Contributions to Zoology, 68 (2) 73-81 (1999)

SPB Academic Publishing bv, The Hague

Phylogenetic relationships of the European newts (genus Triturus ) tested with

mitochondrial DNA sequence data

I. Zajc ¹ & J.W. Arntzen²

Department of Zoology, Leicester University, Leicester LEI 7RH, United Kingdom

Present addresses: 'National Institute ofBiology, Vecna pot 111, P.O. Box 141, 1001 Ljubljana, Slovenia

department of Zoology and Anthropology, Faculty of Sciences, University of Porto, CECA/ICETA/UP.

Campus Agrdrio de Vairdo, 4480 Vila do Conde, Portugal

Keywords : 12S rRNA, ATPase, mitochondrial DNA, newt, phylogeny, Triturus

Abstract Introduction

Phylogenies are a cornerstone to the study of evo- European newts (genus Triturus) are widely studied, but their lution: without them we are unable to reconstruct phylogeny is not yet unambiguously resolved. Fragments of and understand and mitochondrial DNA experiencing different rates of evolution pattern process of evolution-

and to (the ATPase 12S rRNA were in order To robust genes) sequenced ary change. obtain a phylogenetic hy- test a phylogenetic hypothesis derived from biochemical and pothesis requires the gathering of multiple inde- behavioral data. Well-supported branches of the existing and data pendent complementary sets, because usu- phylogeny gainedsupport in our study. The monophyletic origin ally no single data set is sufficiently powerful to ofthe hypothesized T. boscai — T. italicus clade remained am-

resolve older and more biguous, whereas strong support was gainedfor the sister-taxon simultaneously recent

of of relationship T. vulgaris and T. montandoni. The position cladogenetic events.

T. sister the T. vittatus as a taxon to marmoratus species group The genus Triturus (European newts) has been was also supported. The phylogenetic position of T. alpestris the subject of extensive phylogenetic analysis with could not be clarified. With an in-group taxon sampling den- various data sets [osteological 1928; ser than in previous molecular phylogenetic studies and under (Bolkay,

a selection of from Rafmski and Pecio, 1989), the priori species the genera Cynops, Neurer- immunological (Busack

gus and Paramesotriton as out-groups, the monophyly ofTriturus et al., 1988), morphological (Giacoma and Balletto, was It cannot be excluded, however, that strongly supported. 1988), biochemical (Rafmski and Arntzen, 1987), the presumed out-group actually belongs to the in-group, render- behavioral and (Arntzen Sparreboom, 1989), cy- ing Triturus paraphyletic as was concluded from recently pub- et al., and is con- lished 12S 16S rRNA data. togenetic (Macgregor 1990)], and sequence sidered by some to be the phylogenetically best

in the studied genus world (Halliday and Arano,

1991). Despite these concerted efforts, its phylog-

Contents eny is not fully resolved and several competing

with hypotheses are available, none of them un-

ambiguous overall support. We take the study by

Abstract 73 Arntzen and Sparreboom (1989) as the basis of

Introduction 73 our work because the phylogeny they present is Materials and methods 74 based on two independent and complementary data Results 75

sets. Moreover, their resolves the ear- Discussion 78 hypothesis

lier well the later events in Triturus Acknowledgements 80 as as the ra-

References 80 diation and is robust under the jack-knife test. 74 I. Zajc & J. W. Arntzen — Phytogeny of European newts

some behavioral synapomorphies; 5) the mono-

phyly of Triturus, traditionally taken for granted,

has been put in doubt by molecular data (Titus

and Larson, 1995).

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 ah, 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

some related among supposedly closely species

in the T. vulgaris species group. Sequence data

for b for the based on available cytochrome produced ambiguous phyloge- Fig. I. Phylogeny genus Triturus of biochemical and behavioral characters. Numbers refer to specific netic results, perhaps due to the comparison non-

addressed in the text. taxa taken questions as Out-group are homologous sequences (Caccone et al., 1997; cf. from and Paramesotriton. the genera Cynops, Neurergus, Zhang & Hewitt, 1996) and were discarded.

