The Lightning Project

The ongoing saga of the PNG Lightning Maroon Clownfish Breeding Project

Browsing Posts tagged mating

A special thanks goes out to Shane & Len at Advanced Aquarist for co-publishing this blog entry there - it seemed like the perfect contribution given the style of material they like to cover.

The Lightning Project

Just to bring you up to speed, it’s been over 2 years since the “Lightning Maroon” from PNG made it’s way to the US and ultimately into my home tank.  It was a long time coming but in the late spring and early summer of 2012, we finally got a glimmer of success with the first and second spawning between the “Lightning” Maroon, and a normally patterned “wild-type” Maroon clownfish.  The pairing below are the two parents responsible for everything we’re about to cover.

The Lightning Maroon Clownfish and her wild-type mate.

The Lightning Maroon Clownfish and her wild-type mate.

The Results Are In

So not only did we get a spawn that made it, but we got roughly 50 juveniles post-settlement in the very first rearing attempt.  Now, more than 2 weeks in, it seems ever more likely that we have a roughly 50% rate of “Lightning” babies in our group of offspring – note I have yet to do an actual headcount, this is just a ballpark guesstimate on the numbers.  Initially the babies showed up with blue “caps”; thicker headstripes that were readily discernible.  As they progressed, they looked more and more like Picasso Percula babies.  While still possibly premature to say conclusively we have “Lightnings”, we’re definitely starting to see signs that the Lightning trait will come through with defining characteristics that will clearly match up with those that the two original wild Lightning Maroons shared.

17 day old Offspring from the Lightning Maroon & a wild type Maroon from the same island in Papua New Guinae

17 day old Offspring from the Lightning Maroon & a wild type Maroon from the same island in Papua New Guinae

Dismissing the Hybrid Hypothesis

Before going into the genetics discussion, I’m going to address one “possibility” that some creative thinkers might propose, either though just being “creative”, or through having read, misread, or misunderstood what someone has posted on some forum somewhere.  The hypothesis is this; the Lightning Maroon is a different species than a normal white stripe maroon.  And thus, are these offspring “hybrids”?

Categorically I firmly believe no, the Lightning Maroon does not in any way represent a species other than Premnas biaculeatus.  In most hybrid scenarios between two species, the initial primary hybridization generally yields a predictable intermediate form between the two parental species – I am sure there are examples of a primary hybrid where the offspring “range” from one parent to the other, but that is far more common in the second generation if it’s going to happen.  Since we have no intermediate forms in the offspring of this pairing, I believe we can safely rule out the “hybrid” hypothesis without further delay (the same cannot necessarily be said if we look at the “White Stripe” vs. “Gold Stripe” Maroon…the more I read and learn and see…leads me to believe these may in fact be two distinct species in the wild).

Let’s talk Genetics, Breeder Style

I’ll state up front that I’m no geneticist, and that I’ve been known to get my terms confused. So I’ve taken the opportunity to run this by Adeljean Ho (a good friend of Dr. Matthew L. Wittenrich, and the scientist who published work in CORAL that suggests a unique genetic basis for the “Red” form of the Green Mandarin).  Hopefully he caught any errors I may have made in attempting to distill and disseminate these ideas.

Remember, I really downplay the “designer” aspects breeding of marine fish with mutations, but taking on the preservation of this wild trait has forced me to learn it.  Understanding the genetics allows a breeder who is working with “designer” fish to quite literally “create” what he or she envisions; the upshot of this knowledge is that it also levels the playing field for breeders, forcing them to turn back to producing QUALITY fish in order to differentiate themselves.  For me, the emphasis on quality, as driven by “open sourcing” the genetics of a fish, is the best route we can go if we must pursue “designer” variants going forward.

In this discussion of possible Lightning Maroon genetics, here are the important terms. We will try not use the term “gene”, because it kind of gets used interchangeably and thus will probably only confuse. The important terms here are “locus” and “allele”. “Locus” being a specific point in the genetic code where a particular pairing of alleles resides; the alleles being the pieces of genetic information, one from the father, and one from the mother, that come together at the locus to form the genetic makeup of the offspring.

We also cannot neglect the terms genotype and phenotype.  Genotype refers to the genetic “code” specifically, which is important because alleles can be present yet not “expressed” in the phenotype.  Yes, the phenotype is the outward appearance as driven by the genetics.  And this is the conundrum; due to the way certain alleles interact with other alleles, there are traits that can be masked, surpressed, or unexpressed, that is to say you won’t know a fish carries a hidden albino gene in its genotype just by looking at it (and seeing it’s phenotype).

The other important terms to remember are “homozygous” and “heterozygous”; all that really means is whether the two alleles in the loci pair are the SAME genetic code (homozygous, such as A/A or B/B), or different (heterozygous, often abbreviated as “het” for short, such as A/B). Considering the entirety of our genetic makeup, it all boils down to loci (plural of locus) and what pairing of “alleles” is inherited at each locus. Obviously, the outward result of these traits is the result of all these separate loci together, and certainly some observable traits may be governed by multiple loci, which makes it difficult to ascertain the genetics and inheritance behind them. By the same token, the possible individual alleles that can be present at a loci are perhaps infantasimal in their variation (for example, ABCDEFGHIJKLMNOPQRUSTUVWXYZ), but only two individual alleles (eg. A/Z, C/C, or B/Q) will be present in any particular locus.

That said, most all of the genetic variations that we’ve come to openly understand in fish seem to be the result of the genetic makeup of an individual loci, and from there, the combination of multiple traits at different loci  is what gives us a well-understood, massive diversity of ornamental fish varieties (as some would call them, “Designer” fish).  Freshwater Angelfish (Pterophyllum scarlare) make the perfect example as they are well understood genetically (see The Angelfish Society’s Phenotype Library). Combining multiple traits from individual loci is how we get a Pearlscale Lace Clown Veil Angelfish. Those names refer to phenotypes; outwardly discernible traits, in this case those names refer to scale structure + dark gene + stripe genetics + fin length. In the Angelfish breeding community, this would get denoted roughly as (p/p) – (D/+) – (Z/S) – (V/+).  Because we know which alleles at which loci contribute to each end result, in theory any breeder can “make” a Pearlscale Lace Clown Veil Angelfish; the breeder just needs to have the proper parents with the proper genotypes.  The breeder also knows that mating two Pearlscale Lace Clown Veil Angelfish together will result in a plethora of unique genetic combinations, 27 to be exact, all of which have their own name.  One example?  Pearlscale Blushing Superveil Angelfish, (p/p) – (+/+) – (S/S) – (V/V).

