Evolutionary Morphology: Rostock Scientists Clarify Phylogenetic Relationships within the Most Diverse Taxon, Arthropoda

February 06, 2014

With over a million described species arthropods, which include spiders, millipedes, crustaceans and insects, are the most diverse taxon on earth. The total number of species of these segmented armored animals is still unknown and many of them are still undiscovered. Scientists estimate the number of arthropods of a total of ten to thirty million species. Such diversity involves an incredibly wide spectrum of different forms. The research group of Professor Stefan Richter at the Institut für Allgemeine und Spezielle Zoologie, University of Rostock focuses its investigations on the evolutionary origin of biological disparity and diversity. The confocal microscope is an indispensable tool for gaining insights into the interrelatedness of different faunal species. For their research in the field of evolutionary morphology (the evolutionary description of the variety of organisms), Prof. Stefan Richter, Dr. Christian Wirkner and Dr. Martin Fritsch observe, visualize and analyze the wide variety of arthropod organ systems such as the nervous, muscular or circulatory system, using "classic" as well as modern and innovative methods.

"Some of these animals are so tiny, and to investigate the circulatory system, for instance, in greater detail, stained substances are injected directly into the arterie vessel system,” explains Fritsch. "The injected specimens are then analyzed in a micro-computer tomograph, to document and to reconstruct the exact course of the hemolymph vascular system".

The main area of Dr. Martin Fritsch’s research is the evolution of the nervous system of Branchiopoda (freshwater crustaceans so called because of the gills on their feet). Branchiopods are the most morphological diverse group of crustaceans and up today 1,200 described species are known.

Evolutionary tree of Branchiopoda

Branchiopods are a closely related group in the evolutionary tree and in a non-taxonomic sense they can be divided into large and small branchiopods. Large branchiopods are Anostraca or fairy shrimps, Notostraca also called as tadpole shrimps, Laevicaudata, Spinicaudata and Cyclestherida. The latter three are formerly known as "Conchostraca" or clam shrimps. Small branchiopods are cladocerans or water fleas. In contrast to the other branchiopods, cladocerans are much smaller and range from 200 µm to a couple of millimeters, some of them might even reach one centimeter in size. The large branchiopods can reach up to ten centimeters in size.  

Fig 1: Larva of the species <i>Ceanestheria</i> sp. (Crustacea, Branchiopoda, Spinicaudata) with stained nervous system (red), neurotransmitters (green), cell nuclei (cyan), cuticle (autofluorescence in blue).

Tracing the ancestry of Cladocera by examining the nervous system

In his dissertation project, Fritsch tried to trace the evolutionary origin of Cladocera. The evolution of water fleas is one of the most fascinating issues in the study of branchiopods. One possible explanation for the origin of the water flea was proposed by Carl Claus in 1876. Claus´ suggests that the animals evolved from a precociously mature larva of a clam shrimp-like ancestor. According to this theory, the ancestors would be able to reproduce sexually in an early developmental stage and every other form of differentiation would be stopped. This process is also known as progenesis. To testify this process it is vital to look at the development of branchiopods. Within the Branchiopoda we find two different developmental strategies. On the one hand, the sexual reproduction of Anostraca, Notostraca, Laevicaudata and Spinicaudata which leads to gamogenetic reproduced resting eggs and hatching of free-swimming and feeding larvae with three pairs of extremities. Cyclestherida and Cladocera, on the other hand, reproduce mainly asexually, (parthenogenetically) producing so-called subitaneous eggs and hatching of non free-swimming embryo-like larvae that develop under the protective carapace of the mother animal.   

On the basis of this theory and the different developmental strategies, Fritsch investigated the development of the nervous system and the external morphology in all the branchiopod taxa. He examined adult animals, larvae and different embryonic stages; this was an extremely laborious process. Fritsch documented the external morphology of the animals with electron microscopy which gave him a detailed view of changes during the growth process. To analyze the development of the nervous system, he used immunohistochemical techniques. "I labeled the nervous system with immunohistochemical markers and then documented the results with the confocal microscope," explains Fritsch. The size of the investigated animals ranges from 250–1,000 µm, perfect for confocal microscopy approaches.

Fig. 2: Penilia avirostris (Crustacea, Branchiopoda, Cladocera) combined confocal microscope image of the ventral nerve cord (yellow) and the external morphology (cyan). Juvenile stage.

To examine larger animals, however, Dr. Martin Fritsch had to dissect them before putting them under the confocal system. “I did this by embedding large branchiopods in a gelatin mixture and making thin sections with the Leica VT1000S vibratome. I have had excellent results with this procedure,” the biologist reports. As a comparative method, the Rostock scientist additionally applied widefield microscopy. "With confocal microscopy we can label a wide variety of structures and see a lot of detail. However, we sometimes get a better overview of all the cell and organ structures under the widefield microscope. To prepare the specimens, we embed the animals in synthetic resin. Then we produce thousands of thin sections with a microtome, record images of them under a light microscope and perform afterwards a digitalized stack of images. This enables us to create a three-dimensional virtual model that gives us a better understanding of the composition, course and function of structures."

Developmental sequence for each species

The problem with comparing all the data Fritsch had collected was that the individual species undergo different developmental strategies, develop at different rates and also go through different developmental stages. As the developmental stages of Anostraca, Notostraca, Laevicaudata, Spinicaudata, Cyclestherida and Cladocera are not really comparable, therefore, the biologist considered each differentiation of a morphological structure during the development as a single event. An event example would bethe appearance of the brain or the presence of the first trunk extremity. "I coded each event with a letter of the alphabet," the biologist explains. "That way I obtained a developmental sequence for each species that I was then able to compare in detail with each of the others."

Fig. 3: Nervous system of the species Leptestheria dahalacensis (Crustacea, Branchiopoda, Spinicaudata) at the third larval stage (red – nervous system [alpha-tubulin], green – neurotransmitters [serotonin]).

"In the course of my research on the development of the nervous system and the external morphology I have found hints, suggesting that the evolution of the water flea was probably far more complex than previously assumed. I could not confirm the theory that water fleas evolved from a precociously mature larva of a clam shrimp-like ancestor. However, my research has shown that the developmental strategies changed during the course of evolution and the development and maturation phase of Cladocera was shortened. This change led to the typical tiny habitus of the water fleas," explains Fritsch. In view of the amazing biodiversity of branchiopods, he is not likely to run out of work in the future.

Fig. 4: Dr. Martin Fritsch in front of the extensive collection of the Institut für Allgemeine und Spezielle Biologie at the University of Rostock.
Fig. 5: The collection also includes various branchiopod specimens.


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