A revised taxonomy of crested newts in the Triturus karelinii group (Amphibia: Caudata: Salamandridae), with the description of a new species
Author
Wielstra, B.
Author
Litvinchuk, S. N.
Author
Naumov, B.
Author
Tzankov, N.
Author
Arntzen, J. W.
text
Zootaxa
2013
3682
3
441
453
journal article
43360
10.11646/zootaxa.3682.3.5
bd3a8c00-de22-4566-ae08-b0d123f817f5
1175-5326
215933
69B9A846-616F-4774-B3F0-B796D2B90431
The name
T. arntzeni
is a junior synonym of
T. macedonicus
Litvinchuk
et al.
(1999)
described the subspecies
Triturus karelinii arntzeni
based on differences in genome size, protein variation and morphological characteristics. Subsequently,
Espregueira Themudo
et al.
(2009)
elevated this subspecies to species level, i.e.
Triturus arntzeni
Litvinchuk, Borkin, Džukiċ and Kaleziċ, 1999
(in
Litvinchuk
et al.
, 1999
). However, concern has been expressed that the
type
locality of
T. arntzeni
—Vrtovaċ, near Pirot in eastern
Serbia
(
Fig. 1
)—in fact represents the species
T. macedonicus
and not
T. karelinii
s. l.
(Arntzen & Wielstra, 2010;
Stoyanov
et al.
, 2011
). We review the species identity of crested newts from Vrtovaċ below. Because the bulk of evidence points towards
T. macedonicus
being the crested newt species occurring at Vrtovaċ, we identify the name
T. arntzeni
as a junior synonym of
T. macedonicus
and conclude that it does not represent a taxon newly distinguished in
T. karelinii
s. l.
Geography.
The population in
Litvinchuk
et al.
(1999)
representing
T. arntzeni
comprises seven different localities (14a-g; see
Fig. 1
), all of which are situated at, or close to, various crested newt contact zones. Note that locality 14c concerns the actual
type
locality of
T. arntzeni
. For the latest overview of
Triturus
distribution, including a database of locality data, see
Wielstra
et al.
(2013b)
. The species composition of the seven
T. arntzeni
populations we evaluate as follows:
Locality 14a, Trbusaċ,
Serbia
is
T. dobrogicus
(
Wielstra
et al.
, 2013b
). Species identification is based upon the phenotype of material deposited at the Institute of Biological Research, “Siniša Stankoviċ”, University of Belgrade (JWA, BW).
Locality 14b, Trešnja,
Serbia
represents the western
T. karelinii
s. l.
nuclear gene pool with
T. dobrogicus
mitochondrial DNA and some limited presence of, or genetic admixture with,
T. dobrogicus
(Arntzen
et al.
, submitted;
Wallis
& Arntzen, 1989
).
Locality 14c, Vrtovaċ,
Serbia
, is positioned inside the
T. macedonicus
range (Wielstra & Arntzen, 2012). Neighboring crested newt species are, in order of increasing distance,
T. cristatus
,
T. dobrogicus
and western
T. karelinii
s. l.
nuclear gene pool (
Fig. 1
).
Locality 14d, Vlasi,
Serbia
, has western
T. karelinii
s. l.
nuclear gene pool and
T. macedonicus
in syntopy (Wielstra & Arntzen, 2012). Species identification is based on examination of animals observed in the field (JWA, BW).
Locality 14e, Vlasina,
Serbia
has
T. macedonicus
(Wielstra & Arntzen, 2012)
. Species identification is based upon the phenotype of material deposited at the Institute of Biological Research, “Siniša Stankoviċ”, University of Belgrade and of animals observed in the field (JWA, BW). The number of rib-bearing vertebrae (details below) reported in this population (n=7 with NRBV = 13, n=25 with NRBV = 14) is typical for
T. macedonicus
(
Crnobrnja-Isailoviċ
et al.
, 1997
)
.