I’ll borrow notation from the freshwater Angelfish world to try to lay out the options for the Lightning Maroons, and I’ll propose that “L” will stand for the Lighting allele. “+” will stand for the wild-type, default state allele (aka. a normally striped fish). Thus, a wild fish, without any “Lightning” genetics, would be represented as (+/+). Note that in this notation, capital letters are normally used for dominant or partially dominant traits, whereas recessive traits are generally denoted using lowercase letters.  I’m going to assume right up front that “Lightning” is a trait that directly controls the “striping” of the fish.  We are going to assume here that there are only two possible alleles involved in what we are seeing, and that the Lightning Phenotype is driven by one specific allele (L in our examples) and is not in fact the result of two unique alleles coming together (eg. Lightning = L/X, wild fish being +/+).  We are also going to assume that the Lightning trait is the result of genetics at one locus only. A brazen assumption, but it seems likely at the moment.

To explain the multiple locus issue another way, we are assuming it does not take the genetics of two (or more) loci to result in the Lightning phenotype.  In the Angelfish world, there are  phenotypes like Platinum that are the resultant combination of two independent loci, and the presence of specific recessive alleles in homozygous pairings, that result in the all white Platinum Angelfish – in this case the recessive gold trait on the “dark” locus, and the recessive Philippine blue trait on the “philippine blue” locus. Independently, you’d have a Gold Angelfish, or a Philippine Blue Angelfish, but “activate” both of those recessive traits through breeding choices, and you wind up with the possibility of all white platinum offspring.  Yes, you can “make” a Platinum out of parents that are not outwardly “Platinums” themselves, and that is the beauty of understanding the genetics.  What you cannot do is use only Platinums to breed back to the wild form of an angelfish – and that is the curse of “designer” breeding (which is one reason why designer-focused breeding can get in the way of conservation minded breeding – the “ornamental” genetics can function as actual genetic contaminants…but that’s for another day).

Angelfish Phenotype Examples

Genetics in action - the large fish in the foreground is a wild Angelfish, generally presumed (+/+) unless it carries hidden recessive alleles, and called a "Silver", which is the default striping pattern in Pterophyllum scarlare. The Blue one at right is a "Blue Ghost", representing 2 doses of the Philippine Blue allele, and a single dose of the partialy dominant stripeless allele, so (pb/pb) - (S/+). The white angelfish in the back is a "Platinum", the result of a fish being both homozygous for the recessive Philippine Blue allele, as well as homozygous for the recessive Gold allele, thus (pb/pb) - (g/g).

Based on the quantity of Lightning Maroons in the very first batch of offspring, there are three possibilities for how the Lightning Maroon trait genetically functions.  It could be a recessive trait, whereby there must be two alleles for Lightning present in order for the Lightning pattern to be observed.  Lightning could in fact be a dominant trait, whereby it only takes a single dose of the Lightning allele to mask the normal stripping pattern.  And the Lightning trait could be the result of a partial (incomplete) or even codominant allele, where a double dose fish will look different than a single-dose fish, which is still different from the wild type, normally-barred fish.
The Case and Implications for “Lightning” being a Recessive Trait

So let’s look at inheritance and expression of the genetics in play. We’ll start with the easiest to understand, a recessive trait like albinism (I think we all understand how albinism works on some basic level). Another good example – the recently discovered Philippine Blue gene in Angelfish is thought to be recessive.  A fish with a single dose of this allele (pb/+) shows no real difference with the wild form.  But put on a second dose, and *Bam*, you have a Philippine Blue Angelfish.

A Blue Silver Angelfish

A Blue Silver Angelfish , (pb/pb). The angelfish breeding community is thoroughly convinced that pb is a recessive trait on its own locus.

This angelfish is a "Silver", and happens to be a sibling to the Blue Silver angelfish shown above. There is a 2/3 chance that this fish has a hidden Philippine Blue allele, denoted as (pb/+), otherwise it is wild-type in every known sense, written as (+/+). If these two fish were mated, and none of the offspring developed into blues, that would prove the 1/3 chance of this fish having no hidden blue allele.

If “Lightning” is a recessive trait (one that requires two “doses” of the Lightning allele), then the Lightning parent could only be homozygous (l/l).  A fish that is heterozygous (l/+) would appear “normal”. Thus, if our (l/l) fish is mated to a wild type (normally barred) fish with no Lightning genetics (+/+), all the offspring would be (l/+). Such a pairing would result in 0% discernible Lightning Maroons, as all offspring are (l/+) (Figure 1).

Recessive Lightning to Wild homozygous Mate = all hets = no Lightnings.

Figure 1. Recessive Lightning to Wild homozygous Mate = all hets = no Lightnings.

Thus, if “Lighting” is recessive, we know that the Lightning Maroon must be (l/l). If recessive, to have found Lightning offspring in the first generation mating, that implies that the standard-barred mate must carry a “hidden” Lightning allele, and thus be (l/+) itself. Mating (l/l) to (l/+) would give you a 50% expression rate IF (and that’s a big if) the Lightning trait is recessive. Mathematically, the door is open for this trait to be recessive (Figure 2).

Recessive Lightning to Wild heterozygous Mate = 50% Lightnings.

Figure 2. Recessive Lightning to Wild heterozygous Mate = 50% Lightnings.

Now, there is an upside if this trait is recessive; it means we got lucky. Primarily, it means I got lucky on the selection of the non-Lightning mate, because there would be no way of knowing it carried a single-dose, non-expressed Lightning gene. It would mean that the game plan of using a mate from the same island paid off. If you find a wild albino fish, you are most likely to find more albinos in the same geographic region because they’d probably be siblings. Not to mention that many of the non-albino siblings in the area could potentially carry a single albino gene as well.