Nine or twelve Triturus species are currently Materials and methods

recognized, depending on the criteria used for spe-

cies recognition. Here we refer to twelve species, Two specimens were selected from each of nine

with four of them (Triturus cristatus, T. carnifex, Triturus species, if possible from different locali-

T. karelinii and T. dobrovicus) grouped together ties. As out-groups to the genus, representatives

in the T. cristatus superspecies (Wallis and Arntzen, of the salamandrid genera Cynops, Neurergus and

1989). The ‘big’- and ‘medium-sized’ newts (T. Paramesotriton were selected, following Arntzen

T. T. T. Triturus cristatus, marmoratus, vittatus and alpestris) and Sparreboom (1989). boscai was taken

are organized in the subgenus Triturus and the as out-group to the T. vulgaris species group. Speci-

‘small-bodied’ newts (T. boscai, T. helveticus, T. mens for which DNA was extracted were sampled

italicus, T. montandoniand T. vulgaris) are placed as follows: T. alpestris from Mayenne, France and in the subgenus Palaeotriton. The detailed con\ Pola de Sierra, Spain (subspecies cyreni)\ T. boscai

figuration is given in Fig. 1, alongside with the from Toledo, Spain; T. cristatus from Limanowa,

For T. from nomenclature that we adopt. the following Poland and Sinaia, Romania; helveticus

sections of the phylogenetic tree the support is Ambleteuse, France and Canterbury, U. K.; T. itali-

limited or contradictory: 1) the sister-taxon status cus from Conversano, Italy; T. marmoratus from

of T. vulgaris and T. montandoni is supported by El Berrueco near Madrid, Spain and Rochechouart,

allozymc data only; 2) the monophyly of T. boscai France; T. montandoni from Ustrzyki, Poland; T.

and T. italicus hinges on the interpretation given vittatus from an unknown locality in Israel and to behavioral characters; 3) the status of T. vittatus from Adapazari, Turkey (subspecies ophryticus) as the sister taxon of the T. marmoratus species and T. vulgaris from Ambleteuse, France. Out-

be called group may into question on the basis of group taxa Cynops ensicauda, Neurergus strauchii

its overall to Paramesotriton from similarity some small-bodied spe- and sp. were obtained the

T. the for trade cies, vulgaris in particular; 4) support pet with no reliable locality information. the position of T. alpestris in the subgenus Triturus Total DNA was extracted from approximately

is weak due the relatively to non-independence of 50 mg of frozen or ethanol-preserved tissue (liver, Contributions to Zoology, 68 (2) - 1999 75

heart or tail muscle) following standard protocols mesotriton, Pleurodeles, Salamandra, Salaman-

(Sambrook et ah, 1989). For PCR-amplification drina, Taricha and Tylototriton. of the 12S rRNA region the forward (L2475) and Phylogenetic reconstruction was performed under reverse (H2897) ‘universal’ primers were used the principle of parsimony, using the PAUP 3.1.1

with the ’branch-and- (Kocher et ah, 1989). The forward and reverse software (Swofford, 1991)

ATPase primers L9858 (s'-CTCCTCCTTAATGA- bound’ search option (the ‘heuristic search’ method

TATGCCACA-3') and H 10307 (3'-TTCACCCCA- was used in the bootstrap runs). Two a priori se-

in ACTACCCAACTATC-s') were designed our lected strategies were followed. First, a uniform laboratory by G. Rowe. Double stranded DNA am- transition - transversion ratio and uniform posi- plifications were performed using standard PGR tional ratio was used, i.e., ‘no weighting’. Sec- protocols (Erlich, 1989) with negative controls to ond, different weights were assigned to transitions test rRNA for contamination. The 12S fragment versus transversions inversely proportional to the was amplified under stringent conditions (anneal- frequency of different substitutions among in-group

ing temperature 65°C, extension time 25 seconds, taxa as traced from the tree obtained with the un- up to 28 cycles), while the ATPase primers re- weighted approach, with 24 as the base number. quired milder conditions (annealing temperature Gap derived character-state changes were given

45-50°C, extension time of 60 seconds, minimum the same weight as transversions, also on an apriori of basis. Positional 30 cycles). The amplified products were sepa- weighting factors for the first, sec- rated and the third codon determined on 1.5% low melting point agarose gels, cut ond, positions were out of the gel and purified by successive phenol, for the protein coding DNA fragment (ATPase)

phenol-chloroform and chloroform-isoamylalcohol in a similar way. Bootstrap scores (Felsenstein,

extractions, followed by ethanol precipitation. Both 1985; Hillis and Bull, 1993) were determined over

