Two or one species? - A 134 year old snakehead story

For some reason I find it interesting when studies aim to sort out difficult problems such as distinguishing different species or resolving the evolutionary history of species, in particular vertebrate species. Therefore I happened to stumble across this study published 24 of June this year PLoS One. They looked at two species of fish within a family called snakeheads Channidae. These fish have a long slender body for fast stealthy attacks sort of like the eurasian and north american Pike (genus Esox) species. One could assume that these fish occupy the same types of niches in the subtropical to tropical waters of africa and south eastern asia. The family consist of two extant genuses Channa (Asia) and Parachanna (Africa). Several of the species of the Channa genus are very difficult to distinguish from each other and theres probably some undescribed species swimming around the rivers of south east asia. 

In the Thailand, Malaysia, Laos, Cambodia and Vietnam region on the mainland and parts of Sumatra, Borneo and Java theres a species named Channa micropeltes (giant snakehead). The giant snakehead is a large fish, hence its name, reaching a maximum size of about one meter. When young they have a nice red stripe along their lateral sides of the body giving them a pleasing look sadly attracting buyers in petshops around the globe not knowing their adult size. In southern india one find a quite similar species, differing at a few morphological points, called Channa diplogramma (Malabar snakehead). The overall look of the juveniles and the adults bears striking similarities to the giant snakehead which have spawned a debate on wether or not they indeed are two separate species or not. The author of the C. diplogramma species,  Francis Day, did actually 13 years after its description in 1878 synonymise it with C. micropeltes possibly due to these similarities. Now 134 years later Benzinger et al. set out to sort this mystery out.

Channa diplogramma picture from wikipedia

In this paper Benzinger and colleges uses morphological and molecular data from specimens of these two species to try to sort out the time of divergence and if these are two species or one. They used data from mitochondrial 16S rRNA and cytochrome oxidase I (COI), the same gene they used in the paper on stingray evolution i reported on previously. What they found using the molecular and morphological data was that the two species are indeed closely related to each other. The divergence time between the two species is somewhere between 9.5 to 22 million years ago depending on which calibration point they use in their calculations. They finally conclude that the most possible reason for the divergence is a separation of the two populations that later lead to the two separate species.

I think this article sort of highlights how modern methods can resolve old problems, and now 134 years later Francis Day's snakehead species from India finally can be unsynonymised (if thats even a word).

Benziger, A., Philip, S., Raghavan, R., Anvar Ali, P., Sukumaran, M., Tharian, J., Dahanukar, N., Baby, F., Peter, R., Devi, K., Radhakrishnan, K., Haniffa, M., Britz, R., & Antunes, A. (2011). Unraveling a 146 Years Old Taxonomic Puzzle: Validation of Malabar Snakehead, Species-Status and Its Relevance for Channid Systematics and Evolution PLoS ONE, 6 (6) DOI: 10.1371/journal.pone.0021272

Nociception and pain in fish, a tough question part I

This thursday the Swedish Centre for Animal Welfare (SCAW) at the Swedish University of Agricultural Sciences published a report dealing with wether or not fish can feel pain and if they can suffer. This is getting more and more relevant as more people become aware of what they eat and do not want to cause unnecessary suffering to the animal being put on the dinner table. In a few blog post starting from this i will discuss the different articles cited in this report about fish welfare. Sadly its only available in swedish but if you know swedish heres the link: http://www.slu.se/Documents/externwebben/centrumbildningar-projekt/scaw/Fiskkonferens/Kan-fiskar-kanna-smarta-och-eller-uppleva-lidande.pdf

The authors of the report discuss several reviews and articles some of them giving contradictory conclusions in this question. Nociceptors seem to be present in some invertebrates and most if not all vertebrates. They are important instruments in sensing and reacting to various harmful stimuli. Pain on the other hand is the "feeling" we often refer to in our daily life. The feeling of pain is processed in the neocortex in humans. Fish which lacks the layered cortex present in mammals are according to some researchers not able to have the "feeling" of pain. This would mean that they would just react to a painful stimuli and not learn to avoid it since it would be more of a reflex response rather than a response that have been processed in the higher brain centers. Is this the case for fish, do they only react by a reflex to a harmful stimuli or do they process such stimuli in the brain and perceive the feeling of pain? 

The authors of the report give several examples of different studies on this and one of them caught my attention. This was a study done by Dunlop & Laming in 2005 showing an activation of several brain areas including telencephalon in both goldfish and rainbow trout as response to given mechanoceptive and nociceptive stimuli. One of the criteria for determining if a animal feel pain is the presence of receptor cells that link to forebrain areas, a criteria which would be fulfilled if their results is correct. To investigate wether or not the nociceptive cells link to forebrain areas of these two species they removed the bone covering the top of the skull on the fish and inserted electrodes in the spinal cord, cerebellum, tectum and telencephalon. Then they gave both mechanoceptive and nociceptive stimuli on the sides of the fish. They recorded responses in all brain areas included to both types of stimuli. In goldfish the nociceptive stimuli yielded a larger response than the mechanoceptive stimuli in the brain while they did not differ in the rainbow trout.

