Mexican cavefish (Astyanax mexicanus): a non-model organism research story with Dr. Claudia Patricia Ornelas-García

Recently, Brent Foster and I published an article on non-model organism (NMO) research, where we interviewed several researchers working across the globe on the challenges, rewards, and the particular questions they investigate while working on (relatively) little-studied organisms. Although we aimed to provide a bird’s-eye view of NMO research in a pot pourri-style article, we found that they each of our interviewees had many interesting things to say that we thought it was a shame not to share with a wider audience. For this reason, we decided to publish the full interviews as well, which we will be doing over the next several weeks.

This interview is with Dr. Claudia Patricia Ornelas-García, investigator at the National Autonomous University of Mexico (Universidad Nacional Autónoma de México, UNAM), who works on the Mexican cavefish (Astyanax mexicanus) and other freshwater fish, looking at their systematics and speciation. The full transcript of the interview is below:

Can you summarise your work in a few sentences?

I work mainly with the systematics and speciation mechanisms in freshwater fish species. Since my PhD, I have worked with Astyanax, a very interesting genus because it is distributed from Argentina [all the way] to the Mexico-US border. During my PhD we reconstructed the evolutionary history of the genus in a particular region identified as Mesoamerica. But in general terms, what we were doing in that project was to analyse four molecular markers, three mitochondrial and one nuclear, to recover the systematics of the group. During the development of the project, I was very interested in the lacustrine systems of Central American Lakes in Nicaragua and Mexico. In these lacustrine systems there are a pair of morpho-species that were sympatric and we were very interested in the correlation between the morphology and the genetic differentiation, because when we were doing this systematic group, they at least seemed to be sharing some haplotypes in the mitochondria. We continued working on the ecological divergence, ecomorphological divergence, and some morphometrics in this pair of species, and more recently with RadSeq Data.

Years later, when I came to Mexico and established my own lab, we started working with cavefish. Actually, when I started, my first job was in Querétaro, very close to the caves in San Luis Potosí. I started working on the caves and I fell in love immediately because the environment is so amazing. It’s very particular. And the system is also very interesting. As you know, there are a lot of genomic resources available nowadays. In the beginning, when I started working with this group, there were maybe 10 or 11 cave populations already analysed from [a] phylogenetic perspective, but not from an evolutionary or developmental perspective. So in the beginning, I just wanted to include as many caves as we could, so we could test these hypotheses of how many times the fish has been able to adapt to the caves. There were several hypotheses, some of them say that it happened only once and there has been a lot of drift. The other says that there’s two independent lineages. I’m in that group that would suggest that there are two independent lineages that came to the caves and adapted to them. Actually, in our most recent paper, by a master’s student of mine, we assess this question, using not only the complete genomes that are already available, but also including some caves that were never analysed before. So we have a very, very exhaustive sampling. And in our results, we have at least three independent colonisation events of the caves, which for some is crazy, it’s not possible. But from our point of view, we are really relying on exhaustive sampling of the caves, and that is what we’re suggesting.

Nowadays, we are starting to move to some developmental analyses, because we were able to capture some fish from the caves, and we are reproducing them here in the lab. So far, we have been able to reproduce five different populations, different from the common ones like Pachón or Tinaja. We are reproducing Escondido, Arroyo, Tigre, Chica, and Pichijumo. Sabinos is common, but it’s less common than others. So far, we are trying to characterise some developmental features.

One of my students is working on the Rad-seq, and we are starting to work with RNA-seq, and we are a trying to compare the genetic convergences across different cavefish lineages, particularly including some less studied populations such as Escondido,  which is from the [second] linage in the Guatemala region. We are trying to compare the differences during early development because we have realised that they have a very particular mutant in some visual pigments, so we are trying to match that variation with the phenotype. We are also exploring the phenotypic convergence with other cavefish in Mexico, like Prietella phreatophila, a catfish in the northern part of Mexico, and also with other families of cavefishes from the southern part of Mexico. We are trying to investigate them for convergences in phenotype, particularly in some processes related with asymmetry that has been reported in Astyanax and in P. phreatophila, and we are trying to check out if it’s consistent across cavefish living in Mexico or only in these two cavefish.

