Contributions to Zoology, 68 (2) 73-81 (1999)
Phylogenetic relationships of the European newts (genus Triturus) tested with mitochondrial DNA sequence data
I. Zajc , J. W. Arntzen
Keywords: 12S rRNA, ATPase, mitochondrial DNA, newt, phylogeny, Triturus
European newts (genus Triturus) are widely studied, but their phylogeny is not yet unambiguously resolved. Fragments of mitochondrial DNA experiencing different rates of evolution (the ATPase and 12S rRNA genes) were sequenced in order to test a phylogenetic hypothesis derived from biochemical and behavioral data. Well-supported branches of the existing phylogeny gained support in our study. The monophyletic origin of the hypothesized T. boscai - T. italicus clade remained ambiguous, whereas strong support was gained for the sister-taxon relationship of T. vulgaris and T. montandoni. The position of T. vittatus as a sister taxon to the T. marmoratus species group was also supported. The phylogenetic position of T. alpestris could not be clarified. With an in-group taxon sampling denser than in previous molecular phylogenetic studies and under the a priori selection of species from the genera Cynops, Neurergus and Paramesotriton as out-groups, the monophyly of Triturus was strongly supported. It cannot be excluded, however, that the presumed out-group actually belongs to the in-group, rendering Triturus paraphyletic as was concluded from recently published 12S and 16S rRNA sequence data.
Phylogenies are a cornerstone to the study of evolution: without them we are unable to reconstruct and understand pattern and process of evolutionary change. To obtain a robust phylogenetic hypothesis requires the gathering of multiple independent and complementary data sets, because usually no single data set is sufficiently powerful to simultaneously resolve older and more recent cladogenic events.
The genus Triturus (European newts) has been the subject of extensive phylogenetic analysis with various data sets [osteological (Bolkay, 1928; Rafinski and Pecio, 1989), immunological (Busack et al., 1988), morphological (Giacoma and Balletto, 1988), biochemical (Rafinski and Arntzen, 1987), behavioral (Arntzen and Sparreboom, 1989), cytogenetic (Macgregor et al., 1990), and is considered by some to be the phylogenetically best studied genus in the world (Halliday and Arano, 1991). Despite these concerted efforts, its phylogeny is not fully resolved and several competing hypotheses are available, none of them with unambiguous overall support. We take the study by Arntzen and Sparreboom (1989) as the basis of our work because the phylogeny they present is based on two independent and complementary data sets. Moreover, their hypothesis resolves the earlier as well as the later events in the Triturus radiation and is robust under the jack-knife test.
Nine or twelve Triturus species are currently recognized, depending on the criteria used for species recognition. Here we refer to twelve species, with four of them (Triturus cristatus, T. carnifex, T. karelinii and T. dobrogicus) grouped together in the T. cristatus superspecies (Wallis and Arntzen, 1989). The ‘big’- and ‘medium-sized’ newts (T. cristatus, T. marmoratus, T. vittatus and T. alpestris) are organized in the subgenus Triturus and the ‘small-bodied’ newts (T. boscai, T. helveticus, T. italicus, T. montandoni and T. vulgaris) are placed in the subgenus Palaeotriton. The detailed configuration is given in Fig. 1, alongside with the nomenclature that we adopt. For the following sections of the phylogenetic tree the support is limited or contradictory: 1) the sister taxon status of T. vulgaris and T. montandoni is supported by allozyme data only; 2) the monophyly of T. boscai and T. italicus hinges on the interpretation given to behavioral characters; 3) the status of T. vittatus as the sister taxon of the T. marmoratus species group may be called into question on the basis of its overall similarity to some small bodied species, T. vulgaris in particular; 4) the support for the position of T. alpestris in the subgenus Triturus is relatively weak due to the non-independence of some behavioral synapomorphies; 5) the monophyly of Triturus, traditionally taken for granted, has been put in doubt by molecular data (Titus and Larson, 1995).
