Scaly-tailed squirrels and SuperTrees: 
The use of different analysis methods for an Ancient DNA problem

Anja C. Schunke^a, Rainer Hutterer^a, Heiko Schmidt^b,c,d,e

^a Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, D-53113 Bonn, Germany
^b Center for Integrative Bioinformatics Vienna (CIBIV), Max F. Perutz Laboratories (MFPL), Dr. Bohr Gasse 9, A-1030 Vienna, Austria; ^c University Vienna, ^d Medical University Vienna, ^e University of Veterinary Medicine, Vienna, Austria

Abstract

The phylogenetic relationships between species of the African scaly-tailed squirrels (Anomaluridae; Rodentia) have so far not been investigated with molecular methods, mainly because of the poor availability of suitable material. In this project cytochrome b fragments from museum material have been sequenced. As the tissue samples were generally not prepared for molecular analyses the data set from this ‘Ancient DNA’ showed numerous gaps. In order to include as many information as possible the data set was analysed with several methods for data combination.
SuperTree and related methods have originally been developed in order to combine large and divergent data sets, mostly published cladograms or sequences from different genes produced in several laboratories for the same organisms or organism group. In this study the applicability of three combination methods has been tested for the anomalurid ‘Ancient DNA’ data set. A cladogram which matches the distribution of morphological characters could be found with ‘medium level’ approaches as well as with a certain, short fragment of cytochrome b.

Keywords: Anomaluridae; Rodentia; cytochrome b; Ancient DNA; SuperTree methods

1. Introduction

The African scaly-tailed squirrels consist of seven species which occur in large parts of the Afrotropical rain, mountain, and gallery forests. At present the family is subdivided in two subfamilies. The larger species are united in the Anomalurinae with Anomalurus derbianus, A. pelii, and A. pusillus and A. beecrofti, who is sometimes placed in a separate genus Anomalurops. The Zenkerellinae contain the smaller species Idiurus macrotis, I. zenkeri, and the enigmatic Zenkerella insignis, the only member of the group without the ability to perform gliding flight. The often peculiar characters of the anomalurids make analyses of the phylogenetic relationships within the group and  an outgroup comparison difficult, because frequently none of the different characters or character states is represented in any other rodent group or it seems to be distributed arbitrarily within rodents. Therefore the use of molecular data was a promising approach to gain additional insight into anomalurid systematics. The analyses were performed with fragments of the cytochrome b gene for two reasons. First, being a mitochondrial gene, it was originally available in high numbers of copies in every cell, so the probability of recovering at least a few templates is much higher than for nuclear genes, and second, this gene has proven useful for analyses of phylogenetic relationships within rodent families in numerous previous investigations. Unfortunately, a direct collection of genetic material was not possible due to the extremely large area inhabited by the group, including regions of civil war, occurrence of diseases like Ebola, and areas where anomalurids are protected. Hence, the analysis had to be based on collection material. Anomalurids are very well represented in museums world wide, so it was not difficult to get tissue samples. The problem was the fact that the majority of specimens were not prepared for DNA analyses which resulted in typical problems like very little DNA content in the samples, the remaining DNA being more or less degraded, a high amount of diverse and frequently not documented chemicals from preparation and conservation, and high risk of (cross)contamination (for review see Pääbo et al., 2004). This made the investigation an ‘Ancient DNA’ project resulting in a fragmentary data set with a high amount of missing data. However, as presently no better sequences are available, it was an interesting attempt to get at least some insights into anomalurid systematics from molecular data.
Three approaches have been described for the combined analysis of data sets with missing data. In the ‘total evidence’ or SuperMatrix approach the data sets are concatenated directly producing one overall alignment, the so-called SuperMatrix, which is used to reconstruct a phylogenetic tree. The second (SuperTree) approach reconstructs one phylogenetic tree for each data set. All these trees serve as input in a subsequent SuperTree analysis which combines all these overlapping trees into one overall tree, the so-called SuperTree. These two approaches can be classified into ‘low level methods’ (SuperMatrix approaches) and ‘high level methods’ (SuperTree approaches), according to the distance of the combination from the underlying data (Schmidt, 2003, chap. 7.; note, that ‘high’ and ‘low’ do not imply quality.)
Recently, a third ‘medium level method’ has been suggested (Schmidt, 2003, chap. 7). The so-called ‘SuperQuartet Puzzling’ method (SQP) first reconstructs all possible quartet trees, i.e. unrooted trees with four taxa, using the maximum likelihood framework (ML, cf. e.g. Felsenstein, 1981) for each of the data sets separately. The different sets of quartets are combined according to their likelihoods into a set of so-called ‘SuperQuartets’ which is then used to reconstruct an overall tree applying a method related to ‘Quartet Puzzling’ (Strimmer & von Haeseler, 1996; for details concerning SQP see Schmidt, 2003, chap. 7).
Although there has been a long debate about whether SuperTree or SuperMatrix methods have to be preferred (see de Queiroz et al., 1995, and Bininda-Emonds, 2002, for review), it has been concluded recently that these methods should not be seen as competitive methods but should be used complementary (Bininda-Emonds, 2004). Accordingly, several analysis methods were tested to extract the maximum possible amount of information from the sequence data set. 
The SuperMatrix of all fragments was analysed with two different ML methods as ‘total evidence’ approach. Furthermore, the most abundant SuperTree approach MRP (Matrix Representation with Parsimony; Baum, 1992; Ragan, 1992) has been used as well as the novel SQP method.