The other way we will be lucky is that IF Lightning is recessive, and if the initial percentage is in fact roughly 50%, it would mean that all the siblings would then have to carry a single, non-expressed Lightning allele (because their only option from the Lightning parent is to receive a Lightning allele). This would mean that every fish in the group if mated would produce 25%, 50%, or 100% Lightning Maroons. To put it in a commercial context; if we definitively knew that this was a recessive trait, then even the normally striped offspring would be tremendously valuable to breeders, because simply mating two of those together yeilds 25% Lightning.  In an interesting twist, it seems most people expected the Lighting trait to be recessive if genetic, and assumed that we would get the results shown in Figure 1, and only in the 2nd generation would we get more Lightnings, as shown below (Figure 3).

Recessive Heterozygous F1 Offspring, Mated together, produce 25% Lightnings.

Figure 3.Hypothetically recessive heterozygous F1 Offspring, mated together, produce 25% Lightnings.

Still, I’d love to hope that this trait is recessive because it means all the siblings would then carry a hidden Lightning allele. In looking at the number of wild Lightning Maroons presumably observed (and thus caught), we know of only 2. This rarity could suggest a recessive trait, as two wild fish with hidden Lighting Genes, mating together, would produce 25% Lightnings. Given that a clownfish pair’s minimal reproductive goal is to produce two replacements, you can quickly see how a single pair of clowns, constantly churning out babies that are 25% “Lightnings”, might only yield a handful at best (remember, marine fish have been shown to suffer massive mortality in the earliest hours and days of their lives – most never even make it to settlement, and most of those, not past their first year).  Lightning Maroon babies truly stand out in the rearing tank while their normally patterned siblings are difficult to see; you can’t help but assume Lightning offspring be much easier for predators to locate. So the rare Lightning making it in the wild would fit well with a recessive trait hypothesis.

But what are the odds that I got “lucky” with the mate I selected? Impossible to say, but Occam ’s Razor suggests that the following scenarios could be more likely.

The Dominant Scenarios for “Lightning”

Let’s deal with straight up dominance. If this is a dominant trait, then you only need one “dose of the gene” to express the trait. To simplify, breeders tend to view dominant traits as being pretty uniform in their expression, and there’s no difference whether you have one dose or two. In other words, a Lightning Maroon Clownfish could either be (L/L) or (L/+) and would look the same. A good example of this, to borrow from the Angelfish community, is a trait called “Zebra”, which adds extra bars and patterning in the fins. There’s no visible difference between a homozygous Zebra (Z/Z) or a heterozygous Zebra (Z/+).

A Zebra Angelfish

A young Zebra Angelfish, straight up dominance means this fish could be (Z/+) or (Z/Z) - the only way to know is through planned and controlled matings and observing the results.

Let’s again weigh the options. If Lightning is dominant, then the non-lightning mate can only be (+/+). Why? Because any fish that is (L/+) is going to be Lightning. So in this scenario, the normal mate can only be (+/+). That leaves the Lightning Maroon to be either (L/+), or (L/L). Now, here’s where it gets interesting. If the Lightning Maroon was (L/L), we would have 100% Lightning Maroons in the offspring, because every fish could only get a ( L ) allele from the Lighting Maroon, and all (L/+) offspring would then be Lightning (Figure 4). Since we don’t have 100% Lightnings in the offspring, we can rule out the Lightning Maroon being (L/L) if this is a dominant trait.

Homozygous Lightning Father X Homozygous Wild-type Mother = 100% Lightning offspring

Figure 4. Dominant Homozygous Lightning Mother X Homozygous Wild-type Father = 100% Lightning offspring

That would leave (L/+) as our only genetic option for the Lightning Maroon, which would thus result in a roughly 50% expression rate in the F1 generation. The inheritance of the ( L ) allele from the Lightning parent is a just a coin toss, 50% of the time they get a +, and 50% a L.   Once again, the rules of genetic expression and inheritance suggest that this is a possible genetic explanation given the initial results we’re seeing (Figure 5).

Dominant Heterozygous Lightning Father X Homozygous Wild-type Mother = 50% Lightning offspring

Figure 5. Dominant Heterozygous Lightning Mother X Homozygous Wild-type Father = 50% Lightning offspring

Now, my problem with this trait being dominant starts immediately from the fact that it requires at least one outwardly visible Lightning Maroon Clownfish to be breeding in the wild in the first place (unless there is a wild-type pair that is predisposed to throwing off the odd “Lightning” mutation once in a blue moon – afterall, these traits can appear spontaneously). If this trait is dominant, then it might also suggest that this mutation ought not to be as rare as we currently are led to believe it is. And to make matters worse, it does seem that we haven’t seen much straight up “dominant” variations in ANY of our designer clownfish to date; it seems all are either recessive or the result various doses of partially dominant traits. And surprise again; looking back at the Angelfish (which happen to Cichlids, which are a closely related family to the Damselfish, and thus to the Clownfish), we see this: 0nly 1 truly straight-up dominant trait. Meanwhile, there are currently 5 known recessive traits, and 7 traits that are either partially dominant or codominant. Dominant traits just don’t seem that common in clownfishes.

What if “Lightning” represents Partial Dominance?

So what if this is a partially dominant (aka. incomplete dominant) or co-dominant trait. The difference is nuanced, but in the angelfish world co-dominance can cause “blending” of traits in certain mixes, dominant expression in other mixes, whereas partially dominant traits present more of an A/B/C result. To draw a parallel, some might say that if the Lightning trait were codominant, then a fish with a single Lightning allele should still show the white stripes “underneath” the lacy pattern of the Lightning.  I’ll dispense with codominance for the time being and just refer to this option as the partial dominance possibility.

Partial (incomplete) dominance is perhaps the most plausible and most exciting of the three options.  As the scenarios are about to play out, they suggest that the Lightning, in a partial dominance scenario, would only be the “first step”.  Partial dominance is well documented in angelfish, and the stripeless allele is a great example.  A normally striped angelfish is Silver (+/+), a single dose is a Ghost (S/+), and a double dose is a Blushing (S/S).  Take a look at a Ghost and compare it to a Blushing that happens to be showing a second partially dominant trait, the “veil” fin trait (impossible to say at this young size whether our example fish is simply veil (V/+) or super veil (V/V)).

A single dose stripeless angelfish, (S/+), aka. a "Ghost".  You can see a "Silver" (wild type, standard barred) Angelfish in the backround at right for comparision.

A single dose stripeless angelfish, (S/+), aka. a "Ghost". You can see a "Silver" (wild type, standard barred, aka. (+/+)) Angelfish in the background at right for comparison.