Fig. 1. Phylogeny for the genus Triturus based on available biochemical and behavioral characters. Numbers refer to specific questions as addressed in the text. Outgroup taxa are taken from the genera Cynops, Neurergus, and Paramesotriton.
We tested the phylogenetic hypothesis of Triturus with a newly generated set of independent data. Two fragments of the mitochondrial (mt)DNA molecule experiencing different evolutionary rates were studied. The slowly evolving 12S rRNA gene (Kocher et al., 1989; Hickson et al., 1996) was partially sequenced to test some of the supposedly earlier events of the Triturus radiation, while the fast evolving ATPase gene (Kumar, 1996) was partially sequenced to test the branching order among some supposedly closely related species in the T. vulgaris species group. Sequence data for cytochrome b produced ambiguous phylogenetic results, perhaps due to the comparison of non-homologous sequences (Caccone et al., 1997; cf. Zhang & Hewit, 1996) and were discarded.
Materials and methods
Two specimens were selected from each of nine Triturus species, if possible from different localities. As out-groups to the genus, representatives of the salamandrid genera Cynops, Neurergus and Paramesotriton were selected, following Arntzen and Sparreboom (1989). Triturus boscai was taken as out-group to the T. vulgaris species group. Specimens for which DNA was extracted were sampled as follows: T. alpestris from Mayenne, France and Pola de Sierra, Spain (subspecies cyreni); T. boscai from Toledo, Spain; T. cristatus from Limanowa, Poland and Sinaia, Romania; T. helveticus from Ambleteuse, France and Canterbury, U. K.; T. italicus from Conversano, Italy; T. marmoratus from El Berrueco nr. Madrid, Spain and Rochechouart, France; T. montandoni from Ustrzyki, Poland; T. vittatus from an unknown locality inIsrael and from Adapazari, Turkey (subspecies ophryticus) and T. vulgaris from Ambleteuse, France. Out-group taxa Cynops ensicauda, Neurergus strauchii and Paramesotriton sp. were obtained from the pet trade with no reliable locality information.
Total DNA was extracted from approximately 50 mg of frozen or ethanol-preserved tissue (liver, heart or tail muscle) following standard protocols (Sambrook et al., 1989). For PCR-amplification of the 12S rRNA region the forward (L2475) and reverse (H2897) primers were used (Kocher et al., 1989). The forward and reverse ATPase primers L9858 (5‘-CTCCTCCTTAATGATATGCCACA-3‘) and H10307 (3‘-TTCACCCCAACTACCCAACTATC-5‘) were designed in our laboratory by G. Rowe. Double stranded DNA amplifications were performed using standard PCR protocols (Erlich, 1989) with negative controls to test for contamination. The 12S rRNA fragment was amplified under stringent conditions (annealing temperature 65 C, extension time 25 seconds, up to 28 cycles), while the ATPase primers required milder conditions (annealing temperature 45-50 C, extension time of 60 seconds, minimum of 30 cycles). The amplified products were separated on 1.5 % low melting point agarose gels, cut out of the gel and purified by successive phenol, phenol-chloroform and chloroform-isoamylalcohol extractions, followed by ethanol precipitation. Both strands were directly sequenced using the dideoxy chain-termination method (Sanger et al., 1977). Multiple sequences were aligned with Clustal-V software (Higgins and Sharp, 1988) with manual adjustments. The ATPase gene sequences were translated into amino-acid sequences and the reading frame was determined by alignment to sequences of the teleost fish Cottus kessleri (Grachev et al., 1992) and human sequences (Arnason et al., 1996) obtained from GenBank. When the sequences for individuals of the same species were not identical, the consensussequence was used in the phylogenetic analysis with variable sites taken as polymorphisms. The percent sequence divergence was calculated following Mindell and Honeycutt (1990). We aligned our sequences to the published salamandrid 12S sequences (Caccone et al., 1994; Hay et al., 1995), that include the in-group taxa T. vulgaris and T. carnifex and out-group taxa from the genera Euproctus, Notophthalmus, Pleurodeles and Salamandra. Furthermore, the sequences were aligned to those published by Titus & Larson (1995), that include T. alpestris, T. karelinii and representatives of the salamandrid genera Chioglossa, Cynops, Euproctus, Neurergus, Mertensiella, Notophthalmus, Pachytriton, Paramesotriton, Pleurodeles, Salamandra, Salamandrina, Taricha and Tylototriton.