What kinds of asymmetry is this?

Astyanax is very interesting because in Pachón there has been described a directional asymmetry, as well as in Ictalurids [such as Prietella], and actually it’s in the same side, left turn of the head. Particularly in a cave called Chica there is one very well characterised Astyanax hybrid population (between the surface [fish] and the cavefish). In that population there is fluctuation asymmetry, [toward the] left or right, [similar to] chiclids, [that have] fluctuating asymmetry in the mouth of a scales-eating species. It’s interesting that when you analyse it in the hybrid population, the asymmetry is different in comparison to the other caves.

I understand that there are a lot more resources for you to study these types of species now. But obviously, it is generally a group of species that is not studied by an enormous number of people around the world. Are there particular challenges associated with that?

Yeah, I think one of the challenges is that there are a very restricted number of people working with these [species]. And in a way, it’s fascinating, because you will find something new for sure. But [from another point of view], in research groups [studying established organisms] it is easier.

We were trying to characterise the microbiome of the fish, we have a paper on that. And it was a little challenging, because there was not a lot of information already published on protocols or how to treat the data. Or when we are trying to set up [experiments], for example, for physiology or for another kind of ecological analysis, it’s sometimes difficult. But in a way, I think it’s very, very interesting. During my Bachelor’s, I was working with mice, the typical model in the lab, and somehow I think that the number of questions sometimes can be very restricted because there’s already so many studies in these animals, that it’s difficult to come up with something new.

You did touch upon this when you’re describing your work, but was there a specific thing that convinced you that these fish are what you want to work on?

I think the main reason was a because I have always been interested in mechanisms driving the evolution of morphology. When I started working on the lacustrine forms, it was like “Why do they have these different teeth, or different heads, or different body shapes?” and in the caves it’s dramatic, the change is impressive. Are these important morphological changes? When I started investigating the environment, it’s fascinating that they can survive under those conditions. That was one of the reasons.

Another important thing to highlight in Astyanax is that we have the Annual Meeting. It is very interesting because there are a lot of young people, together with senior researchers, and the community is very open. Because it’s not a model organism, [everyone is] really [willing] to talk about the system in a very open way, and include new researchers. Particularly for me, when I was finishing my PhD, this was a very dramatic point, because I saw a potential in the system that I can be included. And I have a lot of things in my favour, I am from Mexico, I can work in the field, I can do a lot of in situ experiments. Even nowadays, there are very few Mexicans working with Astyanax. It just happened that there were a lot of things that made me realise that there was a lot of potential in the [Astyanax] system for me.

Would you say there’s a particular question in your field that you find really fascinating? Or a finding or a result that you really weren’t expecting?

Yeah, as a systematic biologist, I have a very, very important question for me, which is how many species [the cavefish are]. This is an important question from two different perspectives. One is conservation. The other is from an evolutionary point of view, because for sure, they can hybridise, even lineages between the two branches can hybridise. So in a way, it’s very difficult to test for the biological concept of a species. Sometimes it’s difficult even for the developmental biologists, because they are really trying to understand the link between the gene and the morphology. We are trying to understand the mechanisms giving rise to this kind of systems, and how easy it is to speciate in this context. So the implicit question regarding these ecomorphs or ecotypes is, are they different species? And how significant is this for the evolutionary history of the model?

So in a way, it was very interesting for me at the beginning, because when I started going to these conferences on Astyanax, it was very easy for me to always think about the systematics, about the phylogeny. And sometimes [other groups] were interpreting some variation in the morphology through local adaptation. But actually, it’s related to the [evolutionary] history of the system, like the number of vertebrae, or various characteristics, actually, they were shared by ancestry, not by local adaptation. So I think that’s very, very important in these kinds of studies, trying to link between the genetics and the morphology. That’s one of the reasons I want to have a very good notion of how many times this [cave dwelling] model has evolved, because if it [evolved] only once, the interpretation of repeated evolution, or whatever we are asking, is different. The other question that is very fascinating for me is how their morphology can be so divergent, even under gene flow circumstances. How is this possible without any barriers [between the putative species]? That’s the reason we work with Astyanax.