2. Material and methods

2.1. Data basis

The material for this study came from dried skins or material stored in ethanol from 10 collections of the following institutes: Museum National d’Histoire Naturelle (MNHN, Paris), Musée Royal d’Afrique Centrale (MRAC, Tervuren), Naturhistorisches Museum Basel (NHMB, Basel), Naturalis/Nationaal Natuurhistorisch Museum (RMNH, Leiden), Naturmuseum Senckenberg (SMF, Frankfurt), Staatliches Museum für Naturkunde (SMNS, Stuttgart), Zoologisches Forschungsmuseum Alexander Koenig (ZFMK, Bonn), Zoölogisch Museum Amsterdam (ZMA, Amsterdam), Museum für Naturkunde (ZMB, Berlin), and Zadock Thompson Natural History Collections, Vermont. 68 tissue samples were taken from all species: A. beecrofti (14), A. derbianus (18), A. pelii (12), A. pusillus (6), I. macrotis (8), I. zenkeri (8), and Z. insignis (2). The African springhare Pedetes capensis was used as outgroup, because this species is considered to be the closest relative of the anomalurids based on morphological and molecular data (e.g. Tullberg, 1899; Winge, 1924; Simpson, 1945; Bugge, 1974, 1985; Montgelard et al., 2001, 2002; Huchon et al., 2002). Additionally, the following sequences from GenBank were used: Anomalurus spec. AJ389526.1, Idiurus macrotis AJ389525.1, Pedetes capensis U59176.1, U59177.1, and U59178.1 and P. surdaster AJ389527.1.

