Anemonefish Evolution — What Does The DNA Say?

To best understand the evolutionary history of an organism, one has to delve into the nitty gritty of its nucleic acids. Fortunately, anemonefishes have been investigated in considerable detail using molecular techniques, providing us with an intimate glimpse into their complex interrelationships. But no two studies ever seem to tell quite the same story, and there are often major discrepancies between what the DNA tells us and what we see in their biogeography. So let’s examine some of the major published works on Amphiprion and attempt to find consensus.

elliot amphiprion phylogeny
Mitochondrial phylogeny, Elliot et al 1999

The first major study on the genus (Elliot et al 1999) made use of two mitochondrial genes (cytB & 16S rRNA) and included just six species, along with a couple other damselfishes. Not surprisingly, the relationships found here have not always been supported in subsequent papers, though some of the “big picture” presented in this pioneering work still holds true.

allen amphiprion phylogeny
Morphological phylogeny of Allen 1972

The most anomalous finding here was the partial support (16S rRNA, but not cytB) for a monophyletic Amphiprion, with the Maroon Clownfish (Premnas biaculeatus) identified as a true sister lineage to the genus. In contrast, the morphological phylogeny of Allen 1972 placed A. biaculeatus as sister to the ocellaris group, with the two forming a highly derived clade within the genus. This relationship has been strongly supported in most molecular studies since Elliot et al and would argue for the need to synonymize Premnas within Amphiprion. But, as we’ll see, molecular data actually favors the clownfish clade as a basal lineage (i.e. the first major split in the evolution of the genus).

quenouille amphiprion phylogeny
Mitochondrial (3 genes) and nuclear (1 gene) phylogeny, Quenouille et al 2004

In a larger study by Quenouille et al 2004 covering the Family Pomacentridae, a variety of novel relationships were found thanks to the inclusion of several new species and the first use of a nuclear gene (RAG1), along with two new mitochondrial genes (ATPase6 & ATPase8). We get the first evidence for the basal ocellaris+biaculeatus lineage, followed by the clarkii group as sister to the remaining species (as in Elliot et al). But then things get weird… like with the inclusion of A. omanensis with the saddlebacks of the  polymnus group rather than with the more-logical choice of the allardi group. We also get the first data for A. chrysopterus (as sister to the skunk anemonefishes of the perideraion group) and A. akindynos (sister to the tomato anemonefishes in the melanopus group). And then there’s the unusual position of A. nigripes, far from its presumed cohorts in the perideraion group. In total, this was an intriguing study that, in retrospect, didn’t resolve things very well.

It wasn’t until Santini & Polacco 2007 that anemonefishes were examined again, this time using 23 species and the same two genes as the Elliot et al study, plus one new gene (mitochondrial D-loop). The resulting tree showed some anomalous relationships: 1) A. nigripes and A. chagosensis are again recovered within their geographic neighbors in the Indian Ocean, the allardi group, despite the strong morphological and ecological evidence that places them within the perideraion group. 2) A. perideraion is grouped with A. sandaracinos, rather than with the phenotypically and ecologically similar A. akallopisos. 3) “Premnasbiaculeatus is NOT shown to form a monophyletic clade with the ocellaris group.

 

santini amphiprion phylogeny
Mitochondrial (3 genes) phylogeny, Santini & Polacco 2007

Some other noteworthy findings here are the isolated position of A. latezonatus, which has been reaffirmed in other studies. The splitting of the traditional clarkii group (sensu Allen 1972) further confirmed the artificial nature of that assemblage in recognizing the distinct evolutionary relationships of the chrysopterus and allardi groups that were first hinted at in Quenouille’s study. And Santini & Polacco 2007 was also the first time that the close relationship of A. chrysopterus and its suspected hybrid A. leucokranos received support. In all, this was an exciting piece of research which has held up surprisingly well over the years.

Easily the single most prolific molecular amphiprionologist out there is Glenn Litsios from the University of Lausanne, who, from 2012-2014, published four hugely important papers on anemonefishes. These are must-reads for any student of Amphiprion biodiversity, but there are still a variety of discrepancies that raise important questions. The earliest study (Litsios et al 2012) features a phylogenetic tree of the entire Pomacentridae based upon six mitochondrial genes and three nuclear genes, potentially providing the most exhaustive and robust examination to date.

litsios 2012 amphiprion phylogeny
Combined mitochondrial (6 genes) & nuclear (3 genes) phylogeny, Litsios et al 2012

The most startling find here was the support for A. latezonatus as the earliest diverging anemonefish lineage, followed by the familiar ocellaris+biaculeatus clade and the generalist clarkii clade. However, this study was soon followed up with another (Litsios et al 2014) that was focused exclusively on anemonefishes. The phylogeny in that paper was built from seven nuclear genes and found A. latezonatus branching off just before the clarkii group (an identical placement as in Santini & Polacco 2007). This sort of cytonuclear discordance between studies is quite prevalent in the genus (Litsios & Salamin 2014) and hints at extensive hybridization throughout the course of anemonefish evolution.