How does data analysis and sharing between labs that use Astyanax and related species differ, in your opinion, compared to studies that use more established model organisms? You said, it’s a fairly open community. But you know, are there any major differences?

I must admit that I don’t know if I have enough experience on this question. All of my experience is with Astyanax. Even though during my Bachelor’s I was working with mice, I’m not familiar with other groups. In the lab where I was working, they were very specific in the questions that they were [asking], and it was not very easy to share information with other labs.

In my opinion, one of the things that the Astyanax model has is that [researchers working on it] are very open. For example, when we were trying to reproduce a fish, we were obtaining a lot of information [from other groups].

From what I know about Astyanax, that there are labs that work on it in the context of heart regeneration, because some have a non-regenerative heart, in contrast to the regenerative zebrafish heart. How large is the community that works on the more phylogenetic aspect of the species compared to those working on developmental or regenerative questions and is there any crosstalk?

Nowadays, a lot of people are trying to [investigate] this model [from] the eco-evo-devo perspective. They have realised [that it is important to distinguish between the different Astyanax lineages], because some of the results that they get are related with the lineage, and not with particularly with the environment. In these terms, there is a growing number of labs wanting to work in the field, know more about the ecology in situ, learn more about the behaviour, the physiological adaptations. Some are is trying to [replicate] experiments in the field, check if the same thing happens in the lab versus in natural conditions. In my opinion, the species gives a very particular opportunity, because it’s not like in other places where it’s very difficult to get access to the cave systems in the in the field. So nowadays, even though regulations are increasing, they are attainable. [Researchers] can [request] permits and get them in a year, which a reasonable time. So it’s more a question of what the interests of the researchers are, because they can really put their questions in different contexts, and navigate between eco, evo, and devo.

Is there anything else that you would like to add?

The problem with non-model organisms is the conservation situation. For sure, nobody will catch Mus musculus from the field, they already have so many reproductive lines in captivity that they don’t have to. The non-model organisms are in the opposite situation. Most of the labs [working on them] want to have more wild lines, more related with what is really happening in the field. And if you have 200 labs working [on cavefish], imagine the impact that we can have on the natural population. These cavefish are not really large [populations]. We published, just at the beginning of 2023, a paper [on] size estimation of the fish population in the caves, and it’s maybe around 1000 fish, or [a few thousands] of fish. It’s not really that large [a] number. And imagine, in the last 10 years, more or less, there have been around 200 fish extracted from the caves. So if you imagine a system that has to recover from 20% of the population being lost only because of scientific sampling, it’s a problematic situation.

When you try to make researchers aware of the situation, [they] really believe that the main extinction drivers of this kind of population are not related with our sampling. Most of us really believe that it’s all global warming, or local people extracting water for drink. I’m very surprised, because normally you have to fight this kind of attitude outside the scientific [world]. So in my position, I’m trying to make people aware of this, but it’s not easy. But none of the above is false. Global warming, and people extracting water for drink, are part of the problem. Thus, it’s important to be part of the solution, and maybe we should consider what we can do to solve the conservation situation.

Some of the most iconic populations, like Pachón, you can find thousands of papers published on it. And it’s in a little bit of a critical conservation situation right now. Many labs have been able to reproduce it. And most [subpopulations] can be easily [obtained] from [other] labs. But even nowadays, to avoid inbreeding, some labs could require to collect wild fish. The Guatemala region has conservation issues, because although they are not easily-accessible caves, most populations are very small, and not well-connected between them, because of the phreatic level of the water, which can lead to extinction more due to their demography than to other causes. So its conservation situation is very different. But in a way, we have the same situation in other places.

And it’s not only in Astyanax, it could happen in other non-model organisms too. For example, Axolotl is also an amazing model system. If 50 labs in the world start working with one particular population, there can really be an impact in the local population. That is why I think we need to be more aware of the impact and do our best to guarantee the prevalence of this model for future generations. [Because] what makes these organisms amazing also makes them vulnerable, in a way.

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