Mem Inst Oswaldo Cruz, Rio de Janeiro, 101 (Suppl.II) December 2006
Original Article

Challenges of phylogenetic analyses of aDNA sequences

Katharina DittmarI,1; Sheila Mendonça de SouzaII; Adauto AraújoII

IDepartment of Integrative Biology, Brigham Young University, Provo, Utah, US
IIDepartamento de Endemias Samuel Pessoa, Escola Nacional de Saúde Publica Sergio Arouca-Fiocruz, Rio de Janeiro, RJ, Brasil

Page: 9-13 DOI: S0074-02762006001000003
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One of the crucial steps of authentication of aDNA sequences is phylogenetic consistency. Amplified sequences should fit into the phylogenetic framework of their supposed origin. An inherent property of aDNA sequences however, is their short sequence length. Additionally, genes for aDNA studies are often chosen by their preservation potential rather than by phylogenetically informative content. This poses potential challenges regarding their analyses, and might result in an inaccurate reflection of the supposed phylogenetic history of the sequence or organism under study. In this paper some fundamental problems of phylogenetic analysis and interpretation of aDNA datasets are discussed. Suggestions for character sampling and treatment of missing data are made. The publication is the result of a talk from the 1st PAMINSA Meeting in Rio de Janeiro, July 2005.

Phylogenies are central to addressing interesting evolutionary questions in aDNA research (Austin et al. 1997a,b , Hofreiter et al. 2001). Just as in a regular analysis with extant data however, conclusions depend on the handling and analysis of the data. Therefore, an increased effort should be spent on conducting thorough analyses. While numerous protocols regarding stringent aDNA laboratory techniques, such as extraction, amplification and sequencing exist (Handt et al. 1994, Austin et al. 1997b, Cooper & Poinar 2000, Hofreiter et al. 2001) limited attention has been paid to the analytical challenges arising from an aDNA dataset.

Ancient DNA is in general physically and chemically damaged (Handt et al. 1996, Poinar et al. 1996, Poinar & Stankiewicz 1999), which results in the fragmented nature of its sequences. Typically, no large sequences can be obtained, and many characters cannot be sampled. Additionally, only multicopy genes tend to survive in ancient material (Hofreiter et al. 2001). Out of this knowledge, the majority of aDNA sequences have been retrieved from the mitochondrial genome, or from other multicopy genes such as nuclear ribosomal genes (Greenwood et al. 1999, Cooper et al. 2001, Dittmar et al. 2003). Therefore, preservation issues severely limit the available phylogenetically informative character space of aDNA.

Because there is little recourse to avoid incomplete taxa in aDNA research, a higher emphasis should be placed on the initial character sampling. This should be guided by a priori knowledge obtained from a thorough analysis of extant taxa. In the following text we will introduce some ideas how to use extant sequences in aDNA sequence sampling. We also briefly test and discuss recent developments in missing data research.


Choosing the right genes/gene region

The essential problem for the aDNA researcher is that the potential character sampling space is limited from the beginning. DNA damage starts to occur immediately after cell death, and is only to increase in the course of time. In the majority of all cases no DNA is preserved, and only under special circumstances (e.g. permafrost), detectable amounts of DNA, albeit highly fragmented, survive. Very often, this DNA is from multicopy ribosomal and mitochondrial genes, which supposedly have a higher chance of survival because of their larger number of copies per cell. Due to the workings of hydrolytic and oxidative damage, the expected sequence length for aDNA does certainly not exceed 500 bps, and most sequences rather range between 150 to 300 bps (Hofreiter et al. 2001). From these multiple, randomly broken pieces only a minuscule amount is sampled. The generation of larger contiguous DNA sequences has been successfully attempted, but samples came from subfossil material [1000-1500 years old] (Cooper et al. 2001). Usually, aDNA researchers are satisfied with the completion of one single short sequence, which is then used in a phylogenetic analysis to confirm "phylogenetic consistency" (Austin et al. 1997b) and to infer phylogenetic history. Chances are, however, that the sequenced piece contains only a limited number of phylogenetically informative characters, if any at all. It is well known that certain genes, depending on their level of sequence divergence, may reflect relationships on different ordinal levels. However, each gene may also contain regions of rapid or unconstrained evolution, leading to saturation, and consequently to what phylogeneticists refer to as random signal or "noise". If the sequenced aDNA pieces were to be composed entirely of random signal, an inaccurate or conflicting (relative to other datasets) reflection of the "true" phylogenetic history of that sequence will result.