Blushing Angelfish

A Blushing Angelfish with two doses of the "Stripeless" allele, (S/S).

If “Lightning” is a partially dominant trait, the results in the offspring push us to only one genetic possibility. Let me step back to explain why. There are currently only 2 forms of observable pattern in the offspring; “Lightnings” and “normal”. Simply put, the Lightning cannot be (L/L) in the partial dominance scenario. If a partially dominant allele is present in a homozygous state (L/L) and mated to a wild type fish (+/+), we should get all (L/+) – something intermediate between the Lightning and the Wild form, and they should all be the same (emphasis again on the fact that there would be no Lightnings, and no normally barred fish either) (Figure 6). We don’t have that result, so (L/L) is ruled out if Lightning is a partially dominant trait.  Or is it?

Partially Dominant Homozygous Lightning X Homozygous Wild Type = 100% Intermediate Offspring

Figure 6. Partially Dominant Homozygous Lightning X Homozygous Wild Type = 100% Hypothetical Intermediate Offspring

The second consideration for parental genetics would be (L/L) x (L/+), but once again here, the (L/+) cannot look like the wild form, as (L/+) represents an “intermediate form”. Someone out there is going to say “but what if (L/+) does in fact look like the wild form?  If it did, then by definition Lightning would be a recessive trait as I described earlier (Figure 2)!  So this scenario is ruled out.

The third consideration would be (L/+) x (L/+), but then again that would mean both mates should be “intermediary” forms and roughly look the same (which they obviously don’t in our pairing). This alone is enough to scrap this mating as a possibility.   But if you’re not convinced, this hypothetical mating would also mean that 25% of the offspring would be (L/L), 50% (L/+), and 25% (+/+) – if the fact that the parents would have to look the same didn’t throw this out for you, consider that there would still have to be THREE (3) phenotypes in this batch of offspring for that proposed genetic combination in the parents to make any sense (which it can’t, because the parents are not the same).

The only way that “Lightning” works as a partially dominant trait is if the Lightning Maroon is (L/+), and the mate is (+/+). This produces a nice occurrence of 50% like the Lightning, 50% like the male parent (Figure 7). This also takes a lot of the “luck” out of the equation; we didn’t have to stumble upon a mate with a hidden allele like we would have in the recessive scenario.

Partially Dominant Heterozygous Lightning Maroon X Wild Type Male = 50% Lightning Maroons

Figure 7. Partially Dominant Heterozygous Lightning Maroon X Homozygous Wild Type Male = 50% Lightning Maroons

This also seems to be how some currently known traits may work (Picasso in Percs, maybe Snowflake in Ocellaris). If you believe that “Black Ocellaris” are a melanistic variation within Ocellaris, then “black” in ocellaris could also potentially be partial dominance…with “Blacks” having “two doses”, and when you mix Black with Ocellaris, you get “Mochas” which in all photos I’ve seen, are muddy intermediaries. The real question to be asked is what happens when you breed 2 Mochas together – do you get 25% Blacks, 50% Mochas, and 25% normal Orange Ocellaris? I don’t know that anyone has done that and tested the results yet (but I also know that I don’t believe they are the same species of fish at the moment either…you have to throw the genetics out the window when you start hybridizing)

But getting back to the Lightning; if this trait is “partially dominant”, then the most exciting part is yet to come, because it would mean that all the fish we’ve seen so far only have ONE dose of the Lightning allele (L/+), and thus, the designer breeders out there will be clamoring to mate two Lightnings together so they can discover what a (L/L) fish is going to look like.

And that’s the crazy part, because there should only be one of two things that could when we mate Lightnings together – either we’ll get 25% being something new, or we’ll just get more Lightnings. If we get 100% Lightnings, we are either looking at a recessive trait or a straight up dominant trait (or, in a less likely case the difference between a partially dominant (L/+) and (L/L) is simply too minimal to discern, and you’d then just treat it as dominant anyways).

The “Lightning Precursor” Hypothesis – Dealing with Horned and Flaked Maroons

I suppose at this point we have to step back and objectively define what we *think* a Lightning Maroon “is”.  What is the phenotype?  We have to consider the two fish that have been given that label to-date (the less familiar one being the first wild-collected Lightning Maroon from 2008).  Well, the best term I’ve heard used lately was to describe the Lightning Maroons as filigreed. Other’s commonly call the patterning “lacey” or “net-like”.  Whatever it is, the most notable place for this Lightning variation is in the headstripe.  The headstripe is dramatically wider in the Lightnings, and it is “pitted” with normal coloration.  “Horned” and “Flaked” maroons fundamentally lack this very distinct patterning and the wider headstripe it takes to make it.

The other part of the Lightning phenotype is the breaking up of the mid-stripe and tail-stripe into the lacey, interconnecting patterns that split apart and at times, reconnect.  None of the “horned” maroons show this patterning that I’ve seen, while many “horned” maroons simply exhibit broken bars or “extensions” trailing off.  Admittedly, only the most recent “Lighting Precursor” was really suggestive of the body stripping seen in the two wild Lightning Maroons, but the stripes showed a more “smooth” outline and did not reconnect (I’ve been told the other side of this fish was unimpressive) – I think this fish is better considered a more extreme form of these “Horned” Maroons being found in PNG waters.

In drafting this genetics rundown, I realized I had one other genetic possibility on the table; the notion of the “Horned” Maroons being collected in PNG potentially represented the “intermediate” form in a partial dominance scenario (eg. the hypothetical heterozygous offspring shown in Figure 6).  One such Maroon recently made the rounds in the internet being called by some a “Lightning Precursor”.

After examining the data provided publicly by EcoAquarims PNG, it seems these aberrant Maroon clowns  appear to be quite common in the waters of PNG, with various atypical Maroon clownfish being caught approximately every 11 days.  We also had other fish like Mike Hoang’s Goldflake Maroons which indeed, as young fish, had me wondering if we’d see Lightning-like traits as they grew up (sadly the best marked offspring were lost, and those that remain look no different than the “Goldflake” Maroons output by Sustainable Aquatics).

