Wednesday, November 21, 2012

Phylogenetic position of turtles: a network view

The evolutionary history of turtles has been difficult to determine. Historically, turtles were thought to be early diverging reptiles (called anapsids), but recent morphological studies have allied turtles with lizards and snakes (squamates) plus tuataras (together, the lepidosaurs). These relationships are indicated at the top left and top right of the first figure, respectively.

Four hypotheses about the evolutionary relationships of turtles.
The figure is adapted from Hedges (2012).

However, most molecular studies support neither of these hypotheses, as shown in the bottom two parts of the figure. To quote from Parham et al. (2012):

  • Recently, several molecular data sets have recovered support for a novel turtle-crocodilian clade [bottom right of the figure] (Hedges and Poling 1999; Mannen and Li 1999; Cao et al. 2000; Shedlock et al. 2007) or a novel turtle-bird clade (Cotton and Page 2002). However, support for these topologies over an alternative where turtles are the sister taxon to a monophyletic Archosauria [birds + crocodiles; bottom left of the figure] is often weak (Cao et al. 2000; Iwabe et al., 2005; Katsu et al. 2009). The majority of recent molecular analyses support a monophyletic Archosauria (Iwabe et al. 2005; Hugall et al. 2007; Alfaro et al. 2009; Katsu et al. 2009; Lyson et al. 2012).

A number of research groups have recently tackled this phylogenetic problem using genome-wide datasets for various representatives of the taxonomic groups, including Chiari et al. (2012), Crawford et al. (2012), and Tzika et al. (2011). Sadly, they come to a diversity of conclusions; and here I use a phylogenetic network to explore why this might be so.

The sequence alignment used by Crawford et al. (2012) is freely available in the Dryad database, and so it provides a good starting point. The objective here is Exploratory Data Analysis (EDA), to investigate the characteristics of the data before the data are used to formally test the above four hypotheses about turtle relationships. For this I have performed a NeighborNet analysis using the SplitsTree program.

NeighborNet analysis of the aligned sequence data
provided by Crawford et al. (2012).

The NeighborNet displays 99.3% of the data, and so almost all of the data patterns are shown in the splits graph. I have numbered the nine best-supported splits in the data, and shown their location in the graph as well as their relative weights. [The weights represent the relative amount of data supporting each split — a greater weight means more support.]

Note that splits 1-6 & 9 are consistent with the hypothesis in the bottom left of the first figure, and none of the other hypotheses are supported by these seven splits. So, these seven splits appear in the tree produced by Crawford et al. (if Human is the outgroup root), and thus they represent the phylogenetic signals detected by the authors.

However, there are two other well-supported splits (7 & 8) that contradict this tree, and thus they create complexity that is not recognized by the authors. Note that split 7 contradicts splits 4, 5 & 6, and that split 8 contradicts splits 4 & 9. These two splits thus represent data that refutes the hypothesis of relationships favoured by the authors, as well as contradicting all three of the other hypotheses. Of course, splits 7 & 8 do not appear in the tree because there is at least one stronger split that contradicts them (eg. split 4).

Of particular note, the complexity created by split 7 involves the relationship between the turtles and the tuatara, while split 8 involves the relationship between the turtles and the crocodilians. This emphasizes just why there are so many different hypotheses about turtle relationships — many contradictory relationships are supported by at least some of the data! This calls into question the strong conclusions reached by the authors from these data.

In this context, it is worth emphasizing that split 9, which supports the Archosaurs, is rather small. This contradicts the quote above from Parham et al. (2012), which indicates that molecular data usually support this group more strongly than alternative phylogenetic arrangements.

However, it is the tuatara relationship that is one of the keys to understanding the complexity of turtle relationships. It is therefore unfortunate that there are no other available datasets to test this relationship further. Those studies with genomic data available do not include the tuatara; and those genomic studies that do include the tuatara apparently do not have their aligned molecular data freely available online (and sometimes both issues apply).

One potentially interesting genome study from the former group is that of Chiari et al. (2012). Their sequence alignment is also freely available in the Dryad database, and so it is possible to perform an EDA here, as well. As above, I have performed a NeighborNet analysis using the SplitsTree program.

NeighborNet analysis of the aligned sequence data
provided by Chiari et al. (2012).

The NeighborNet displays 99.0% of the data, and so almost all of the data patterns are shown in the splits graph. I have shown the same nine splits where they are supported by the data. Note that (i) splits 5 & 7 are missing because the tuatara is absent from the dataset, and (ii) split 8 involves crocodile and painted turtle, both of which are also absent from the data.

Once again, split 9 (supporting the Archosaurs) is very small, thus confirming this part of the results of Crawford et al. However, in this case there is a more strongly supported split, labelled X, that contradicts split 9. This second dataset is therefore consistent with the hypothesis in the bottom-right of the first figure; and this is reflected in the rooted tree produced by Chiari et al. Split X does exist in the NeighborNet of the data of Crawford et al., but it has a weight 0.00003, and so it is almost impossible to detect visually in the graph.

So, these two datasets apparently support two different hypotheses of turtle relationships. However, both datasets also provide incompatible data patterns within themselves, as discovered by the EDAs, and so they do not necessarily provide strong support for any one hypothesis of turtle relationships. It seems that we need more data for the tuatara, so that it can be incorporated into datasets such as that of Chiari et al.

I will finish by noting that the genome study of Tzika et al. (2011) provides no resolution of this situation. Their Figure 4a matches the result of Chiari et al. (turtle + crocodile) and their Figure 4c matches the result of Crawford et al. (birds + crocodile)! The multi-gene study of Shen et al. (2011), on the other hand, supports the Archosaurs (birds + crocodiles).


Chiari Y., Cahais V., Galtier N., Delsuc F. (2012) Phylogenomic analyses support the position of turtles as the sister group of birds and crocodiles (Archosauria). BMC Biology 10: 65.

Crawford N.G., Faircloth B.C., McCormack J.E., Brumfield R.T., Winker K., Glenn T.C. (2012) More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs. Biology Letters 8: 783-786.

Hedges S.B. (2012) Amniote phylogeny and the position of turtles. BMC Biology 10: 64.

Parham J.F., et al. (2012) Best practices for justifying fossil calibrations. Systematic Biology 61: 346-359.

Shen X.-X., Liang D., Wen J.-Z., Zhang P. (2011) Multiple genome alignments facilitate development of NPCL markers: a case study of tetrapod phylogeny focusing on the position of turtles. Molecular Biology Evolution 28: 3237-3252.

Tzika A.C., Helaers R., Schramm G., Milinkovitch M.C. (2011) Reptilian-transcriptome v1.0, a glimpse in the brain transcriptome of five divergent Sauropsida lineages and the phylogenetic position of turtles. EvoDevo 2: 19.

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