To avoid this problem, aDNA researchers should adopt a more problem oriented character sampling approach on the outset of their analyses. Rather than randomly sequencing pieces of aDNA, and "hoping for the best", researchers can actually make use of extant data to explore character congruence or informational content.

In order to reconstruct the phylogenetic history of an organism on a molecular level, multiple sources of character information are available in the form of different genes. Today it is widely accepted that phylogenies should be reconstructed with all available information in a combined analysis, because it provides the greatest possible explanatory power (Nixon & Carpenter 1996, Wiens 1998, Pickett et al. 2005). Additionally, combined datasets have been shown to reveal hidden support even for relationships in conflict among analyses of individual markers (Gatesy et al. 1999). Therefore, in an aDNA study, the sampling of different genes (e.g. mitochondrial and nuclear) should at least be attempted. For instance, chances are, that if mitochondrial cytB sequences can be obtained, then fragments of all other mitochondrial genes also survived.

Phylogenetically informative characters are distributed throughout the entire gene sequence. This distribution is not random, since functional and structural constraints on different parts of the gene selectively govern the evolution of different regions within a gene. Thus, this mechanism also accounts for the fact that most genes contain both more conservative and more variable regions, which might either be informative on different ordinal levels, or not informative at all. Therefore, an exploration of extant homologous sequences could identify potentially problematic regions, which then could be excluded a priori from the aDNA study. Or, in other words, potentially informative regions could be identified, and selectively included into the analysis. Several methods to analyze the quality of a dataset concerning information content or character incongruence have been proposed in the past. One approach was introduced by Lyons-Weiler et al. (1996) in form of the program RASA (relative apparent Syna-pomorphy analysis). This method tests (under the assumption of an hierarchical structured dataset) if the proportion between potential synapomorphies and potential informative similarity for any two taxa increases above the expected by chance alone. While this method has been used by several researchers, Simmons et al. (2002) tested a number of hypothetical and empirical datasets with strong phylogenetic signal under these criteria, and reported a failure to detect phylogenetic signal for both matrices. Another approach is a comparison of the homogeneity of signal in different genes through a series of partition homogeneity tests (PHT) (Farris et al. 1994, Swof-ford 2002). The PHT, also known as incongruence length difference test (ILD, Farris et al. 1994) examines the relationship between incongruence and phylogenetic accuracy. This test distinguished between cases in which combining the data generally improved phylogenetic accuracy (P > 0.01) and cases in which accuracy of the combined data suffered relative to the individual partitions (P < 0.001). Applied to an extant gene, divided into defined sub-regions, potentially incongruent and congruent sub-regions could be defined a priori, and aDNA sequencing could be adjusted accordingly. As a caveat however, it has been shown, that noise itself can generate highly significant results in the ILD test (Dolphin et al. 2000). Additionally, Dowton and Austin (2002) showed that the ILD test is unlikely to be an effective measure of congruence when two datasets differ markedly in size.

In order to test the phylogenetic utility of potentially available aDNA characters, we propose an approach that uses extant sequences, and is rooted in the comparison of partitioned Bremer support (PBS) values (Bremer 1995, Lambkin et al. 2002). Usually, PBS has been used to compare the phylogenetic utility of different genes in a combined analysis, and to assess localized character incongruence (DeSalle & Brower 1997, Gatesy et al. 1999). We however want to extend the application of PBS to an a priori assessment tool, identifying particular regions of good character support within a gene. In a similar way, PBS has been applied to mitochondrial genome data (Cameron et al. 2004). The idea is to calculate PBS values for particular predefined regions within a gene, based on an alignment of extant sequences (Fig. 1). The most obvious ways to partition single genes in a replicable manner is by codon positions, and secondary or domain structure. Since we aim to choose topologically sequential characters for sequencing (one cannot sequence every second nucleotide for 150 consecutive base pairs) only the latter two options apply to aDNA. Another way of replicable partitioning is to apply a sliding window (Fig. 1), thus portioning the data into n subsets of a particular length (e.g. 150 bps), which then could be subjected to a PBS analysis (TreeRot v2b, Sorenson 1999). One of the criticisms of PBS is that values are not comparable across analyses, because they are dependent on the specific dataset they were computed for. For instance a Bremer Support value of 5 might be high for a particular dataset, but low for another. However, since we use it as a comparative metric within a dataset, PBS is perfectly suitable for the selection of approximate gene regions that contribute more support than others.