Let’s deal with the “Goldflakes” of the world first.  It turns out that abnormally spotted and overbarred Maroon Clownfish (what I’m calling “Flaked” here) are indeed commonplace in captive culture.  So far, these fish have seemed to elude genetic categorization, apparently really behaving fundamentally more like “misbarring” in other species of clownfish.  Most recently German breeder Sylvio Heydenreich shared some videos depicting some highly overbarred Maroons on the MBI website; when asked about these fish, he stated quite directly that, “Die Fehlzeichnungen lassen sich ganz leicht über die Wasser Qualität steuern.”  Or as Google likes to translate it, “The failure drawings can be controlled easily through the water quality.”  Failure drawings of course, being what is probably a literal translation for “misbarring”.  And to that end, we already are aware that misbarring in clownfish has environmental causes, not genetic causes. So as much as we like these “Goldflakes”, all observations to date suggest we think of this type of patterning as a likely non-genetic occurance.

Meanwhile, those “Horned” Maroons coming out of PNG had all of us, even me, convinced that the Lightning Maroon could be a homozygous (double-dose) example for a partially dominant trait. Simply put, the breeding results don’t really suggest this possibility because we lack the intermediaries (I would’ve expected the 100% “horned” batch to show up, like Figure 6).  Still, I do have two normally barred fish that show spots.

Note the extra spot on the back of this normally barred juvenile.

Note the extra spot on the back of this normally barred juvenile.

Is this baby a “Horned” Maroon?  Well, here’s the kicker.  There are only two ways you get hypothetical intermediates (intermediates being the proposed placement of the “horned” Maroons).  You either get 100% in the F1 batch, or the male parent has to be an intermediate itself, in this case, a “Horned” Maroon. And this is where there’s still an outside chance – the male has a single broken tailbar.  But…if this was in fact an “intermediate”, what genetics must we get in the offspring?  75% Lightnings, and 25% intermediates – NO wild-types.  Again, let me be explicitly clear – for “Horned” Maroons to be “Lightning intermediates” or “Lightningprecursors”, I would have had to encounter “Horned” Maroons  in the offspring and at a rate of 25% -or- 100%.  So…if the babies all wind up showing extra horned bars and spots as they grow up over the next few months, and the ratio of Lightnings to non-lightnings is 3:1, there could still be “hope”.  Otherwise, we have probably closed the book on the “Lightning Precursor” hypothesis that tried to link the Horned Maroons to the Lightning, at least for now.

All of that said, what I really think we’re seeing here is something much more fundamental in the Horned Maroons.  We are seeing this “flake” overbarring, a commonplace occurring in captive-bred maroons, showing up on a few random offspring.  You wouldn’t notice it in the Lightning offspring because it’s just “painted over”, but you can see it in the normally barred fish.  Years ago, breeders would have destroyed these types of fish as “culls”…that’s when the 3-bar wild-type fish was considered something to aspire to as a breeder, and not “common” and “boring” as many hobbyists may consider a wild-type clownfish today.  Given that we know of a possible causal relationship between “overbarring” and “environment”, perhaps there is something environmentally going on in the waters of PNG to show us more “environmentally overbarred”, aka. “Horned” Maroons, than perhaps we might expect in other parts of the ocean.

Or, and this is still a possibility; the “Horned” Maroons of PNG could yet represent another, distinct genetic variation.  It’s certainly possible – breeding them could give us the answers, although it may be difficult in the face of commonly-occurring “flaked overbarring” potentially giving you a fish with the same basic phenotype.

The Odds on the Lightning Pair’s Genetics

Let’s get back to the Lightnings.  If we give equal weight to all three possibilities for the interaction of the “Lightning” allele, we are left with three scenarios for the genetics of the parents.  Once again, notation here…(female first) X (male second).

Recessive, where we have (l/l) X (l/+)
Dominant, where it can only be (L/+) X (+/+)
Partial Dominance, where it must be (L/+) X (+/+)

By this alone, each has a 1/3 chance of being right.  There is a 2/3 chance, or 67% roughly, that the Lightning is (L/+).

However, for the sake of doing something interesting, what if I used the genetic ‘spread’ in Angelfish to derive an alternate baseline for the odds of a trait being dominant, recessive, or partial/codominant within the clownfish family?

Recessive = 5/13, or roughly 38%
Dominant = 1/13, or roughly  8%
Partial/Codominant = 7/13, or roughly  54%

If this was at all representative of the odds for trait expression in clownfish (and it’s really probably not, it’s just a fun way to think about it), then we have a 62% chance that the Lightning Maroon is (L/+), and within that 62%,  it would then represent a 87% chance  that the trait would be partially dominant (again, roughly 54% overall).

Overall, whether we weight the system or not, the odds remain in the rough territory of 2:1 that the Lightning Maroon is (L/+), and the mate I used is (+/+), vs the only possible alternatives of (l/l) and (l/+).  The kicker for me is when you move beyond “probability” alone, and put in the observations and the way mother nature seems to work.  I’ll get to my prognostication in a minute, but first, I must point out that this puzzle can be solved.

How are breeders going to help figure it out?

In a nutshell, this project will soon turn to the massive “cloud computing”, or in this case “crowd breeding” effort of marine aquarists who get these offspring.  It has always been my intention to get the F1 fish out to other breeders to both diversify the risk, but also to leverage the collective efforts of breeders to provide for rapid, definitive answers.  In a nutshell, anyone breeding with my offspring, you have my formal request to track your project at the MBI, and to do so openly.  You also have my request that you must track your offspring numbers and take photos of each one on both sides, because it is the headcounts and photos that will help determine the genetics in the end.  Here’s how we’ll do it (again, assuming that “Lightning” is the result of a single locus and a single allele).

We can determine (or rule out) a recessive trait by mating the non-lightning siblings together; if recessive, 2/3 of the F1 babies will carry a hidden Lightning gene. This means that picking any random 2 fish, the odds are roughly 40% that both are (L/+), so four out of 10 random pairings would yield Lightning offspring to the tune of 25%, if this is a recessive trait. The only way you get Lightnings out of pairing 2 normally-barred siblings is if this trait is recessive.

We can also determine this trait to be recessive by matings of Lightning Maroons to their non-lightning siblings. In this scenario, 2/3 of the pairings would produce 50% Lightning offspring, while the remaining 1/3 would produce nothing but normally striped fish.