Phylogenetic reconstruction was performed under the principle of parsimony, using the PAUP 3.1.1 software (Swofford, 1991) with the ’branch-and-bound’ search option (the ‘heuristic search’ method was used in the bootstrap runs). Two a priori selected strategies were followed. First, a uniform transition - transversion ratio and uniform positional ratio was used, i.e., ‘no weighting’. Second, different weights were assigned to transitions versus transversions inversely proportional to the frequency of different substitutions among in-group taxa as traced from the tree obtained with the unweighted approach, with 24 as the base number. Gap derived character-state changes were given the same weight as transversions, also on an a priori basis. Positional weighting factors for the first, second, and the third codon positions were determined for the protein coding DNA fragment (ATPase) in a similar way. Bootstrap scores (Felsenstein, 1985; Hillis and Bull, 1993) were determined over 5000 replications to obtain an impression of the strength of support from the data for the phylogenetic tree showing maximum parsimony. Templeton’s (1983) non-parametric test was used to test one-sided for the significance of differences between documented phylogenies and the newly derived alternatives.
Aligned sequences represent 212 bp of the ATPase gene and covers the short stretch of overlap between regions coding for ATPase 6 and ATPase 8 (Fig. 2). The first nucleotide corresponds to position 256 in the Cottus kessleri ATPase sequence (Grachev et al., 1992). Aligned sequences represent 301 bp of 12S rRNA gene. The first nucleotide corresponds to position 233 in the Mertensiella luschani 12S rRNA sequence (Titus and Larson, 1995). No insertions or deletions (‘indels’) had to be inferred to align the ATPase sequences. Alignment of the 12S sequences required two indels at one position among the Triturus species and two indels covering four positions in the out-group taxa. More indels on nine positions were required when published sequences were added to the data set, lengthening the fragment to 305 positions, with no regions of ambiguous alignment (sensu Titus and Larson, 1995).
One-hundred and sixty-one of the 212 ATPase nucleotide positions were identical across the four species studied and 38 variable positions differed by a single substitution in one taxon (Fig. 2). This left 13 (6.1 %) nucleotide positions with potentially, phylogenetically relevant information. The sequence difference between T. boscai and in-group taxa ranged between 18.9 % and 19.3 %. Within the vulgaris species group the distances were considerably lower, ranging from 3.3 % between T. montandoni and T. vulgaris to 10.4 % between T. montandoni and T. helveticus. Taking the short stretch of sequence overlap between both ATPase genes into account, the transition: transversion ratio among in-group taxa was 3.2 and the number of substitutions at the first, second, and third codon position were 5, 3, and 17, respectively. Following these relationships, the weights assigned to transitions versus transversions were 3 and 21 whereas the weights applied to the first, second and third codon positions were 8, 14, and 2. At the level of amino acid codon usage, 16 variable characters were found with nine synapomorphic character states for the T. helveticus - T. montandoni - T. vulgaris clade, four autapomorphic characters states for T. helveticus and three synapomorphies were found for T. montandoni - T. vulgaris. The DNA sequences of T. vulgaris and T. montandoni differed only by silent substitutions.
Two-hundred and thirty-five of the 301 nucleotide positions on the 12S fragment were identical across the 12 taxa studied and 25 variable positions differed by a single substitution in one taxon, leaving 41 (13.6%) phylogenetically informative sites (Fig. 2). Half the number of variable sites are found in one-third of the 12S fragment, at the 3’-end. The sequence difference between out-group and in-group taxa ranged between 6.0 % and 14.0 %. Within the genus Triturus the distances were considerably lower, ranging from 0.7 % between T. montandoni and T. vulgaris [which is less than that found within T. alpestris (3.0 %) and T. vittatus (1.7 %)] to 10.6 % between T. cristatus and T. alpestris. The average sequence difference between in-group species and an out-group taxon ranged from 7.4 % (Cynops) to 11.2 % (Neurergus). The transition:transversion ratio among in-group taxa was 2.8 and weights determined were 4 and 20.
