One omitted feature is fertilisation mode: internal or external. This is a crucial feature in research on reproductive strategies relating to certainty of paternity and male competition. Another aspect largely omitted is parental care behaviour. Parental care can be defined as non-gametic investments in offspring that incur a cost to the parent, but which provide a benefit to the offspring. Parental care in amphibians is discussed in Croaking Science (date to come). The new RM classification  explicitly excludes parental care on the grounds that parental care information is lacking for too many species. However, many kinds of parental care are actually included: for example, the provision of trophic eggs to larvae (character 10 above); while others such as larval transportation by adults are omitted. Another omitted feature which I find surprising is the differences in anuran spawn characteristics: single non-adhesive eggs, eggs in clumps, eggs in strings. It is likely that these differences are evolved characteristics important to reproductive success, so should be included in a classification of RMs. Another omission is the diversity of larval forms: there is huge diversity in tadpole form and behaviour, related to the habitats they live in: this may go beyond the usual definition of an RM, but is an important aspect of reproductive success. There are also occasional inconsistencies: phyllomedusine tree frogs wrap their egg clutches in leaves, and this is classed as a constructed nest (character 6 above); newts wrap their eggs individually in leaves, but this behaviour is not acknowledged as a kind of nest construction.

One excellent point made by the authors is about plasticity: i.e. individuals within a species may vary their RM, depending on circumstances. One example I’ve observed is the giant tree frog Boana boans. These frogs generally construct nests, as basins in gravel or sand (character 6 above), just beyond the edge of streams. However, where there is no suitable ‘beach’, the eggs are deposited at the water surface amongst emergent vegetation.

I’m sure that this new RM classification will stimulate discussion and research, and that later versions will include more species and modes. The authors hope that their work will stimulate the development of RM classifications for other taxa: how about reptiles?

References

Breder and Rosen (1966). Modes of Reproduction in Fishes. Natural History Press, New York.

Duellman and Trueb (1986). Biology of Amphibians. Johns Hopkins University Press, Maryland.

Haddad and Prado (2005). Reproductive modes in frogs and their unexpected diversity in the Atlantic forest of Brazil. Bioscience 55, 207-217.

Nunes-de-Almeida et al. (2021). A revised classification of the amphibian reproductive modes. Salamandra 57, 413-427.

Salthe and Duellman (1973). Quantitative constraints associated with reproductive modes in anurans. Pp 229-249 in: Vial (ed.) Evolutionary biology of the anurans. University of Missouri Press, Columbia.

Croaking Science: Caecilians – unusual reproductive ecology

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Croaking Science: Caecilians – unusual reproductive ecology

Caecilians, or blind snakes, as they are also often called, are limbless, elongate amphibians which inhabit tropical regions, with hotspots in South America and Asia (Kupfer et al., 2016; Gomes et al., 2012). Of all the groups of amphibians, caecilians are the most poorly understood, which is in part due to their secretive, fossorial lifestyle (Figure 1). They belong to the order Gymnophiona, one of three orders which comprises the Amphibia. There are currently 209 recognised species, which represents just 3% of the total 7,950 amphibian species worldwide (AmphibiaWeb, 2018). Caecilians are all ground-dwelling tropical amphibians which exhibit a wide range of reproductive modes, from laying eggs which hatch into aquatic larvae to those which hatch into miniature terrestrial juveniles. Little is known of their diverse reproductive modes but in recent years research has elucidated more information on their unusual breeding ecology.

Figure 1. Caecilians have an unusual appearance, being primarily fossorial and elongate in form. [Photo credit: Will Brown – Brown-snouted Blind Snake (Ramphotyphlops wiedii or nirgrogrescens) 3, CC BY 2.0,https://commons.wikimedia.org/w/index.php?curid=71399151]
Oviparous caecilians lay eggs, which are usually laid in a small terrestrial nest (Figure 2). The females of all these species exhibit extensive parental care, guarding their clutches of eggs against predators (Gomes et al., 2012). Eggs will hatch into either free-swimming larvae which develop in small water bodies, or terrestrial juveniles which are independent of their parents. Ichthyophis kohtaoensis inhabits the tropical forests of northern Thailand and belongs to the second largest caecilian family. In this species, breeding coincides with the rainy season with clutches of eggs laid in damp soil at the onset of the first rains (Kupfer et al., 2004). Females lay their eggs in small chambers near to temporary and permanent ponds, pools and slow flowing brooks and rivers. This allows the free-swimming larvae to readily hatch into the water to complete their aquatic development. Research suggests that females eat little or no food during the three month incubation period since the body condition of females attending young clutches is higher than those with older clutches (Kupfer et al., 2004). This, along with the small clutch size, indicates that there are costs to parental investment and attending eggs. In addition, there is a correlation between female body length and clutch size with older females being longer than young females (Kupfer et al., 2004). This implies that older, more experienced females produce larger clutches and more offspring (Kupfer et al., 2004).