We don’t need to use the siblings to specifically test for a recessive trait, but non-sibling fish present a conundrum – you have less insights onto what their genetics could possibly be.  Still, you can simply mate Lightings to unrelated white stripe maroons (and breeders out there, I will work as hard as I can to produce offspring from the other PNG White Stripe pair in the house so we have a clean PNG bloodline which we can outcross to, and Dale Prichard in the UK hopefully can contribute more, or you can look to the other PNG maroons being exported from EcoAquariums PNG now).  If the trait is recessive, then you have to consider the unknown odds that any randomly-selected, unrelated fish, could be carrying a single hidden copy of the recessive allele.

However, if the trait is partially dominant, any Lightning paired with a wild-type sibling, or any outcross (mating of a Lightning to unrelated normal fish) should yield a percentage (50%) of Lightnings in the offspring. Conversely, again, if the trait is recessive, these outcrossed matings will produce nothing but normally barred fish UNLESS, once again, you get “lucky” to stumble upon a fish with a hidden allele. But that’s the rub – you’re far more likely to find that hidden gene in the normally barred siblings.

If we get something “new” out of the Lightning X Lightning mating, it should be 25% of the “new” variety, and that would convincingly clinch the genetics as partial dominance. Sounds far-fetched? Well, in Percula, Picasso X Picasso is where we get Platinums from.  If mating Lightning X Lightning simply makes 100% Lightnings, then the trait easily falls into the category of a straight up dominant trait.

I’m a betting man if the wager is bragging rights…so my guess is…

…partial dominance.  Ultimately, my gut call is for partial dominance because it seems to be the most commonplace type of genetic trait we’ve seen in our designer clownfish, and it’s the most prevalent in a widely cultivated and well-documented group of related fish (the freshwater Angelfish). The odds also do slightly favor partial dominance.  Partial dominance may also be one of the easiest to prove – just mate two Lightnings together and see what you get. Partial dominance (and in this case, straight dominance) also requires less luck to have had the outcome I seem to have had with my initial pairing. If ever there was a project that had just about everything except “luck” on its side, it is The Lightning Project.

One last wonderful caveat – every possibility laid out above could wind up being 100% wrong.  Until we get those second generation fish produced, and aquarist start gathering the data and sharing it, we simply won’t know.

A special thanks to Adeljean Ho for acting as a sounding board and editor on this piece.  I am sure Adeljean, with his strong interest in genetics, was probably as excited about this as I am!  Thanks!

Yes, that “surprise” that happened today was the honestly unanticipated 2nd spawning of the Lightning Maroon.  The massive flooding in Duluth the last couple days prevented Barb from making the trip to teach me how to do a skin scrape on the Lightning Maroon; had she come yesterday as originally planned, I very well may have a) fished out the male, or possibly even the Lightning Maroon herself for a skin scrape and thus b) probably delayed or prevented this very spawn from happening.  Serendipity at it’s finest.  It was still the plan to skin scrape the fish today to rule out any external parasites, but when Barb called to say she was on her way, I had to tell her that the fish had started spawning behavior, and that maybe it wasn’t a good idea to touch them today.  This was shot right after I got off the phone with her:

By the time she and Heidi had arrived, things were looking pretty serious, so much so that I had to excuse myself from being a good host to immediately film the fish’s behavior.  Lucky thing I did, because I caught the very first few eggs being laid as it happened (left the audio commentary on, if you can imagine me shooting while talking and pointing to the tank…)

Afterwards, believe it or not, I actually tore myself away so that we could do some skin scrapes on some other fish downstairs just so I could learn how to do it.  Turns out it was really easy, but honestly, it’s one of those things that I think you simply want to see done the first time, just so you know you’re doing it right.  And between my two microscopes, I really a) don’t have one powerful enough to look at the sample and b) wouldn’t necessarily know what to look for, whereas Barb has that experience.  The samples we took from the fish downstairs were clean (no pathogens noted).  Always a nice thing to hear ;)

After Barb and Heidi departed, I sat down and took some additional video of the post-spawn behavior.  You can clearly see both the mark on the male right between the eyes, as well as the inflamed tissue around the left eye of the female.

So that’s where we’re at.  A new batch of eggs, and new hope that maybe, just maybe, I can take some of the pressure off if things go our way and we get some baby Lightning Maroon Clownfish.  Wouldn’t that be great?  If we have success, it’s realistic that 1-2 months from now we might have our first ideas at what we’re looking at.  Of course I’ve just now done the math and realized – I will be speaking in Boston the weekend these eggs are due to hatch.  THANKFULLY I have not one, but TWO local hobbyists here in town who have both hatched and reared maroon clownfish.  Looks like I’ll be asking both Mike Doty and Jay Hansen to do some pretty serious fish sitting next weekend!!!


So 24 hours later, not a new scratch on the smaller PNG Male, and the larger PNG female White Stripe Maroon has been tolerating her new mate. They both share the Red Bubble Tip Anemone that is in the tank (Entacmaea quadicolor). Big step forward indeed. The biggest change I noticed when observing them this evening? The smaller male is now really chasing those 6 Percula culls around. None are showing damage, but they are definitely getting his attention, whereas before he largely ignored them. The larger “female” seems uninterested in any of her tankmates now…a dramatic change from times gone by.

But what was responsible for the attitude change? Was it the 6 culled perculas that I added to the tank? Or was it the isolation of the female, allowing the male to roam “her” territory freely? Or both?

While hardly a scientific study, the best course of action might be to sequester the Lightning Maroon in a breeder net or drilled specimen cup (like the one I’m currently using for the male), and allow the smaller PNG male to roam the Ecoxotic for a while. After maybe 2-3 days of that, freeing the Lightning and careful observation should suggest whether it works or not. This is hardly a “new” technique, but in fact it is standard practice to isolate overly aggressive fish for a short period of time, allowing newcomers to settle in. It works with mean Tangs and Angelfish, and so to, it now seems with a mean female PNG White Stripe Maroon. Will it work with the Lightning?

Today I released the “male” PNG Maroon into the Lightning Maroon’s tank once more. For most of the afternoon, NOTHING happened, because the Lightning Maroon spent the evening hiding it it’s cave, and the PNG Maroon mostly wallowed in the ORA Red Goniopora at the far opposite end of the tank. However, in the middle of their night, the Lightning finally realized what was up, and started harassing the smaller male PNG Maroon. Once again, I returned the PNG Maroon to the safety of the specimen cup.