5000 obtain strands were directly sequenced using the dideoxy replications to an impression of the

chain-termination method (Sanger et ah, 1977). strength of support from the data for the phyloge-

with Clustal-V netic maximum Multiple sequences were aligned tree showing parsimony. Temple-

software (Higgins and Sharp, 1988) with manual ton’s (1983) non-parametric test was used to test

one-sided for the of differences be- adjustments. The ATPase gene sequences were significance

translated into the read- documented and the amino-acid sequences and tween phylogenies newly

alternatives. ing frame was determined by alignment to se- derived

quences of the teleost fish Cottus kessleri (Grachev et ah, 1992) and human sequences (Arnason et

ah, 1996) obtained from Genßank. When the se- Results

quences for individuals of the same species were

not the used in 212 of the ATP- identical, consensus sequence was Aligned sequences represent bp the phylogenetic analysis with variable sites taken ase gene and covers the short stretch of overlap

as between polymorphisms. The percent sequence diver- regions coding for ATPase 6 and ATPase

gencewas calculated following Mindell and Honey- 8 (Fig. 2). The first nucleotide corresponds to posi-

cutt We tion 256 ATPase (1990). aligned our sequences to the in the Cottus kessleri sequence published salamandrid 12S sequences (Caccone (Grachev et ah, 1992). Aligned sequences repre-

et ah, the 1994; Hay et ah, 1995), that include sent 301 bp of 12S rRNA gene. The first nucle-

ni-group taxa T. vulgaris and T. carnifex and out- otide corresponds to position 233 in the Mertensiella

group taxa from the genera Euproctus, Notoph- luschani 12S rRNA sequence (Titus and Larson,

thalmus, Pleurodeles and Salamandra.Furthermore, 1995). No insertions or deletions (‘indels’) had to

the the sequences were aligned to those published by be inferred to align ATPase sequences. Align-

Titus & Larson that include T. of the 12S indels (1995), alpestris, ment sequences required two at T. karelinii and representatives of the salamandrid one position among the Triturus species and two

genera Chioglossa, Cynops, Euproctus, Neurergus, indels covering four positions in the out-group taxa.

Mertensiella, Notophthalmus, Pachytriton, Para- More indels on nine positions were required when 76 /. Zajc & J. W. Arntzen - Phytogeny ofEuropean newts

data for 12S rRNA ATPase and ATPase amino acid Fig. 2. mtDNA sequence (top panel), (middle panel) sequences (lower panel),

with between ATPase DNA are strands, from 5' to 3' end. The taxa studied ensicauda. overlap genes. sequences light are : 1) Cynops

T. Paramesotriton Triturus T. T. cristatus, T. helveticus, T. italicus, 9) 2) Neurergus strauchii, 3) sp., 4) alpestris, 5) boscai, 6) 7) ,8)

T. Asterisks indicate the termination codon for the ATPase 8 marmoratus, 10) T. montandoni, II) 72 vittatus, and 12) vulgaris. gene.

added the data studied and 38 variable differed published sequences were to set, species positions

in This lengthening the fragment to 305 positions, with by a single substitution one taxon (Fig. 2).

no regions of ambiguous alignment (sensu Titus left 13 (6.1%) nucleotide positions with poten-

The and Larson, 1995). tially, phylogenetically relevant information.

T. boscai and One-hundred and sixty-one of the 212 ATPase sequence differencebetween in-group

18.9% 19.3%. Within nucleotide positions were identical across the four taxa ranged between and Contributions to Zoology, 68 (2) - 1999 77

the vulgaris species group the distances were con- siderably 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

3.2 and the number of among in-group taxa was substitutions at the first, second, and third codon position were 5,3, and 17, respectively. Follow- ing 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 16 variable usage, characters found nine were with synapomorphic character states for the T. helveticus— T. montandoni

— T. vulgaris clade, four autapomorphic character

Fig. 3. Composite phylogeny for the genus Triturus, constructed states for T. helveticus and three synapomorphies from DNA data. lines mitochondrial sequence Thin and thick were found for T. montandoni— T. vulgaris. The represent branching order as reconstructed from 12S rRNA and DNA of T. and T. montandoni sequences vulgaris ATPase with sequence data, respectively, bootstrap replication silent substitutions. differed only by scores indicated along branches.