Locality 14f, Stracin,
Macedonia
has the western
T. karelinii
s. l.
nuclear gene pool and
T. macedonicus
in syntopy (Wielstra & Arntzen, 2012). Species identification is based upon the phenotype of material deposited at the Institute of Biological Research, “Siniša Stankoviċ”, University of Belgrade (JWA, BW). Note that a population of
T. macedonicus
is located c. 4 kilometers to the west (Rugince,
Macedonia
in Wielstra & Arntzen, 2012).
Locality
14g
, Berovo,
Macedonia
is western
T. karelinii
s. l.
nuclear gene pool. Species identification was confirmed based upon the phenotype of material deposited at the Institute of Biological Research, “Siniša Stankoviċ”, University of Belgrade and of animals observed in the field (JWA, BW) and conforms to species identification by
Kaleziċ and Hedgecock (1980)
. Another confirmed
T. karelinii
s. l.
nuclear gene pool locality is located 9 kilometers to the northwest (Mitrašinci,
Macedonia
in
Wielstra
et al.
, 2013a
).
Throat and belly pattern.
In the field crested newts are best identified by morphotype (see introduction) and by their coloration pattern (
Arntzen, 2003
;
Arntzen &
Wallis
, 1999
) as follows:
Triturus karelinii
s. l.
: Stocky build. Heavily white-stippled sides. Ventral surface yellow-orange with many small to medium-sized, frequently angular black spots and continuous on the throat.
Triturus carnifex
: Medium
stocky build. Little or no white stippling on sides. Throat color variable with white stipples. Ventral surface yellow with few large, roundish, ill-defined and muddy-gray to black spots.
Triturus macedonicus
: Medium
stocky build. Sides densely white-stippled. Throat dark black or a muddied mix of black and yellow with many, medium sized white stipples. Ventral surface yellow to orange-yellow with a dense pattern of small, irregular spots.
Triturus cristatus
: Slender
build. Heavily white-stippled sides. Throat a muddied mix of black and yellow with fine white stippling. Ventral surface yellow-orange with irregular black spots.
Triturus dobrogicus
: Very
slender build. Heavily white-stippled sides. Black throat with large angular white spots (especially in males), ventral surface deep orange with many sharp, roundish black spots.
For a picture overview of throat and belly patterns of the different crested newt species, see
Arntzen and
Wallis
(1999)
. Pictures of the throat and belly pattern of the
holotype
and
paratypes
of
T. arntzeni
(online Appendix 1), preserved material stored at the Institute of Biological Research, “Siniša Stankoviċ”, University of Belgrade, and live newts from the
type
locality as observed by several of us (JWA, BW) most resemble
T. macedonicus
and not
T. karelinii
s. l.
In particular we point to the absence of the distinctive
T. karelinii
s. l.
throat pattern, with small angular dark spots surrounded by yellow, similar to the pattern on the belly. These spots are also absent in the
holotype
of
T. arntzeni
. To ascribe the Vrtovaċ newts to
T. macedonicus
by coloration pattern alone is not straightforward: as noted by
Freytag (1988)
,
T. macedonicus
overlaps considerably with
T. cristatus
in patterning and coloration.
Number of rib-bearing pre-sacral vertebrae.
The
Triturus
morphotypes were initially distinguished with the help of the ‘Wolterstorff index’, a measure of overall shape, defined as ‘forelimb length divided by inter-limb distance’ (
Wolterstorff, 1923
).
Arntzen and
Wallis
(1994)
found the number of rib-bearing pre-sacral vertebrae (NRBV) in
Triturus
to represent a taxonomically discriminating character superior to the Wolterstorff-Index (the two are strongly negatively correlated across species). Accordingly, the
Triturus
morphotypes are characterized by a
modal
number of rib-bearing pre-sacral vertebrae (NRBV) (
Arntzen &
Wallis
, 1999
). Modal NRBV count increases from
12 in
T. marmoratus
–
T. pygmaeus
, via
13 in
T. karelinii
s. l.
,
14 in
T. carnifex
–
T. macedonicus
and
15 in
T. cristatus
, to 16/
17 in
T. dobrogicus
(
Arntzen, 2003
;
Arntzen &
Wallis
, 1999
).