Meanwhile, the pair downstairs has made progress. Once again, I released the larger FEMALE PNG Maroon into the tank with the male, and once again, the male went through massive body contortions to appease the female. After a few hours with virtually no aggression witnessed, and no visible signs of attack, I’ve decided to take a big chance and let them spend the night together. Hopefully, come morning, I have a good stable pairing, and not a tattered, or worse murdered, PNG Maroon.

Keep your fingers crossed. If I can get that PNG Maroon pair to spawn downstairs, it offers yet another viable method for me to get a “male” to the Lightning Maroon. While I suspect the main issues with the pairing I’m attempting is the relative similarity in size, it is conceivable that in 6 month’s time, I could get a juvenile PNG Maroon raised up from the other pair, and paired with the Lightning Maroon. Of course, that means that PNG pair needs to get their butts in gear and start spawning immediately, and not a year from now (which is probably more likely).

And again, sorry for no pictures or videos – my wife still has our good camera on vacation!

As I turned the specimen cup over on the male PNG Maroon this afternoon, the fish struggled not to leave the protective confines of its plastic prison. It took only 5 seconds to understand why – it was immediately met with violent attacks from the Lightning Maroon Clownfish. After a minute or two of watching, it was pretty clear that there was no interest on the part of either fish to be in close contact. It wasn’t even hard to scoop the little male PNG Maroon Clownfish back into the safety of the specimen cup.

Hardly what I had been hoping for, but not terribly surprised. Later today I may once again reintroduce the “female” PNG Maroon to her male downstairs in the 10 gallon, hoping that goes as well as their last date. If it does, it will provide yet another avenue for me to try with the Lightning, that is, sequestering the Lightning Maroon in the specimen cup for a day or two might change the relationship dynamic. But I’m not going to try it until I have a proof of concept working, maybe two.

And sorry, no vids or pictures – my wife has the good camera and is on vacation until Friday!

So that didn’t go well…

I tried the “diversion” / “dither fish” pairing tactic with the other PNG Maroons I’ve been trying to pair up.  I initially added three small Onyx Percula culls, and then released the male PNG Maroon from his “cage”.  I was shooting video, but the battery ran out so I’m not sure I got any.  For about the first 3-5 minutes, the interactions looked positive.  The male would tail stand, tremble, clamp fins…all signs of submission.  And then, it got to the point where the male turned to flee and the female just lashed out.  I watched patiently, knowing that this is not uncommon.  Still, after a few more minutes, the male’s rear dorsal and anal fin were missing chunks.  The female would periodically hunt the male down, whereever he was, and give chase.  If she could get her mouth on him, she did, even pulling him backward through the water as she tore a chunk from his tail.

I did get the video, but really, there’s nothing to watch.  None of the interactions happened until after the camera died :(

And meanwhile, the Perculas garnered almost no attention, so I upped the number from three to six.  That only did slightly better, causing the female to occasionally bully her way through the group, but not really apply any serious aggression towards them.  Indeed, she solely sought out the male maroon…relatively speaking she could’ve cared less about the other, smaller clownfish.

So in a change of events, I caught the female and put her in the cage.  The male will now get to recouperate in the main tank, and the female will be returning to the male’s territory, which I can only guess whether it will magnify, or reduce, her aggression towards the “intruder” when she is reintroduced.

This all suggests that maybe my next attempt should be to use Sanjay’s method, where he utilized 2 maroons.  I have a small male from another pair I could use to try this technique with these PNG maroons, and hopefully the female will pick the larger vs. smaller male being offered.   With a family weekend ahead of me, probably no news until next week.

Pairing remains at a standstill for the Lightning Maroon, in large part given my desire to sit in front of the tank for an extended period and keep a watchful eye over the interactions so I can intervene if need be.

Meanwhile, I’ve been digging through my circle of fish friends and trying to see what other tricks are out there that we hadn’t yet tried.  Sanjay Joshi suggested something that had worked with his Gold Stripe Maroons.  To summarize, he provided two possible males to a female, and let the female choose the mate she preferred.  Of course, this makes’s tough for the female to fend off both suitors, so instead, she teams up with one to drive the other away.

My main concern over trying this is that a) you don’t control which fish she chooses and b) you risk both fish.  Since more PNG Maroon Males/Juveniles are not available, I have to be cautious and protective of both of them.  But Sanjay’s recommendation reminded me of a very similar technique described to me by Chicago-area clownfish breeder Mitch May, better known as Booyah on most reef forums.  Mitch’s technique works on the same basic principal of encouraging the natural behavior of teamwork in a pair to drive away third party interlopers.  It’s also a bit safer perhaps, and I’m happy to share this concept with Mitch’s permission.

In a nutshell, the application is to take a “Female” and the desired “Male” and place them together with a few additional fish.  Per Mitch’s instructions, he’ll take one additional fish that is 50% of the size of the desired male (who is the second biggest of all the fish after the females).  Then, we’ll also add 2 more clownfish juveniles, these at 25% of the size of the desired male.  So it works out like this – 3″ female, 2″ male, 1″ juveniles and 2 0.5″ juveniles.  This actually mimics the natural social structure of many clownfish species, although in most cases this unit can’t be easily replicated in captivity.  Generally speaking, attempts like this usually end up with lots of aggression directed at the smaller fish, and even death as a possibility.  However, when trying to drive the top two fish together, the presence of the other three juveniles focuses their aggression away from each other, and instead towards the juvenile intruders.

It may sound cruel, but the best fish to use are those that would be culled.  Since they are never going to be sold, and since humane euthanasia or becoming food for a Lionfish are the most likely results of their existence, their sad lives might actually at least have a redeeming purpose.  If they are killed in the process, while the path to their death was likely more violent, the outcome was ultimately the same.  Of course, I’m not condoning that this simply be a commonplace practice, or that you don’t intervene should the aggression become more violent than just the normal social threats and displays.

So to that end, and knowing I have dozens of culled Perculas sitting in tanks that I simply haven’t put down, I’m going to try this technique out.  I’ll first do it with the two White Stripe pairings downstairs that aren’t working out.  If it works there, then it will get applied to the Lightning Maroon and her would-be mate.