2.2. DNA extraction and sequencing

Initially several DNA-extraction methods were tested, but the following protocol has generally shown to give the best results. Small pieces of tissue were placed in 200 µl Wilson-Buffer (3.025 g Tris, 0.9 g EDTA, 1.4 g NaCL, 250 ml ddH_20, pH 8.0, store at room temperature),  incubated at room temperature for 1 to 4 hrs and homogenated with a micropestle. Than another 200 µl Wilson-Buffer and 2 µl RNAse (10 mg/ml) were added and incubated at room temperature for 5 min. After adding 20 µl Proteinase K (25 mg/ml) and 40 µl SDS solution (10%) the sample was incubated at 55°C for 2 to 48 hrs. If the tissue dissolves badly, 2 to 5 µl of Proteinase K were added every 6 to 12 hrs. DNA was extracted by adding 600 µl Phenol/Dichlormethane/3-Methyl-1-butanol 25:24:1, vortexing and centrifuging for 10 min at 13000 rpm. The upper phase was transferred to a new tube, 600 µl of Dichlormethane/3-Methyl-1-butanol 24:1 added and after vortexing centrifuged for 10 min at 13000 rpm before transferring the upper phase carefully to new tube (avoid any traces of Dichlormethane/3-Methyl-1-butanol). For precipitation 40 µl Sodium acetate solution (3 M) and 1 ml Ethanol (96%) were added and carefully mixed. If no DNA was visible, the sample was kept at -20°C for 15 min to 2 hrs. The tube was centrifuged for 10 min at 13000 rpm, the supernatant discarded, 400 µl Ethanol 70% added and again centrifuged for 5 min at 13000 rpm before discarding the supernatant. DNA was dried at room temperature and dissolved in 20 to 50 µl ddH_20.
The study was started with a set of primers taken from Da Silva & Patton (1993). With combinations of these it was possible to obtain sequences of the first ca 800bp of the cytochrome b gene. On this basis it was possible to design several primer pairs for fragments of ca 200-300bp. The three primer pairs that worked best and were used for the study were the following: AnoL1 CTAATGACCAATATACGAAAAAC and AnoH1 AATGAATAGGCAGATGAAGAA (fragment a), AnoL2 AACTCACAAYTGCCGAGACG and AnoH2 RATAGCCGATAGGAGGTTT (fragment b), and MVZ 23 TACTCTTCCTCCACGAAACAGGITC and MVZ 16-H AAATAGGAAYTATCAVTCTGGTTTYAT (fragment c, primers modified after Da Silva & Patton, 1993; Fig. 1). PCR and cycle sequencing reactions were conducted either in a Biometra T-Gradient or Applied BioSystems Gene Amp PCR System 2700 thermocycler with the same primers. The PCR was carried out using a PCR-Kit from Sigma. One 25 µl reaction contained 3.0 or 5.0 µl MgCl_2 (25 mM) and 17.2 or 15.2 µl ddH_2O (depending on primer combination), 2.5 µl 10x Buffer, 0.5 µl dNTP mix (2 mM), 0.3 µl of each primer (20 pM) and 0.2 µl Taq-Polymerase (5 U/µl). The PCR was run with 5 min at 95°C for initional melting of the DNA, 35 cycles of 20 s at 94°C, 20 s at 49°C and 45 s at 72°C and a final elongation of the DNA for 8 min at 71°C. The results were checked by gel electrophoresis. If the PCR products were weak, the PCR was repeated up to five times and the products pooled before continuing. PCR products were cleaned with a Qiagene Gel Extraction Kit according to the manual and up to 12 µl cleaned PCR product dried in reaction tubes for the cycle sequencing reaction with ABI Prism® Big Dye™ Terminater Cycle Sequencing Kit. 2.0 µl ReadyMix, 1.0 µl MgCL (12,5 mM), 1 µl Primer (20 pM) and 6.0 µl ddH_20 were added. The cycle sequencing reaction was run with 2 min at 94°C, 25 cycles of 15 s at 92°C, 15 s at 50°C and 2 min 30 s at 60°C and 8 cycles of 20 s at 93°C and 15 s at 60°C. Cycle sequencing products were cleaned by transferring the product to a new sample tube and adding40 µl ddH_20, 5 µl Sodium acetate (3 M) and 125 µl Ethanol (100%) and centrifuging 20 min at 13000 rpm. The supernatant was discarded and the pellet washed with 300 µl of freshly diluted Ethanol (70%), again centrifuged for 5 min at 13000 rpm, the supernatant discarded and the pellet dried at room temperature prior to dissolving in 4 µl of stop buffer (5 ml Formamid, 1 ml Blue-Dextran ??). The products were used for direct sequencing in an ABI Prism 377-96 Sequencer. All samples were used at least two times with every pair of primers, generally more often, especially when already one fragment had been amplified.