Two-hundred and thirty-five of the 301 nucle- otide positions on the 12S fragment were identi- ond group contains T. alpestris and all small-bod-

cal ied with across the 12 taxa studied and 25 variable Triturus species, a low bootstrap support

= Within this the of positions differed by a single substitution in one (Ph 59%). group, monophyly taxon, leaving 41 (13.6%) phylogenetically infor- the T. vulgaris species group is strongly supported

mative sites (Fig. 2). Half the number of variable (Ph = 100%), whereas the relationship of T. al-

sites 12S T. and italicus were found in one-third of the fragment, pestris, boscai, T. among themselves at the 3' is resolved < On end. The sequence difference between poorly (Ph 55%). the basis of

12S out-group and in-group taxa ranged between 6.0% sequence data no firm conclusion can be drawn and 14.0%, Within the distances the order within the the genus Triturus regarding branching vulgaris

were from 0.7% the ATPase data considerably lower, ranging species group. However, sequence between T. montandoni and T. vulgaris [which is provided strong support for the sister taxon status less than that found within T. alpestris (3.0%) and of T. montandoni and T. vulgaris (Pb = 96, Fig. 3) T. vittatus (1.7%)] to 10.6% between T. cristatus and the same bootstrap value was observed when and T. The difference amino-acid data instead alpestris. average sequence sequence were analyzed between in-group species and an out-group taxon of the DNA sequence. Under the weighting scheme ranged from 7.4% (Cynops ) to 11.2% (Neurergus). topologically similar results were obtained, with

The transition transversion ratio three reservations: : among in-group 1) with the ATPase gene frag-

taxa 4 of = for was 2.8 and weights determined were and ment, a score Pb 100 was observed the 20. vulgaris montandoni clade, while not with the Given Cynops, Neurergus and Paramesotriton 12S rRNA gene fragment; 2) generally less phylo-

as the of Triturus resolution and lower out-groups, monophyly appears genetic bootstrap replication

to be with strongly supported a 89% bootstrap scores were observed on well-established branches; replication score (Pb) (Fig. 3). Two major in-group and, 3) an equally parsimonious solution was found dusters are formed. The first, with Pb = 85%, in which the genus Triturus is paraphyletic. Se-

consists of T. vittatus sister the T. derived from the as a taxon to quences same or congeneric spe-

marmoratus = The cies different authors et species group (Pb 98%). sec- by (Caccone ah, 1994; 78 I. Zajc & J. W. Arntzen - Phytogeny ofEuropean newts

4. Molecular for salamanclrid salamanders reconstructed from 12S rRNA data Fig. (A) phylogeny sequence as published by Cl,

Caccone et al. (1994); C2, Caccone et al, (1994: 523); H, Hay et al. (1995); TL, Titus and Larson (1995) and PS, present study. (B)

Schematic for salamandrid derived from 12S and 16S rRNA data from Titus and phylogeny genera as sequence (adapted Larson,

1995). Note the possible paraphyly ofthe genus Triturus.

Titus & Larson, 1995; Hay et al., 1995 and present difference between the shortest tree and the tree

similar another and conse- in which Triturus is study) are very to one monophyletic was significant

quently the taxa they represent are placed close (P < 0.05).

together in the phylogenetic tree that has maxi-

mum parsimony (i.e., Cynops sp., Neurergus strauchii, Notophthalmus viridescens, Paramesotri- Discussion

ton sp., Pleurodeles waltl, Salamandrasalamandra,

Triturus alpestris, T. cristatus superspecies and T. We have tested an existing phylogeny of Triturus

vulgaris). against independent molecular data. Two PCR-

With wide of included in of the molecule with a range out-groups amplified fragments mtDNA

the the of the Triturus low and rates of analysis, monophyly genus relatively (12S rRNA) high evo-

was not supported. The sister clade to the subge- lution (ATPase) were employed to test some of

nus Palaeotriton wouldnotbe the subgenus Triturus the earlier and more recent speciation events of