The other technique is one to encourage spawning.  Two breeders have definite experience with this technique.  Mitch May calls it “doubling down”, based on the gambling concept of taking “one good hand and turning it into two good hands”.  Chad Vossen of St. Cloud, MN, calls it the less glitzy, but more straightforward “egg fostering”.  In either case, the premise is the same.  For a pair that is going through the motions but never producing, a clutch of eggs is taken from a spawning pair and placed with the non-spawning pair.  The presence of the eggs kicks in the male’s nest tending instincts and seems to also kick the female into egg production mode.  While I won’t be using this technique just yet, it may come in hand down the line if the Lighting is paired but not moving towards spawning.

Hopefully I’ll get the test pairings restarted downstairs tonight…videos will be shot of course!

I know you’re all excited…well…there WAS a PNG Maroon Clown involved, but it was NOT a “Lightning Maroon”.  No, the spawn I found today was between the large Sumatran Gold Stripe Maroon female from Jonica, and one of the small male PNG White Stripe Maroons which I had placed with the GSM to ensure it stayed a male.  I’d been noticing that the male would occasionally stay hidden in back, not coming out to feed.  This behavior seemed periodic.  Nothing else seemed out of the ordinary.  The female GSM never became noticeably ripe with eggs.  Her aggression level has not increased at all (as you are about to see).  I observed NO cleaning behavior other than the female’s constant digging.  No, the ONLY thing that changes is the male is becomes “shy”.  Being the third or fourth time I’ve observed this periodic shyness, I decided to upturn my live rock, carefully, to see what was going on.  Surprise, we have eggs.

Now, even if I raise these, I will do so only to get some practice rearing Premnas larvae.  After I’m assured I have it nailed down, any babies I raise from this pairing will be destroyed.  It may seem cruel, but the reality is that such “hybrid” larvae are likely to be intermediary between the two variants at best.  Some breeders would argue that you could use such methodology to introduce Lightning genetics into a Gold Stripe variety…ultimately creating Lightning Maroons that have YELLOW bolts on them vs. white.  No doubt, someday, someone may try that.  More realistically, some breeders might argue that such a cross could be used to breed more “docile” genetics into a white stripe form, which is generally deemed more aggressive.  While this is true, you lose the natural form along the way – the fish that are uniquely adapted to a location like the reefs of PNG is lost, and you have a man-made creation in its place which might fail to flourish in the wild for who knows what reason.  Or if reintroduced fish did survive, they could inadvertently introduce a latent gold stripe gene and one day all of the Maroon Clownfish in PNG would be gold stripes.  It’s a slippery slope when we start ignoring the unique traits that make up variants.  To take this “cross” to the most extreme, there are indeed people who believe that the Gold Stripe Maroon Clownfish represents a different species from the White-Striped forms (Premnas biaculeatus).  There is even a different scientific name that gets floated for the Gold Striped form – Premnas epigramma – by people who believe this.  Of course, this name is not currently valid in any sense.

While we may never know exactly where  the name Premnas epigramma came from, it is likely a slight derivation of another name, Premnas epigrammata, published by Fowler in 1904.  If in fact this name was applied to the Gold Striped Maroon Clown from Sumatra, it could someday become resurrected should someone research, and determine, that the form described by Fowler is in fact distinct from all the other forms.  Of course, if Fowler used the name on fish other than the Sumatran Gold Striped form, then that might leave the door open for an entirely new species name to be applied.  Ah…the joys of taxonomy.  There are actually at least 5 names out there for Premnas species, but all are considered synonymous with Premnas biaculeatus – check ‘em out on Fishbase.

Nevertheless, scientists often have it wrong, or perhaps more appropriately, incomplete.  Consider Amphiprion barberi from Fiji, which was at first considered a population of the western Australian A. rubrocinctus, but then treated for a considerable time as a color variant of the Cinnamon or Dusky Clownfish, A. melanopus.  Only recently was this species recognized as distinct through genetic analysis, which confirmed the truly unique nature of the species.  Of course, this multi-decade “misidentification” calls into question the “pedigree” of every fish in the trade as “A. rubrocinctus“, not to mention the voracity of some captive bred Tomato (A. frenatus) and Cinnamon Clownfish which could very well be hodgepodges of 2 or even 3 species of fish.  Heck, there area already breeders who knowingly sell what should be called Percularis (the hybrids of the Common Clownfish (A. ocellaris) X the Percula Clownfish (A. percula)) as run of the mill common Ocellaris Clownfish.  The worst breeders do so intentionally.  Others may be mislead by vendors who can’t even tell the two species apart and don’t even bother to make corrections when the problems are pointed out to them (for history’s sake…that link points to a pairing of what I strongly believe is a female Percula with a male Ocellaris, being sold as a “True Percula Breeding Pair” and yes, I emailed the vendor about those and other mislabled pairs over a week ago – I am disappointed).

Interestingly though, talented hobbyists and breeders sometimes see differences where a scientist focused on morphology does not.  One of the best examples is the Darwin Black Ocellaris, which is so fundamentally different in breeding from the common orange Ocellaris Clownfish that we treat this variant as a separate entry in a more difficult class for scoring in the Marine Breeding Initiative.  The Maroon Clownfish actually shows some similarities in this regard.  Gold Stripes are generally relatively more peaceful, whereas White Stripes are generally considered downright vicious by most hobbyists and breeders (who have hard times keeping the juveniles from rendering all the fish in the batch too torn up to sell!).  Interestingly, through my own informal observations, it seems that Gold Stripe Maroons are particularly prone to losing their stripes as they mature, and this pattern of loss seems consistently from the bottom up.  It seems conversely that White Stripe Maroons may in fact NOT lose their stripes as they age, but be more more prone to darkening of the stripes.  It is differences such as these that are suggestive that in fact, there may be more subtle differences yet to be uncovered, and in fact, we may find we’re dealing with 2 species, and not one.

So…enough of my ranting for now.  For the record, the female Gold Stripe Maroon was brought in on 7-10-2010, and it is safe to say she spawned by maybe 11-28-2010, although I suspect there had been a few spawns prior…maybe as far back as 2 months ago.  Still, it just goes to show you that even when the fish are mature, clownfish are not “quick” to spawn.  And thus, the Lightning Project continues to be a big, long waiting game.

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