2.3. Data analysis

The obtained sequence fragments were aligned using the CLUSTAL W program (Thompson et al., 1994) and corrected manually using BioEdit 5.0.9. (Hall, 1999).
Network analyses have been performed applying the NeighborNet method (Bryant & Moulton, 2004) implemented in the SplitsTree program (version 4beta, Huson, 1998) with default parameters.
For all different data sets as well as for the SuperMatrix, ML trees have been reconstructed using two ML methods, Quartet Puzzling as implemented in the TREE-PUZZLE package (Schmidt et al. 2002) and IQPNNI (Vinh & von Haeseler, 2004). The following options were used in the TREE-PUZZLE analysis: HKY model (Hasegawa et al., 1985) as model of evolution, 5000 puzzling steps, exact maximum likelihood estimation. For the IQPINNI reconstructions HKY model (Hasegawa et al., 1985) was used to model the evolutionary process. At least 100 iterations were run before the ‘stopping rule’ was applied to estimate the stopping time (Vinh & von Haeseler, 2004).
Missing parameters were estimated from sequence data. The ML trees constructed with IQPNNI were used as input trees for the MRP SuperTree analyses (Baum, 1992; Ragan, 1992). Tree topologies have been encoded to the so-called ‘matrix representation’ using the program SuperTree version 0.85b by Salamin et al. (2002) and applying the coding scheme by  Ragan (1992) and Baum (1992) as well as the scheme by Purvis (1995). The first encodes each branch of a rooted input tree by assigning a ‘1’ to all taxa of the respective branch. All other taxa in that tree get a ‘0’ and all missing taxa a ‘?’. Purvis (1995) encodes only sister groups, assigning ‘1’ to the taxa of one sister group ‘0’ to the other. Taxa in the rest of the tree and missing taxa are assigned ‘?’.
From the resulting binary matrix representations, trees were constructed as described by Ragan and Baum (both 1992) using PAUP* version 4.0b10 (Swofford, 2002).
The ‘medium level analyses’ have been performed using the implementation of the SQP method as implemented in an upcoming version of TREE-PUZZLE.

3. Results

The quality of the molecular data was strongly influenced by the bad condition of most tissue samples. Consequently it was only possible to amplify very small fragments of the cytochrome b gene. For samples with good DNA the entire 822 bp sequence could be obtained, for the others fragments of 187 to 247 bp were amplified. In spite of the very short length of fragments most of the samples gave no results. For the others the PCR product was as a rule weak and frequently had to be repeated up to five times and the products were pooled before cleaning. In several cases only one or two of the fragments could be amplified (Tab. 1).

3.1. Low level combination or total evidence

In a first analysis the complete data set was tested with a NeighborNet analysis (Fig. 2). The results show that specimens clustered according to the available fragments rather than to the species. An analysis of the complete data set with PAUP produced a badly resolved cladogram, where only the specimens of A. pusillus are considered monophyletic (results not shown). However, when the cladogram is forced into complete resolution with IQPNNI the results correspond to the currently accepted taxonomy of the anomalurids (Fig. 3).
For the next step three separate analyses were made for each of the fragments (Fig. 4). The resulting cladograms were better resolved due to the completeness of data within the subsamples, but those for the first two fragments showed several outliers. A single specimen of A. pelii was found within A. derbianus, and single specimens of A. beecrofti and Idiurus clustered within other (but different) species. Apart from these outliers the species were generally found monophyletic. The best resolution was found with the shortest fragment c, where the cladogram also matched the generally accepted taxonomy of the group. Idiurus is found monophyletic, although the two species could not be resolved. The Anomalurinae are also found monophyletic with Anomalurops being the sister group of the three Anomalurus species and A. pusillus the sister group of the unresolved A. derbianus and A. pelii.

3.2. High level combination or SuperTree

The following analyses were run with two different combinations of the available data with regard to the treatment of the overlapping region o of the first two fragments (Fig. 1). In the first case all three complete fragments a, b, and c were used, thus using the overlapping part twice, if both fragments were sequenced for one specimen (‘3 fragments’). For the other case the overlapping part was used as an individual fragment and the two flanking fragments were shortened appropriately, so the overlapping region was only used once (‘4 fragments’). The analyses were run with Baum/Ragan coding as well as with Purvis coding leading to the four trees presented in Fig. 5. For the analysis with 3 fragments the resolution was bad, with one I. macrotis and two I. zenkeri being the sister group of the unresolved rest. Within this unresolved branch most of the species were found either monophyletic or unresolved, except for single outliers, and a mixed branch for A. derbianus and A. pelii. When 4 fragments were used the results were better resolved, but contradict each other. With the Baum/Ragan coding A. beecrofti appeared as the sister group of a clade mixed of A. derbianus and A. pelii, while A. pusillus is found as sister group of Idiurus. With the Purvis coding A. beecrofti shows up within Idiurus while A. derbianus, A. pelii and A. pusillus build the other branch.