(i.e., T. vittatus, T. marmoratus, and the T. cristatus the Triturus radiation, respectively. The chosen

Triturus superspecies), but the subgenus plus fragments showed evolutionary rates that were

Taricha and the sister Notophthalmus and clade anticipated and that were appropriate for address-

this would be to group composed of Cynops, Eu- ing phylogenetic questions at this taxonomic level. proctus, Neurergus, Pachytriton, and Paramesotri- Faster evolving protein-coding ATPase genes can

ton indicated the be reliable of (Fig. 4A). Templeton’s test that tracers evolutionary history among Contributions to Zoology, 68 (2) - 1999 79

close relatives (as within the T. vulgaris species Innocenti et al., 1983), morphological (Giacoma

group), where silent, third codon position substi- and Balletto, 1988) and immunological data (Busack

tutions account for most variation (Zardoya and et al., 1988). According to these studies, T. vittatus

the other if the is T. the small-bodied Meyer, 1996). On hand, saturation more similar to alpestris or of transitions is suspected, it is imperative to re- newts of the subgenus Palaeotriton. Immunologi-

duce the emphasis on, or even eliminate, this class cal data (Busack et al., 1988) suggested a sister of T. T. substitutions from phylogenetic analysis (Disotell taxon relationship for v. vulgaris and helve-

et and The ATPase al., 1992; Knight Mindell, 1993). Although ticus. sequences provide strong sup- intuitively appealing, differential weighting may port for the monophyly of T. vulgaris and T.

downweight informative transitions in conserved montandoni, in line with allozyme data (Rafmski regions and upweight transversions primarily and Arntzen, 1987). present in more variable regions of the molecule, The rate of 12S rRNA molecular evolution in

thereby obscuring phylogenetic relationships (Titus some Palearctic salamandrids was calibrated as

and Mindell mA of Larson, 1995; and Tacker, 1996). approximately 3 lineage independence per

Given the observed transition saturation and 7 mA bias, was percent sequence divergence per per-

not prevalent among the compared sequences. The cent sequence divergence when only transversions

considered observation that with weighting the bootstrap sup- are (Caccone et ah, 1994). Applying

port for congruent sections of competing phylo- these rates provides an estimate of 12-19 mA for

solutions and the the of the T. the T. genetic dropped phylogenetic age vittatus lineage (versus

resolution decreased, supports this view. marmoratus species group) in the subgenus Triturus

Our and estimate of mA for the T. analysis presents a hypothesis that is con- an 14-19 boscai

cordant with established the T. within well-supported areas in the lineage (versus vulgaris species group)

Triturus phylogeny. As to the specific questions the subgenus Palaeotriton. This is in line with

we be solved while others estimates from molecular and raised, some appear to fossil, biochemical,

14-15 mA are not. The new data do not help to elucidate the biogeographical data that converge to

relationship between T. boscai and T. italicus. Their for the T vittatus lineage and to 13-15 mA for the

T. monophyly was suggested by allozyme data and, boscai lineage (reviewed in Oosterbroek and

ambiguously, by behavioral data (Arntzen and Arntzen, 1992).

of Sparreboom, 1989). The observation of‘flamenco’ The monophyly the genus Triturus, although behavior in the sexual repertoire of T. helveticus widely accepted, is defined on the basis of some- (M. Faria, pers. comm.) and T. marmoratus pyg- what vague character state descriptions (‘a suite

of behavioral character states’ and level maeus (T. Halliday, pers. comm., M. Sparreboom, ‘a high

boscai - Pers. comm.) further erodes the support for T. of sexual dimorphism’ Halliday, 1977) that are

T. the — italicus monophyly. Similarly, current study not explicit synapomorphies. Moreover, a feature does not convincingly clarify the position of T. such as the potential for interbreeding (Wolterstorff

alpestris. The phylogenetic position of this spe- and Herre, 1935) is explicitly plesiomorphic. The

cies could also of has not be satisfactorily resolved from monophyly the genus Triturus recently behavioral data (Arntzen and Sparreboom, 1989). been put into question in a molecular phylogenetic

it in third with of the and Bolkay (1928) placed a subgenus study family Salamandridae (Titus

intermediate characteristics, in between Triturus Larson, 1995) and in a molecular biogeographical and Palaeotriton. study of Euproctus (Caccone et al., 1994). In both