The problem with missing data

In order to place aDNA sequences in a phylogenetic context, an alignment with extant sequences is necessary. By their very nature, extant sequences tend to be much larger in size. In a sequence alignment the unsampled aDNA characters would be coded as missing, and the sequence would be incomplete (Fig. 2A). Numerous empirical and theoretical studies have shown that the inclusion of highly incomplete taxa (> 50% missing data) makes it difficult to place these on the overall topology (Huelsenbeck 1991, Nixon and Wheeler 1992, Anderson 2001). As a result, tree topologies or parts of the tree will most likely be unresolved. Potentially, highly incomplete taxa (e.g. aDNA) also influence phylogenetic inferences on the more complete taxa (e.g. extant taxa).

The most common way to deal with a majority of missing data values for one taxon would be its elimination from the analysis. This is of course not feasible in this particular case, because it would defy the very purpose of aDNA work. Another option would be the selective inclusion of alignment sections with complete data across all taxa (Fig. 2B, cutting approach). This however, would lead to extremely short alignments, since the regular aDNA sequence amounts to roughly 150 to 500 bp length. In the past decade however, several researchers challenged the common notion of inherent difficulty of incomplete taxa, and suggested that there is not necessarily a direct relationship between the degree of incompleteness and the resulting phylogenetic placement of a taxon (Wiens 2003, 2005, Philippe et al. 2004). In fact, recent computer simulations clearly showed that an accurate placement of highly incomplete taxa is possible provided a sufficient number of overall characters are present (Wiens 2003, 2005). This would result in the complete inclusion of the highly incomplete taxa (Fig. 2C, missing data approach).

We used a sample dataset of complete 18S rDNA (1866 bp) to test the two approaches under a simple absolute congruence scenario. Five ingroup (Hymenoptera) and two outgroup taxa (Ephemeroptera) were included in the analysis. First, the complete dataset was analyzed under exhaustive searches under both maximum parsimony and maximum likelihood (PAUP*, Swofford 2002). Nonparametric bootstrap values (1000/100 bootstrap replicates, 100/10 heuristic random addition replicates, heuristic search) were calculated using PAUP* 4.0b10 (Swofford 2002). Both analyses converged on the same topologies (Fig. 3).



Then each one of the ingroup taxa was cut down to 150 bp in two arbitrarily chosen places in sequential order. Both datasets of this particular taxon were subjected to the cutting, and the missing data alignment approach, bringing the number of total analyses to 40 [5 taxa ´ 2 subsets ´ 2 alignments ´ 2 optimality criteria]. Resulting topologies were compared for topological congruence with the tree derived from the complete dataset. In case of alternative relationships, trees were subjected to the Shimodaira-Hasegawa test (SH test, 1999) by comparing them to the single "best" ML tree from the complete dataset. The SH test was performed with 10,000 replicates, resampling the partial likelihoods for each site (RELL model) using PAUP*.

In the overwhelming majority of cases (16 out of 20), the missing data approach recovered the original topology under both optimality criteria (absolute congruence), whereas the cutting approach only reached this result four times (all Maximum Likelihood, Table). Both optimality criteria seemed to perform equally well under the missing data approach. All other hypotheses were significantly different from the best ML topology in the SH test (Table). Therefore aDNA analyses should be conducted with the maximum number of extant characters possible, and the trimming of the dataset to exclude missing characters should be avoided.



Before setting out to obtain aDNA sequences that are to be used to answer specific questions related to the phylogenetic history of that particular taxon, researchers should keep the following in mind: (1) inherently, aDNA sequences will always contain high amounts of missing data; (2) combined analyses of different genes have been shown to provide greater explanatory power. Therefore, multiple genes should be targeted for an aDNA analysis; (3) exploratory analysis of available extant taxa can provide a priori clues as to which gene regions to sequence; (4) generally, alignments containing aDNA sequences should not be cut as to avoid missing data, but should be included into a substantial dataset of extant sequences with un-obtainable characters coded as missing.



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Received 20 July 2006
Accepted 16 October 2006


1 Corresponding author and present address: University of Wyoming, College of Agriculture, Department of Molecular Biology, 1000 University Ave., Laramie, Wyoming, 82071, US.

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