A disadvantage of NRBV (similarly applying to the Wolterstorff index) is that, although informative at the population level, it is not unambiguous at the individual level, due to intraspecific variation. Moreover, intermediate scores due to interspecific hybridization may point to a third species not involved (
Arntzen &
Wallis
, 1994
). We determined NRBV for 63 individuals from the
T. arntzeni
type
locality (the
holotype
and 12
paratypes
, deposited at the Zoological Institute, Russian Academy of Sciences, St. Petersburg;
46 specimens
deposited at the Institute of Biological Research, “Siniša Stankoviċ”, University of Belgrade and four specimens deposited at Naturalis Biodiversity Center, Leiden, see online Appendix 2). Three specimens with different NRBV (13 versus 14) on either sides of the body were ignored. Eleven individuals (18.3%) have an NRBV count of 13, NRBV is
14 in
n=44 (73.3%) and NRBV is
15 in
n=5 (8.3%). The distribution of modal versus non-modal NRBV scores in the Vrtovaċ population is not significantly different from that of
T. carnifex
/
T. macedonicus
(G-test of independence, d. f. = 1, P>0.05; reference data in
Arntzen, 2003
). The NRBV count of 15 shown by the
holotype
of
T. arntzeni
is indicative since this value fits
T. macedonicus
better than it does
T. karelinii
s. l.
Genome size.
The size of the genome may be a source of relevant taxonomic information (
Green & Sessions, 2007
;
Kron
et al.
, 2007
).
Litvinchuk
et al.
(1999)
observed a difference in genome size between
T. arntzeni
and
T. karelinii
s. l.
, with non-overlapping confidence intervals. We here interpret the genome size of
T. arntzeni
from Vrtovaċ (n=13) not as ‘different from other
T. karelinii’
(as in
Litvinchuk
et al.
, 1999
), but as ‘not different from
T. macedonicus’
(
Fig. 2
; constituent data in online Appendix 3). It is important to note that these genome sizes also differ from those of
T. cristatus
and
T. dobrogicus
. In contrast to
Litvinchuk
et al.
(1999)
we do not invoke convergent evolution or introgressive hybridization to explain the data but conclude that newts from Vrtovaċ actually are
T. macedonicus
. The genome size of the
holotype
of
T. arntzeni
in particular falls within the range of
T. macedonicus
but outside of the range of
T. karelinii
s. l.
The fourteenth specimen labeled as
T. arntzeni
in
Litvinchuk
et al.
(1999)
is from Tresnja (population 14b, see above) and has a genome size that is intermediate of
T. karelinii
s. l.
and
T. dobrogicus
; it is excluded from
Fig. 2
.
Allozyme data.
Litvinchuk
et al.
(1999)
presented data for nine allozyme loci and analyzed these at the population level. We here re-analyze the data at the level of the individual and in a phylogenetic framework for 42 individuals with a complete dataset and 48 individuals with data missing at one locus (online Appendix 4). Unfortunately the dataset for the
holotype
of
T. arntzeni
is highly incomplete, but several
paratypes
can be included in the analysis. We first determine the number of distinctive gene pools that is best supported by the data probabilistically using BAPS v.5.3 (
Corander
et al.
, 2008
). The recognized groups were clustered in a dendrogram on the basis of Rogers’ genetic distance with BIOSYS-1 (
Swofford & Selander, 1981
).
All ten included
T. arntzeni
individuals and three
T. cristatus
are in one group. Other BAPS groups are composed as follows: all
T. karelinii
s. l.
individuals with representatives of all three
T. karelinii
s. l.
nuclear gene pools, the n=3 included
T. macedonicus
individuals, two
T. cristatus
groups and four
T. dobrogicus
groups and, finally, a mixed group with one
T. cristatus
and one
T. dobrogicus
(online Appendix 4). In the UPGMA dendrogram (
Fig. 3
) the BAPS group that contains all
T. arntzeni
clusters with
T. cristatus
and it does not cluster with either
T. karelinii
s. l.
or
T. macedonicus
. A distance-Wagner tree yields the same result (data not shown). We further note that the two loci considered to distinguish
T. arntzeni
from
T. karelinii
s. l.
by
Litvinchuk
et al.