The 12S rRNA data the of them Triturus were involved. sequence support place- only two species of ment T. vittatus as the sister taxon to the T. With just the representatives of the genera Cynops,

marmoratus species group in the subgenus Triturus, Neurergus and Paramesotriton as out-groups, a

as was inferred from its breeding behavior (Arntzen denser in-group taxon sampling does not challenge and Sparreboom, 1989). This challenges the in- the hypothesis of Triturus monophyly. However, ferences from the phenetic analyses of osteologi- 12S and 16S mtDNA sequence data indicate that

cal (Rafmski the and Pecio, 1989), karyological (Bucci- genera Cynops, Neurergus andParamesotriton 80 I. Zajc & J. W. Arntzen - Phytogeny ofEuropean newts

be relative TR.1977. The courtship of European newts: an evo- themselves may in-group taxa to Triturus Halliday lutionary perspective. In: Taylor DH, Gutman SE, eds. The (Fig. 4B). In the light of these results, their a priori reproductive biology of amphibians. New York: Plenum choice as out-groups to the genus Triturus may Press, 185-232. have been unfortunate. Haliiday T, Arano B. 1991. Resolving the phylogeny of the

European newts. Trends Ecol. Evol. 6: 107-117.

Hay JM, Ruvinsky I, Hedges SB, IMaxson LR. 1995. Phyloge-

Acknowledgements netic relationships ofamphibian families inferred from DNA

of mitochondrial 12S and I6S ribosomal RNA sequences

Mol. Biol. Evol. 12: 928-937. genes. We acknowledge the laboratory facilities provided by Prof. T. Hickson RE, Simon C, CooperA, Spicer GS, Sullivan J, Penny Burke, the assistance of Dr. D. Thomaz, and the constructive

D. 1996. Conserved sequence motifs, and sec- and alignment, comments of Drs. A. Zuiderwijk an anonymous reviewer. ondary structure for the third domain of animal 12S rRNA.

Mol. Biol. Evol. 13: 150-169.

for Higgins DC, Sharp PM. 1988. CLUSTAL; a package per- Literature forming multiple sequence alignment on a microcomputer.

Gene 73: 237-244.

\mason U, Xu X, Gullberg A. 1996. Comparison between the Hillis DM, Bull JJ. 1993. An empirical test ofbootstrapping as

mitochondrial DNA of Homo and method for confidence in complete sequences the a assessing phylogenetic analy-

based nonchimeric J. sis. 42: 182-192. common chimpanzee on sequences. Syst. Biol.

Mol Evol. 42: 145-152. Knight A, Mindell DP. 1993. Substitution bias, weighting of

Arntzcn M. 1989. A of the Old DNA evolution and the of JW, Sparrcboom phylogeny sequence phylogenetic position

World Triturus biochemical and behavioural Pea’s Biol. 42: 18-31. newts, genus : viper. Syst.

data. J. Zool. (Land.) 219: 645-664. Kochcr TD, Thomas WK, Meyer A, Edwards SW, Paabo S,

Bolkay SJ. 1928. Die Schadel der Salamandrinen, mil beson- Villablanca FX, Wilson AC. 1989. Dynamics of mitochon-

derer Zeitschr. drial DNA in Riickssichtaufihre systematische Bedeutung. evolution animals: Amplification and sequenc-

Anal. Entwickelungsgesch. 86: 259-319. ing with conserved primers. Proc. Natl. Acad. Sci. U.S.A.

Bucci-Innoccnti S, Ragghianti M, Mancino G. 1983. Investi- 86: 6196-6200.

gations of karyology and hybrids in Triturus boscai and T. Kumar S. 1996. Patterns of nucleotide substitution in mito-

of the chondrial of Genetics 143: vittatus with reinterpretation species groups within protein coding genes vertebrates. ,

Triturus (Caudata: Salamandridae). Copeia 1983: 662-672. 537-548.