3.3. Medium level combination

The medium level combination produced with 3 fragments a well resolved tree with monophyletic A. beecrofti, A. pusillus, Idiurus and a group mixed of A. derbianus and A. pelii (Fig. 6). The cladogram from 4 fragments had a very bad resolution, but it shows no contradictions with the first one.

4. Discussion

4.1. Comparison of the results

The results for the different methods differ in several aspects. As expected the total evidence analysis yielded a very bad resolution due to the high amount of missing data (more than 50%) in the data set. The network showed that in the SuperMatrix the availability of fragments for a specimen had a similar influence like the species it belongs to. Consequently the TREE-PUZZLE cladogram from the SuperMatrix is also badly resolved, although the resolution with IQPNNI brought results that matched the morphological classification.
In separate analyses of the three sequenced fragments the resolution is much better, but the results contradict each other. Additionally, the cladograms from the first two fragments contradict previous morphological and taxonomic results, while the tree found with the shortest fragment matches the currently accepted classification on the subfamilies and genera level. When the trees for each fragment were combined the resulting cladograms were badly resolved when 3 fragments were used and appearingly better resolved but contradicting with 4 fragments. Finally the medium level combination resulted in a relatively well resolved cladogram matching the morphological classification.
In summary the SuperTree analysis seems to be least suitable for this Ancient DNA data set, when the morphological classification is regarded as reliable. Cladograms matching this classification were obtained with IQPNNI for the SuperMatrix, TREE-PUZZLE for the shortest fragment c, and SQP for 3 fragments. The latter is not completely resolved, while in the cladogram from the shortest fragment some individuals are missing in cases where this fragment could not be amplified for the specimen.

4.2. Methodological remarks

In the anomalurid data set on the one hand basal splits are found less reliably than relationships between closely related taxa, on the other hand there are some cases of obviously closely related species that could not be resolved. The following points seem to have a great impact of the analysis of this kind of data: For every species there should be at least one complete sequence with all fragments, otherwise specimens tend to be grouped according to the fragments available rather than to their systematic position. Additionally, the smaller the given data set, the more problems are caused by sequences with numerous ambiguities. Finally, fragments used for analyses should not be too short because of lack of phylogenetic information (e.g. fragment o).
The shortes fragment c with less than 200 bp seems to have a remarkably high amount of phylogenetic information in this group. Since it is also relatively easy to amplify it should be tested for other taxa too, because it might proof useful for analyses with old collection material, from which it is not possible to obtain longer sequences.

4.3. Relationships of anomalurid species

Some of the taxa are clearly better supported than others. Though no problem with morphological data the separation of A. derbianus and A. pelii is obviously difficult with the sequenced fragments, but together they appear as monophyletic in the majority of cladograms. Also very stable is the species A. pusillus, which is one of the most reliably found taxa. A. pusillus is generally found as the sister group of the clade A. derbianus and A. pelii, and A. beecrofti as the sister group of the three Anomalurus species. The sister group of the four larger species, which are also considered as subfamily Anomalurinae, is Idiurus, member of the subfamily Zenkerellinae. Like A. derbianus and A. pelii the species I. macrotis and I. zenkeri are mixed in all cladograms. However, there is a clear congruence between the cladograms resulting in the phylogram given in Fig. 7. Unfortunately it had not been possible to get a sequence from Z. insignis, the only species not able to perform gliding flight, which is generally placed together with Idiurus in the subfamily Zenkerellinae.