litsios salamin amphiprion phylogeny
Mitochondrial (6 genes) versus nuclear (7 genes) phylogenies, Litsios & Salamin 2014

Another good example can be seen with the position of the A. akindynos and A. mccullochi. Recall that Allen’s morphology based hypothesis had A. mccullochi as belonging to the melanopus group, while A. akindynos was classified within a broad concept of the clarkii group. When we examine mitochondrial data, we find that both species group together with the melanopus group, but this changes when nuclear data is used, as the akindynos clade instead belongs alongside the chrysopterus group. If we assume that the geographic overlap of the akindynos and melanopus groups precludes them from belonging to the same lineage, it would appear that the nuclear dataset is the more reliable of the two. Perhaps what the mitochondrial data is showing us here is evidence for past hybridization between the akindynos and melanopus lineages?

And we find even more discordance with the placement of the melanopus group itself. Using mitochondrial data, this clade nestles deeply within the tree, whereas nuclear data suggests an earlier position, forming a sister relationship with the clarkii group. The latter hypothesis is more in keeping with morphological studies, which placed both together in the same subgenus; however, that had more to do with these groups lacking the diagnostic traits of other lineages rather than any obvious shared similarities between the two. And it’s hard not to be struck with the vast ecological differences between them, as A. clarkii is the ultimate anemone generalist, while A. melanopus and its ilk are only ever found in the Bubbletip Anemone.

Another strange result can be seen in the skunk clade. The biogeography, ecology and morphology of the Orangefin Skunk Anemonefish (A. sandaracinos) all suggest that it should be the sister group to the more widespread and diverse A. perideraion lineage, but, for the most part, neither mitochondrial nor nuclear data seem to agree here. The only exception to this was the phylogeny presented in Litsios et al 2012, which placed A. sandaracinos in a basal position among the skunk anemonefishes; however, follow-up studies instead grouped it with the enigmatic A. pacificus. And let’s not forget that the Central Indian Ocean members of this group, A. nigripes and A. chagosensis, have never once been shown to group alongside their skunky brethren in any genetic study, despite the compelling evidence to the contrary. Clearly, we have much left to understand about these fishes and their inscrutable genetics.

li amphiprion phylogeny
Mitochondrial genomic phylogeny, Li et al 2015

The most recent study to investigate Amphiprion was that of Li et al 2015, which used entire mitochondrial genomes (rather than just single or multiple genes) to build their phylogeny. Unfortunately, the authors were missing several major lineages in their analysis—latezonatus, akindynos, chrysopterus—and thus there is little clarity added to our understanding. The results are in fact identical to those found by Litsios & Salamin 2014, helping to confirm the backbone of the mitochondrial phylogeny for Amphiprion.

What’s needed next is to expand the mitochondrial dataset to include the genomes of some of the important missing taxa, but this will only tell us part of the story. To really understand Amphiprion, we’ll need far more of the nuclear genome incorporated into phylogenetic studies, and with the cost for next-gen sequencing techniques dropping rapidly, we can likely expect this to happen fairly soon. Anemonefishes are simply far too interesting not to investigate. Answering the many questions concerning their speciation and biogeography will undoubtedly prove informative for understanding how reef fishes of all kinds diversified in the Indo-Pacific.

  • Elliott, J.K., Lougheed, S.C., Bateman, B., McPhee, L.K. and Boag, P.T., 1999. Molecular phylogenetic evidence for the evolution of specialization in anemonefishes. Proceedings of the Royal Society of London B: Biological Sciences, 266(1420), pp.677-685.
  • Li, J., Chen, X., Kang, B. and Liu, M., 2015. Mitochondrial DNA Genomes Organization and Phylogenetic Relationships Analysis of Eight Anemonefishes (Pomacentridae: Amphiprioninae). PloS one, 10(4), p.e0123894.
  • Litsios, G., Pearman, P.B., Lanterbecq, D., Tolou, N. and Salamin, N., 2014. The radiation of the clownfishes has two geographical replicates. Journal of biogeography, 41(11), pp.2140-2149.
  • Litsios, G., Sims, C.A., Wüest, R.O., Pearman, P.B., Zimmermann, N.E. and Salamin, N., 2012. Mutualism with sea anemones triggered the adaptive radiation of clownfishes. BMC evolutionary biology, 12(1), p.212.
  • Litsios, G. and Salamin, N., 2014. Hybridisation and diversification in the adaptive radiation of clownfishes. BMC evolutionary biology, 14(1), p.245.
  • Quenouille, B., Bermingham, E. and Planes, S., 2004. Molecular systematics of the damselfishes (Teleostei: Pomacentridae): Bayesian phylogenetic analyses of mitochondrial and nuclear DNA sequences. Molecular phylogenetics and evolution, 31(1), pp.66-88.
  • Santini, S. and Polacco, G., 2006. Finding Nemo: molecular phylogeny and evolution of the unusual life style of anemonefish. Gene, 385, pp.19-27.