Given Cynops, Neurergus and Paramesotriton as out-groups, the monophyly of Triturus appears to be strongly supported with a 89 % bootstrap replication score (Pb) (Fig. 3). Two major in-group clusters are formed. The first, with Pb = 85 %, consists of T. vittatus as a sister taxon to the T. marmoratus species group (Pb = 98 %). The second group contains T. alpestris and all small-bodied Triturus species, with a low bootstrap support (Pb = 59 %). Within this group, the monophyly of the T. vulgaris species group is strongly supported (Pb = 100 %), whereas the relationship of T. alpestris, T. boscai, and T. italicus among themselves is poorly resolved (Pb < 55 %). On the basis of 12S sequence data no firm conclusion can be drawn regarding the branching order within the vulgaris species group. However, the ATPase sequence data provided strong support for the sister taxon status of T. montandoni and T. vulgaris (Pb = 96, Fig. 3) and the same bootstrap value was observed when amino acid sequence data were analyzed instead of the DNA sequence. Under the weighting scheme topologically similar results were obtained, with three reservations : 1) with the ATPase gene fragment, a score of Pb = 100 was observed for the vulgaris - montandoni clade, while with the 12S rRNA gene fragment; 2) generally less phylogenetic resolution and lower bootstrap replication scores were observed on well-established branches; and, 3) an equally parsimonious solution was found in which the genus Triturus is paraphyletic. Sequences derived from the same or congeneric species by different authors (Caccone et al., 1994; Titus & Larson, 1995; Hay et al., 1995 and present study) are very similar to one another and consequently the taxa they represent are placed close together in the phylogenetic tree that has maximum parsimony (i.e., Cynops sp., Neurergus strauchii, Notophthalmus viridescens, Paramesotriton sp., Pleurodeles waltl, Salamandra salamandra, Triturus alpestris, T. cristatus superspecies and T. vulgaris).
With a wide range of out-groups included in the analysis, the monophyly of the genus Triturus was not supported. The sister clade to the subgenus Palaeotriton would not be the subgenus Triturus (i.e., T. vittatus, T. marmoratus, and the T. cristatus superspecies), but the subgenus Triturus plus Taricha and Notophthalmus and the sister clade to this group would be composed of Cynops, Euproctus, Neurergus, Pachytriton, and Paramesotriton (Fig. 4A). Templeton’s test indicated that the difference between the shortest tree and the tree in which Triturus is monophyletic was significant (P < 0. 05).
We have tested an existing phylogeny of Triturus against independent molecular data. Two PCR-amplified fragments of the mtDNA molecule with relatively low (12S rRNA) and high rates of evolution (ATPase) were employed to test some of the earlier and more recent speciation events of the Triturus radiation, respectively. The choosen fragments showed evolutionary rates that were anticipated and that were appropriate for addressing phylogenetic questions at this taxonomic level. Faster evolving protein-coding ATPase genes can be reliable tracers of evolutionary history among close relatives (as within the T. vulgaris species group), where silent, third codon position substitutions account for most variation (Zardoya and Meyer, 1996). On the other hand, if the saturation of transitions is suspected, it is imperative to reduce the emphasis on, or even eliminate, this class of substitutions from phylogenetic analysis (Disotell et al., 1992; Knight and Mindell, 1993). Although intuitively appealing, differential weighting may downweight informative transitions in conserved regions and upweight transversions primarily present in more variable regions of the molecule, thereby obscuring phylogenetic relationships (Titus and Larson, 1995; Mindell and Tacker, 1996). Given the observed transition bias, saturation was not prevalent among the compared sequences. The observation that with weighting the bootstrap support for congruent sections of competing phylogenetic solutions dropped and the phylogenetic resolution decreased, supports this view.
