Following up my article on marsupial frogs, I have acquired a copy of William Duellman’s book, Marsupial Frogs. Gastrotheca and Allied Genera published in 20151. Book hardly does the work justice because it a complete account, based on five decades of research by the author, particularly on their taxonomy and evolution, of this remarkable group of South American amphibians. As well as general chapters on aspects of their biology, each species is described in detail. Duellman began his book as follows:
What are marsupial frogs, and why are they so particularly worthy of study. These are the only frogs in which males fertilize the eggs out of the water and then place them in a pouch on the back of the female. The developing embryos of marsupial frogs and their allies—i.e. the amphibian family Hemiphractidae—have large external bell-shaped or sheet-like gills that are unlike those of any other lineage of frogs. These behavioral and developmental features are unique to marsupial frogs and their allies.
The first point I wanted to check was what he had to say about the identification of frogs exported from South America in the 1950s, presumably through the animal trade. These animals were the basis of early work on reproductive behaviour and the use of the dorsal sac to hold fertilized eggs. As I stated in my previous article, the frogs are now thought not to have been Gastrotheca marsupiata or marsupiatum as they were labelled at the time, but G. riobambae from Ecuador. Duellman in his book repeats the sorting out of this misidentification but also adds that another species, G. pseustes, very similar in appearance to G. riobambae, also occurs in the Andes surrounding Quito; that species may have been also have been involved in the exports and studies.
Getting the species right is important in the these frogs (69 species recognised in the genus Gastrotheca at the time Duellman’s book was written) because the stage of development at which the young emerge from the pouch on the mother’s back differs. In a few species, incidentally, the pouch is on the side and even extends via a slit into the abdominal cavity. Some hemiphractid species, all in genera other than Gastrotheca, have no fully enclosed pouch: some have a dorsal patch to which eggs adhere; others have a basin-like structure while in one genus, the eggs are enclosed but the two edges of the skin flaps forming the cover do not fuse.
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| From Duellman's book |
In species such G. riobambae, G. marsupiata and G. pseustes the tadpoles when they hatch and shed their external gills leave the pouch (assisted by the toes of the female being inserted into the pouch) and continue their feeding, development and metamorphosis in ponds. The tadpoles, it should be noted, hatch at a more advanced stage than in species like the Common Frog. By contrast in some other species of Gastrotheca, the young hatch and emerge as fully-developed froglets; metamorphosis takes place in the egg capsule within the mother.
This is where things get physiologically more interesting. Marsupial frog tadpoles develop large external gills connected to the tadpoles by stalks. The gills have a large surface area and form, by analogy with mammals, the fetal side of the placenta. The brood pouch is formed from skin but internally loses loses much of the structure typical of skin when eggs are in the pouch. The epidermis becomes thin and produces well vascularized extensions that partially envelop the egg. In other words, the skin forms the analogue of the maternal side of the mammalian placenta. Between the two sides is the egg capsule across which any exchange or transfer of materials between mother and egg/tadpole must occur. It is obvious that oxygen must pass from mother to egg/tadpole with carbon dioxide moving in the reverse direction. The form of the nitrogenous waste produced by the tadpoles shows an interesting adaptation. Free-living tadpoles usually resemble fish in that they produce ammonia—they are ammonotelic—which being toxic is swiftly excreted into the surrounding water; at metamorphosis terrestrial amphibians switch to produce urea. However, marsupial frog tadpoles produce urea—they are ureotelic—and not ammonia. Not all the urea though leaves the egg capsule. It has been suggested that the urea could serve some osmotic function (as in the Crab-eating Frog) and preventing the loss of water. This aspect required further study but the inclusion of urea in the culture medium made it possible to keep early embryos of G. riobambae alive after they were removed from the pouch; the usual saline solution was not sufficient.
Much of this work on the developmental biology of marsupial frogs has been done by Eugenia del Pino in Quito2; she has worked extensively on G. riobambae, and the aspects of her research described here only skim the surface of her studies done over the past 50 years on these fascinating frogs and their adaptations. Her research as a developmental biologist therefore complements that of Duellman, centred on evolution and taxonomy, over a similar period of time.
The big question, of course, with marsupial frogs is whether or not the developing tadpoles draw on maternal nutrients to support their growth and development in addition to the nutrients provided at the time of laying in the yolk—the eggs of marsupial frogs are very large and therefore contain a lot of yolk. In the tadpole-producing G. riobombae there is evidence that the developing tadpole relies solely on the yolk until it hatches and leaves the pouch. The weight of dry matter in the egg capsule was found not increase during development. By contrast, a more recent study3 on a froglet-producing species, G. excubitor, has suggested that maternal nutrients could be transferred to the developing young. However, having read the paper and having looked at the supplied raw data I remain unconvinced. The number of animals studied was very small at each stage of development; the embryos were only studied at early stages of development and the results of feeding stable carbon and nitrogen isotopes to prey insects, while indicating that carbon and nitrogen compounds (which could be simple products of metabolism like carbon dioxide or urea) pass from mother to embryo through the egg capsule, are not a demonstration of NET transfer of nutrients.
There do appear to be adaptations associated with producing froglets rather than tadpoles. Duellman provided a diagram showing that froglet-producing species have fewer, larger eggs (thereby providing evidence that the yolk is sufficient for development to froglet); eggs in the pouch are arranged in a single layer such that they can be completely enveloped in maternal blood vessels from both sides. By contrast, tadpole producers have more, smaller eggs, stored as a double layer in the pouch. All these findings are consonant with the increased requirements for oxygen in the later stages of growth and development by the larger tadpoles and metamorphs of the froglet producers.
In conclusion, the question remains—as it does for other frogs in which eggs are held in various body cavities until newly-metamorphosed froglets emerge—do maternal nutrients contribute to the metabolism and growth of the developing tadpole? The simplest way of answering that question would be to weigh the dry matter of the egg capsule and its contents right through the period of development in the pouch, as has been done in the tadpole-producing G. riobambae. We would then know if then know if the egg capsule with its rich supply of blood vessels on both sides acts in a manner more comparable with the mammalian placenta or if it just, as it says on the tin, just a gill. Molecular biologists might want to just have a look at whether or not transporters for glucose and amino acids occur in the egg capsule.
The question of whether the external gill acts as a gill or an analogue of the mammalian placenta takes me full circle to my start of the first article and why Amo was fascinated by marsupial frogs and had a film made of their reproductive behaviour. There is still much of interest to do, with key questions still waiting to be answered, 65 years later.
1. Duellman WE. 2015. Marsupial Frogs. Gastrotheca & Allied Genera. Baltimore: Johns Hopkins University Press.
2. del Pino, EM. 2018. The extraordinary biology and development of marsupial frogs (Hemiphractidae) in comparison with fish, mammals, birds, amphibians and other animals. Mechanisms of Development 154, 2-11.
3. Ware RW, Catenazzi A. 2016. Pouch brooding marsupial frogs transfer nutrients to developing embryos. Biology Letters 12: 20160673. http://dx.doi.org/10.1098/rsbl.2016.0673
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