(1999)
are actually represented by the same alleles in
T. cristatus
and
T. macedonicus
. However, the reference sample size is small, in particular for unquestionable
T. macedonicus
(n=3) and western
T. karelinii
s. l.
nuclear gene pool
Nuclear DNA sequence data.
Wielstra
et al.
(2013a)
provided a framework to cluster
Triturus
newts to species based on three nuclear DNA markers developed by
Espregueira Themudo
et al.
(2009)
. Unfortunately, our attempts to obtain nuclear DNA sequence data from the
type
material (the
holotype
and 12
paratypes
) of
T. arntzeni
were not successful, presumably because of degraded DNA. However,
Wielstra
et al.
(2013a)
presented sequence data for another five individuals from Vrtovaċ (voucher ID
2533-37
stored at Naturalis Biodiversity Center, Leiden). These individuals were all assigned to
T. macedonicus
(see Appendix S
1 in
Wielstra
et al.
, 2013a
).
FIGURE 2.
A comparison of the genome size (pg) of
Triturus arntzeni
with that of other groups of
Triturus
newts. From left to right, the groups are
T. pygmaeus
(n = 1),
T. marmoratus
(n = 2),
T. dobrogicus
(n = 115),
T. cristatus
(n = 183),
T. carnifex
(n = 3),
T. macedonicus
(n = 6),
T. arntzeni
(white; n = 13), and western (n = 2), central (n = 2) and eastern (n = 58)
T. karelinii
s. l.
nuclear gene pool (colors correspond to those in Fig. 1). Shown are median, quartiles and range. Data are taken from (Litvinchuk
et al.
, 1999; Litvinchuk
et al.
, 2007) and provided in online Appendix 3. Note that the genome size of
T. arntzeni
is dissimilar to
T. karelinii
s. l.
and is not different from that of
T. macedonicus
and
T. carnifex
.
Mitochondrial DNA.
As
noted above we did not manage to obtain usable DNA extract and were not able to sequence mitochondrial DNA for the
type
material. However, we note that mitochondrial DNA could not be used to identify these newts anyway. This is because
T. macedonicus
in and around
Serbia
contain asymmetrically introgressed western
T. karelinii
s. l.
mitochondrial DNA (the area for which this phenomenon has been documented is detailed in Wielstra & Arntzen, 2012). Similarly, the nearest Serbian
T. cristatus
populations contain asymmetrically introgressed western
T. karelinii
s. l.
mitochondrial DNA (Wielstra & Arntzen, 2012;
Wielstra
et al.
, 2013b
). So, from the perspective of mitochondrial DNA,
T. macedonicus
and
T. cristatus
are indistinguishable in the Vrtovaċ region.
FIGURE 3.
Genetic similarity among crested newts (
Triturus cristatus
superspecies
) based on the allozyme data presented in Litvinchuk
et al.
(1999). Similarity among ten groups of crested newt, identified by Bayesian clustering, is expressed in an UPGMA dendrogram built with Rogers’genetic distance (for details see text and for data see online Appendix 4). Pie sizes reflect sample size. Color codes are as in Fig. 1 and individuals from the population of special interest (the type locality of
T. arntzeni
—Vrtovać) are shown in white. Note that this group does not cluster with
T. karelinii
s. l.
In conclusion, our analyses confirm that newts from Vrtovaċ, the
type
locality of
T. arntzeni
, including the
holotype
of
T. arntzeni
, are dissimilar to
T. karelinii
s. l.
and resemble
T. macedonicus
(NRBV, genome size, nuclear DNA sequences) or
T. cristatus
(allozyme data). The throat and belly pattern suggest they are
T. macedonicus
, but we acknowledge that the distinction with
T. cristatus
is difficult. On balance, we consider the population from Vrtovaċ to represent
T. macedonicus
(with perhaps some influences of
T. cristatus
). Consequently, we consider the name
T. arntzeni
to be a junior synonym of
T. macedonicus
and we conclude that the name
T. arntzeni
should not be used for the central/western
T. karelinii
s. l.
species.