Busack SI), Jericho BG, Maxson LR, lizzcll T. 1988. Evolu- Macgrcgor HC, Sessions SK, Arntzcn JW. 1990. An integra-

of salamanders in the Triturus: tive of of tionary relationships genus analysis phylogenetic relationships among newts

The view from 44: 307-316. the Triturus immunology. Herpetologica genus (family Salamandridae), using compara-

Caccone A, Milinkovitch MC, Sbordoni V, Powell JR. 1994. tive biochemistry, cytogenetics and reproductive interactions.

Molecular biogeography: using the Corsica - Sardinia micro- J. Evol. Biol. 3: 329-373.

plate disjunction to calibrate mitochondrial rDNA evolu- Mindell DP, Honeycutt RL. 1990. Ribosomal RNA in verte-

tionary rates in mountain newts (Euproctus). Erratum. J. brates: Evolution and phylogenetic implications. Annu. Rev.

Evol. Biol. 7: 227-245, 523-524, Ecol. Syst. 21: 541-566,

Caccone A, Milinkovitch MC, Sbordoni V, Powell JR. 1997. Mindcll DP, Tackcr CE. 1996. Rates of molecular evolution:

Mitochondrial DNA rates and biogeography in European phylogenetic issues and applications. Annu. Rev. Ecol. Syst.

newts (genus Euproctus). Syst. Biol. 46: 126-144. 27: 279-303.

Disotcll T R, Honeycutt RL, Ruvolo M. 1992. Mitochondrial Oostcrbrock P,Arntzen JW. 1992.Area-cladograms ofcircum-

DNA phylogeny of the Old-World monkey tribe Papionini. Mediterranean taxa in relation to Mediterranean palaeoge-

Mol. Biol. Evol. 9: 1-13. ography. J. Biogeogr. 19: 3-20.

Erlich HA (cd.). 1989. PCR Technology. Principles and Appli- Rafinski J, Pecio A. 1989. Craniometric studies on the species

cations DNA New York: Stockton Press. of the 1815 Sala- for amplification. genus Triturus, Rafinesque, (Amphibia,

Felscnstcin J. 1985. limits An Folia Biol. 37; 131-150. Confidence on phylogenies: ap- mandridae).

proach using the bootstrap. Evolution 39: 783-791. Rafinski J,Arntzen JW. 1987. Biochemical systematics ofthe

Giacoma Ballctto E. 1988. of the salamandrid Old World Triturus: data. C, Phylogeny newts, genus allozyme Herpeto-

genus Triturus. Boll. Zool. 55: 337-360. logica 43: 446-457.

Grachev MA, Slobodyanyuk SJ, Kholodilov NG, Fyodorov Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular clon-

SP, Belikov SI, Sherbakov DY, Sidcleva VG, Zubin AA, ing: A laboratory manual. New York: Cold Spring Harbor

Kharchenko VV. 1992. Comparative study of two protein- Laboratory Press.

coding regions of mitochondrial DNA from three endemic Sanger F, Nickeln S, Coulson AR. 1977. DNA sequencing with

sculpins (Cottoidei) of Lake Baikal. J. Mol Evol. 34: 85- chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A.

90. 74: 5463-5467. Contributions to Zoology, 68 (2) - 1999 81

- W. 1935. Die der Wasser- Swofford DL. 1991. PAUP phylogenetic analysis using par- Wolterstorff W, Herre Gattungen

simony, version 3.1. Champaign: Illinois Natural History molche der Familie Salamandridae. Archiv Naturgesch. 4:

Survey. 217-229.

Templeton AR. 1983. Phylogenetic inference from restriction Zardoya R, Meyer A. 1996. Phylogenetic performance of mi-

endonuclease site with reference tochondrial in cleavage maps particular protein-coding genes resolving relationships

the evolution of humans and Evolution 37: 221- vertebrates. Mol. Biol. Evol. 13: 933-942. to apes. among

244. ZhangD-X, Hewitt GM. 1996. Nuclear integrations: challenges

molecular for mitochondrial DNA markers. Trends Ecol. Evol. 11:247- Titus TA, Larson A. 1995. A phylogenetic perspec-

tive on the evolutionary radiation ofthe Salamander family 251,

Salamandridae. Syst. Biol. 44: 125-151.

Wallis GP, Arntzen JW. 1989. Mitochondrial DNA variation

in the crested Limited newt superspecies: cytoplasmic gene

Evolution 43: 88-104. Received: 4 March 1998 flow among species.