Our analysis presents a hypothesis that is concordant with well-supported areas in the established Triturus phylogeny. As to the specific questions we raised, some appear to be solved while others are not. The new data do not help to elucidate the relationship between T. boscai and T. italicus. Their monophyly was suggested by allozyme data and, ambiguously, by behavioral data (Arntzen and Sparreboom, 1989). The observation of ‘flamenco’ behavior in the sexual repertoire of T. helveticus (M. Faria, pers. comm.) and T. marmoratus pygmaeus (T. Halliday, pers. comm., M. Sparreboom, pers. comm.) further erodes the support for T. boscai - T. italicus monophyly. Similarly, the current study does not convincingly clarify the position of T. alpestris. The phylogenetic position of this species could also not be satisfactorily resolved from behavioral data (Arntzen and Sparreboom, 1989). Bolkay (1928) placed it in a third subgenus with intermediate characteristics, in between Triturus and Paleaotriton.
The 12S rRNA sequence data support the placement of T. vittatus as the sister taxon to the T. marmoratus species group in the subgenus Triturus, as was inferred from its breeding behavior (Arntzen and Sparreboom, 1989). This challenges the inferences from the phenetic analyses of osteological (Rafinski and Pecio, 1989), karyological (Bucci-Inocenti et al., 1983), morphological (Giacoma and Balletto, 1988) and immunological data (Busack et al., 1988). According to these studies, T. vittatus is more similar to T. alpestris or the small-bodied newts of the subgenus Palaeotriton. Immunological data (Busack et al., 1988) suggested a sister taxon relationship for T. v. vulgaris and T. helveticus. The ATPase sequences provide strong support for the monophyly of T. vulgaris and T. montandoni, in line with allozyme data (Rafinski and Arntzen, 1987).
The rate of 12S rRNA molecular evolution in some Palearctic salamandrids was calibrated as approximately 3 mA of lineage independence per percent sequence divergence and 7 mA per percent sequence divergence when only transversions are considered (Caccone et al., 1994). Applying these rates provides an estimate of 12-19 mA for the age of the T. vittatus lineage (versus the T. marmoratus species group) in the subgenus Triturus and an estimate of 14-19 mA for the T. boscai lineage (versus the T. vulgaris species group) within the subgenus Palaeotriton. This is in line with estimates from fossil, biochemical, molecular and biogeographical data that converge to 14-15 mA for the T. vittatus lineage and to 13-15 mA for the T. boscai lineage (reviewed in Oosterbroek and Arntzen, 1992).
The monophyly of the genus Triturus, although widely accepted, is defined on the basis of somewhat vague character state descriptions (‘a suite of behavioral character states’ and ‘a high level of sexual dimorphism’ - Halliday, 1977), that are not explicit synapomorphies. Moreover, a feature such as the potential for interbreeding (Wolterstorff and Herre, 1935) is explicitely a plesiomorphy.The monophyly of the genus Triturus has recently been put into question in a molecular phylogenetic study of the family Salamandridae (Titus and Larson, 1995) and in a molecular biogeographical study of Euproctus (Caccone et al., 1994). In both of them only two Triturus species were involved. With just the representatives of the genera Cynops, Neurergus and Paramesotriton as out-groups, a denser in-group taxon sampling does not challenge the hypothesis of Triturus monophyly. However, 12S and 16S mtDNA sequence data indicate that the genera Cynops, Neurergus and Paramesotriton themselves may be in-group taxa relative to Triturus (Fig. 4B). In the light of these results, their a priori choice as out-groups to the genus Triturus may have been unfortunate.
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We acknowledge the laboratory facilities provided by Prof. T. Burke, the assistance of Dr. D. Thomaz, and the constructive comments of Drs. A. Zuiderwijk and an anonymous reviewer.