Figure 2. A female caecilian guarding her brood of eggs from predators. [Photo credit: Davidvraju, Creative Commons Attribution-Share Alike 4.0 International, https://commons.wikimedia.org/wiki/File:Caecilian_guarding_its_eggs.jpg]
The ringed caecilian, Siphonops annulatus, is an unusual oviparous species inhabiting South America. The eggs hatch into poorly developed terrestrial young which feed on the skin of the female. The young possess specialised teeth which allow them to scrape skin from their mother’s back immediately after hatching (Wilkinson et al., 2008). The feeding behaviour of the young is described as being “quite frenetic with the young frequently tearing pieces of skin by spinning along their long axes and sometimes struggling over the same piece of skin” (Wilkinson et al., 2008) (Figure 3). This maternal dermatophagy is also known from a second, distantly related African caecilian species, Boulengerula taitana (Kupfer et al., 2008), which suggests that skin feeding is an ancient form of caecilian parental care and may have persisted for more than 100 million years (Wilkinson et al., 2008). In addition, Wilkinson et al. (2008) report a previously undocumented behaviour which involves the female raising herself upward, exposing her vent and releasing a clear fluid, which the young consume. This appears to be a form of feeding the young, but further studies are required to ascertain the function of this behaviour.

Figure 3. A caecilian mother with eggs which will hatch into young which feed off her skin. [Photo credit: Wilkinson M, Sherratt E, Starace F, Gower DJ (2013)]
Viviparous caecilians, which give birth to live young, represent another unusual reproductive mode for amphibians. During development inside the female, the foetuses initially obtain their nutrition from a yolk, similar to egg-developing species. After this, the developing young feed on a uterine milk, secreted from the oviducts of the female. The young obtain this through possessing specialised embryonic teeth which they use to scrape at the oviduct wall (Gomes et al., 2012). The evolution of viviparity in caecilians is poorly understood, but since both viviparous and oviparous species possess specialised teeth for scraping (either skin or oviduct milk), this suggests that all these species evolved from a common, oviparous ancestor (Kupfer et al., 2006). Gegeneophis seshachari is the only known species of viviparous caecilian from the Indo-Seychellean region (Gower et al., 2008). In this area the rainy season is short and unpredictable such that laying eggs which hatch into offspring that rely on the presence of water or a damp environment may be highly risky. Water bodies may dry up rapidly before larvae can fully develop. Therefore, Gower et al. (2008) propose that viviparity may have evolved in this species in response to short, unpredictable rainy seasons and the lack of suitable locations to lay and incubate their eggs.

Caecilians possess interesting and unusual reproductive modes, much of which is poorly understood. In addition, many of these species are threatened or Data Deficient and in regions which are undergoing large habitat loss and fragmentation. More primary data are required to fully understand caecilian reproductive ecology and evolution, which will help inform much needed improvements in conservation assessments for these species.

 

References

AmphibiaWeb (2018) https://amphibiaweb.org. University of California, Berkeley, CA, USA. Accessed 15 Nov 2018.

Gomes, A.D., Moreira, R.G., Navas, C.A., Antoniazzi, M.M. and Jared, C. (2012). Review of the reproductive biology of caecilians (Amphibia, Gymnophiona). South American Journal of Herpetology, 7 (3): 191-202.

Gower, D.J., Giri, V., Dharne, M.S. and Shouche, Y.S. (2008) Frequency of independent origins of viviparity among caecilians (Gymnophiona): evidence from the first ‘live-bearing’ Asian amphibian. Journal of Evolutionary Biology, 21: 1220–1226.

Kupfer, A.K., Nabhitabhata, J. and Himstedt, W. (2004) Reproductive ecology of female caecilian amphibians (genus Ichthyophis): a baseline study. Biological Journal of the Linnean Society, 83: 207-217.

Kupfer, A., Müller, H., Antoniazzi, M.M., Jared, C., Greven, H., Nussbaum, R.A. and Wilkinson, M. (2006) Parental investment by skin feeding in a caecilian amphibian. Nature, 440 (13 April 2006), doi:10.1038/nature04403.

Kupfer, A., Wilkinson, M., Gower, D.J. Müller, H. and Jehle, R. (2008) Care and parentage in a skin-feeding caecilian amphibian. Journal of Experimental Biology, 309A: 460–467.

Kupfer, A., Maxwell, E., Reinhard, S. and Kuehnel, S. (2016) The evolution of parental investment in caecilian amphibians: a comparative approach. Biological Journal of the Linnean Society, 119: 4–14.

Wilkinson, M., Kupfer, A., Marques-Porto, R., Jeffkins, H., Antoniazi, M.M. and Jared, C. (2008) One hundred million years of skin feeding? Extended parental care in a Neotropical caecilian (Amphibia: Gymnophiona). Biological Letters, 4: 358–361.

doi:10.1098/rsbl.2008.0217