What their study show is that the response to nociceptive stimuli in these fish are not based solely on reflexes alone since the forebrain, not only the spinal cord, is activated upon stimulation. In telencephalon some of the structures suggested to be homologous to hippocampus and amygdala of tetrapod brains are located, which would imply a processing of these stimuli in the brain to in the future avoid these harmful experiences. Other studies have shown that fish seem to remember and do not bite a fishing hook a second time other than if it is food deprived. Then it might be the only way for the fish to find food at the moment and they have to bite the hook a second or third time. Taken together this data would suggest at least that fish are somewhat able to "feel" pain and remember it to avoid being in this situation in the future.

In my next blog post I will continue to discuss fish welfare, and focus a bit of stress and how that affect fish during sport fishing.

References:

Dunlop R, & Laming P (2005). Mechanoreceptive and nociceptive responses in the central nervous system of goldfish (Carassius auratus) and trout (Oncorhynchus mykiss). The journal of pain : official journal of the American Pain Society, 6 (9), 561-8 PMID: 16139775

Freshwater stingray facts part II: Speciation in realtime?

In my previous blog post i touched upon the occurrence of hybridizations between the different species of the genus Potamotrygon, which suggests a close relationship between the species. Most of the around 18 described* species in this genus show a large range of coloration patterns. Giving a example, there are several variants of P. motoro (just searching on Google for pictures reveal the range). there are everything from plain motoro rays with a dark brown background color and orange spots to the marbled variants with all their amazing patterns. Given that the other species in the genus also show this polychromatic appearance make it difficult sometimes to draw lines between the different species. This makes it important to use new tools to be able to establish the evolutionary origins and relationships between the different species in the genus and also within the Potamotrygonidae family.  

Figure 1. My old pair of P. motoro with the male showing courting behavior.  This pair was wild caught, the male was from Colombia and the female from Peru. 

The Potamotrygonidae family is the only family of batoids that all included species are fully freshwater. The closest extant relatives of Potamotrygonidae rays are sea living pacific stingrays of the genus Himantura. Therefore the proposed origin of this family is that a group of Himantura rays from the pacific got landlocked and ended up in what now is the amazon area of south america. Another hypothesis is that a group of these rays wandered into freshwater from the north down into the amazon area at the time before the emergence of the isthmus of Panama. 

A study by Daniel Toffoli and colleges published 2008 used a method called DNA barcoding to resolve the evolutionary relationship between 10 of the described species of Potamotrygonidae stingrays with a Himantura ray, another ray and a shark as outgroups. The point of this method is to in a quick and easy way be able to identify what species of an organism you are dealing with when the morphological data are conflicting. They wanted to test if this method would work to distinguish these species from one snother. They extracted, amplified and sequenced a gene called mitochondrial cytochrome oxidase I (COI) from several specimens of several different localities from each species. They used the sequences to produce phylogenetic trees using three different methods, Neighbour Joining, Maximum Likelihood and Bayesian Likelihood. 

Using these methods the authors show that it is, using this gene, impossible to sort out the interspecies relationship in this family other than that what they call the rosett spot clade is a well supported clade of more closely related species. The trees also show that Plesiotrygon iwamae, P. shroederi, P. sp 1, P. henlei, P. falkneri, P. leopoldi and P. cf. motoro all seem to form distinct monophyletic species using samples from the different locations. The three other species form all together a clade where it is hard to determine the species of each individual from molecular data alone, ie. they seem to share haplotype in this gene. Thus the data seem to show P. motoro as a paraphyletic species as seen in the simplified sketch below(figure 2). 

Figure 2. A simplified sketch of the ML and NJ trees presented in the article. The Bayesian  likelihood tree show a similar topology, some nodes that are collapsed in the tree above have stronger support in the BL tree. P. henlei are located on either branch depending on the method used as seen in the figure. The dotted NJ branches do not have enough bootstrap support in the other analyses, but present in the NJ tree with high support. P. motoro is shown two times because they are from different localities and it shows that this species seem to be paraphyletic when one look at this gene locus. (after Toffoli et al. 2007)

There could be many reasons for this strange appearance in the tree where a individual of one species seem to be closer genetically to another species than to its own kind. One possible reason is a poor identification of the individuals, however P. scobina and P. orbignyi differ quite much from P. motoro in colours and patterns so this seem unlikely. A second possibility is that this gene is so well conserved in this linage so that there isn't enough informative sites to produce a reliable phylogenetic tree. The third option is that the divergence of these three species is so recent in time that they haven't yet completely formed their own distinct haplotype. In this scenario P. motoro could be considered the ancestral species of the other two as the authors suggests. This is an intriguing scenario are we actually looking at speciation while it is taking place in realtime? A final scenario is that the Potamotrygon genus actually represents the full spectra of a highly polychromatic species, however this may be the least parsimonious explanation . 