Acknowledgements

Staff members of the following institutions kindly allowed access to the collections and taking tissue samples from the specimens: Michel Tranier, Jacques Cuisin and Jean-François Ducroz, Muséum National d’Histoire Naturelle (Paris), Wim Van Neer and Wim Wendelen, Musée Royal d’Afrique Centrale (Tervuren), Raffael Winkler and Urs Rahm, Naturhistorisches Museum Basel, Chris Smeenk, Naturalis (Leiden), Dieter Kock, Naturmuseum Senckenberg (Frankfurt), Miguel Vences and Adri G. Rol, Zoölogisch Museum Amsterdam (Amsterdam), Manfred Ade, Irene Thomas, and Detlef Willborn, Museum für Naturkunde (Berlin), and ??, Zadock Thompson Natural History Collections (Vermont). Financial support by the Deutsche Forschungsgemeinschaft to A.S. and R.H. (DFG, HU 430/1-1, 430/1-2) and the Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF) to H.A.S. is greatfully acknowledged. The visit of A.S. to the MNHN was funded by the TMR Programme of the European Commission (Parsyst Paris).

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Tab. 1. Cytochrome b fragments that could be amplified for the available tissue samples.

Species	Specimen number	sequenced fragments	GeneBank accession numbers
A. beecrofti	MNHN 1880-1460	
		MNHN 1955-659	a
		MNHN 1992-1206
		MNHN 1992-1211
		MNHN 1993-611
		MNHN 1993-1862
		MNHN 1994-598
		NHMB 7353
		NHMB 7354		a
		RMNH 38260
		ZFMK 64.501				c
		ZFMK 69.412
		ZFMK N.N.
		ZTNHC 1516		a	b	c
A. derbianus	MNHN 1939-73
		MNHN 1970-114
		MNHN 1984-1163
		MNHN 1992-1208
		MNHN 1992-1209
		MNHN 1992-1210
		MNHN 1992-1212
		MNHN 1995-3304
		MNHN 1998-971
		NHMB 7341
		NHMB 7345		a	b
		RMNH 25771
		ZFMK 61.957			b
		ZFMK 64.491
		ZFMK 64.817
		ZFMK 73.363		a	b	c
		ZFMK 68.1314
A. pelii		MNHN 1962-1175
		MNHN 1983-578			c
		MNHN 1983-579
		MNHN 1984-1160
		NHMB 3326
		ZMA 21.273				c
		ZMA 21.312
		ZMA 21.318
		ZMA 21.347				c
		ZMA 21.856		a	b	c
		ZMA 21.858
		ZMA 21.862
A. pusillus	MNHN 1965-191
		MNHN 1983-582	a	b	c
		MNHN 1992-1213
		NHMB 7349		a	b	c
		NHMB 7351				c
		SMNS N.N.		a	b	c
I. macrotis	MNHN 1908-61
		MNHN 1985-220	a		c
		NHMB 7357				c
		NHMB 7359
		SMNS 43325		a	b	c
		SMF 89949		a	b	c
		SMF 89950		a	b	c
		N. N.			a	b	c
I. zenkeri	MNHN 1966-1946		b
		MNHN 1992-1214		b
		NHMB 7360				c
		NHMB 7362		a		c
		ZMB 22747				c
		ZMB 36375
		ZMB 22762
		ZMB 22745
Z. insignis	MNHN 1970-112
		MRAC 28806

Fig. 1. Position of sequenced fragments on the cytochrome b gene.

Fig. 2. Network for all samples obtained with NeighbourNet method (Bryant & Moulton, 2004) as implemented in SplitsTree using default parameters (a, b, c: sequenced fragments for the specimens).

Fig. 3. Total evidence cladogram calculated with IQPNNI (a, b, c: sequenced fragments for the specimens, scale bar 0.1 mutation 100 pos., see text for details).

Fig. 4. TREE-PUZZLE trees for each of the fragments a, b, and c separately (numbers: puzzle support values, see text for details).

Fig. 5. Results of the MRP SuperTree analyses with Baum/Ragan (a, c) and Purvis (b, d) coding and 3 (a, b) and 4 (c, d) fragments respectively (a, b, c: sequenced fragments for the specimens, see text for details).

Fig. 6. Medium level trees obtained with SuperQuartet Puzzling for 3 (a) and 4 (b) fragments (a, b, c: sequenced fragments for the specimens, numbers: puzzle support values, see text for details).

Fig. 7. Most likely relationships between anomalurid species as derived from molecular and morphological characters (see text for details).

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