This paper leads you to very interesting questions. To fully resolve these questions one would have to look at more genes and possible more individuals (as the authors also say) to fully confirm the reason for this tree topology. If this is the case, that P. motoro is a species which are undergoing a divergence into the three species, this could explain the possible hybridization of the different species that are known to occur in aquariums. 

*It remains a few species waiting to be described!


References:

Toffoli, Daniel, Tomas Hrbek, Maria Lúcia Góes De Araújo, Maurício Pinto De Almeida, Patricia Charvet-Almeida, and Izeni Pires Farias. “A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes).” Genetics and Molecular Biology 31, no. 1 (2008): 324-336. 

Freshwater stingray facts: Different chromosome number between males and females in two Potamotrygon species (P. aff. motoro and P. falkneri)

One of my main interests when it comes to fish are the freshwater stingrays of south america. A few years ago I had two adult P. motoro rays and a adult of a unidentified species, both from south america. The pair of motoro rays were one female and one male and they had baby stingrays a few times during their time in my posession. The baby i had for the longest was a female. On the outher appearance of stingrays and other Chondrichthyes it is very simple to distinguish between male and females, males have one clasper on each pelvic fin and females have nothing but their normal fins. However what cytological differences underlie the sexes of the freshwater stingrays of south america?

My two old adult P. motoro rays with the male in the background.

This question Vanessa Paes da Cruz and colleages answered in a paper published Neotropical Ichthyology march 31 this year. They collected 30 P. aff. motoro and 34 P. falkneri specimens from different rivers in Brazil.

They found that in both species females have a diploid number of 66 chromosomes while males have 65 chromosomes. This is interesting, males have an uneven number of chromosomes! When they looked further into what chromosomes that differed between males and females it appeared to be the sex chromosomes that differed. They found that the sex chromosome system in these two species consisted of this arrangement: X1X1X2X2/X1X2Y. Possible differences in number of chromosomes have previously been proposed in other Potamotrygon species and the reason for this difference they attribute to a ancestral system similar to the one seen in humans XX/XY where the Y once during evolution have fused to an autosomal chromosome. This then resulted in a new Y chromosome and the X2 chromosome. This later resulting in the "new" X1X1X2X2/X1X2Y system seen in these two species. 

One thing that came into my mind while reading this article was if this system also could be the result of a hybridization of two Potamotrygon species a long time ago. Many of the species of the Potamotrygon genus have to my knowledge been known to hybridize in aquarium. However the reason for this arrangement it is certainly interesting!

/Simma lungt!

Ref.

Vanessa Paes da Cruz, Cristiane Kioko Shimabukuro-Dias, Claudio Oliveira and Fausto Foresti, (2011), Karyotype description and evidence of multiple sex chromosome system X1X1X2X2/X1X2Y in Potamotrygon aff. motoro and P. falkneri (Chondrichthyes: Potamotrygonidae) in the upper Paraná River basin, Brazil., Neotropical Ichthyology, 9(1):201-208

Animal of the week: Zebrafish (Danio rerio)

"-The zebrafish a model organism on the rise"

This is one of the most common introductions in scientific articles relating to zebrafish research for the last 20 years. But what is a zebrafish?

The zebrafish is a small, 2-4 cm, cyprinid fish from the genus Danio. It has its origin in the southeastern himalaya region in asia with its main distribution in India. It has five blue horizontal stripes running laterally on its body hence the name zebrafish.

Zebrafish are omnivorous and forms small shoals of 6-10 individuals. They do not take care of their offspring and they spawn in the middle of the water column during the early morning. The eggs fall down to the bottom and lay in the substrate for a few days until they hatch. The first few days after hatching the young zebrafish are attached to stones and other things on the bottom substrate using a few specialized cells on the head. The stick to the bottom until they have consumed the yolk sac. When its consumed they let go of the substrate and make their way to the surface to engulf a small amount of air to fill their swim bladder. When thats done they swim around up in the water column feeding on various small crustaceans and other small creatures until they are large enough to spawn, after apporx. 3 months. 

The short generation time of about 3-4 months is one of the reasons for its popularity in scientific research. This mean that it is quick and rather easy to create different strains and mutants for research. Another reason is that the zebrafish have had its genome sequenced, so molecular studies on zebrafish is easy.