Abstract
Iberian populations of large-bodied newts, with Triturus marmoratus in the north and T. pygmaeus in the south of the peninsula, were studied for external morphology, mitochondrial dna and for a panel of single nucleotide polymorphisms. This confirmed the species’ low level of interspecific hybridization and their parapatric, mosaic-like mutual range border across the peninsula. The genetic data also revealed substantial variation within T. pygmaeus, with narrow (0.43–35.2 km) clinal transitions in the very centre of Portugal. Similar clines were observed for body size and colouration pattern. Pygmy newts in the west of Portugal are larger, with a more striped (less reticulated) green dorso-lateral colouration pattern than those in the east and south of the country. The western group of populations is described as a new species, Triturus rudolfi sp. nov., on account of a long, ca. 2.5 Ma, independent evolutionary history and limited hybridization with its sister-species T. pygmaeus, suggesting selection against hybrid offspring. The range of the newly described species may be restricted to the wider Lisbon Peninsula, stretching northwards along the Atlantic coast to the river Vouga estuary. Inland, the range border may be set by the lower Tejo River, or by the currently wide area of agricultural land at either side of that river, that may accommodate a residual hybrid zone. The close contact between both pygmy newt species is effectively limited to a ca. 20 × 40 km area directly north of the town Entroncamento, where T. rudolfi sp. nov. is sandwiched in between T. marmoratus and the river Tejo.
Introduction
We do not know, and probably never will know, the number of species in Iberia or Siberia. Apart from essentially unsolvable conceptual questions of ‘what is a species?’, or what criteria to employ for species recognition and delimitation (de Queiroz, 2007; Wilkins, 2011; Burbrink & Ruane, 2021), a frequently overlooked issue is that we are dealing with a moving target, with standards now set higher than before, procured by technological innovation. Once the ‘low hanging fruit’ of species was harvested in the early days – say from Aristotle and Theophrastus (Mayr, 1982), through Linnaeus (1758) to Mayr (1969) – increasingly species are described that are morphologically cryptic, with small ranges and from difficult-to-explore parts of the planet, or that are (or were) problematic to survey such as cave organisms and internal parasites. While, for example, Mertens and Wermuth (1960) might have thought that their checklist of European amphibians and reptiles was pretty complete, recognized species numbers have more than doubled (N close to 300; Speybroeck et al., 2020) and the end is not in sight (e.g., Bassitta et al., 2020; Recknagel et al., 2023).
Recent taxonomic advance is often achieved through phylogenetic and phylogeographic studies that employ multi-locus nuclear genetic data. Such studies are, however, prone to ‘taxonomic inflation’ because exhaustive datasets tend to resolve consistent divergences deep within species boundaries (Arntzen and Bauer, 1996; Sukumaran and Knowles, 2017; Leaché et al., 2019; Burbrink et al., 2022; Dufresnes et al., 2023). In response, it has been advocated to subject candidate species to population genetic tests, increasing the workload even further. One acid test would be hybrid zone analysis, to affirm that candidates experience some degree of reproductive isolation, consistent with selection against hybrid offspring (Mayr, 1942; Dufresnes et al., 2020; Burbrink and Ruane, 2021).
Much of the increase in documented species richness is in the southern Holarctic – thus more in Iberia than in Siberia – in line with the well understood ‘southern richness, northern purity’ paradigm (Hewitt, 1996, 2000). The present study follows on from analyses that deal with morphological and genetic differentiation of large-bodied newts (genus Triturus) in southwestern Europe, i.e., France, Spain and Portugal (Arntzen, 2018, 2024). The data so far show the existence of three, spatially coherent, morphologically differentiated groups. Briefly, to the north of ca. 40 degrees northern latitude (northern) marbled newts are large and robust, with dark coloured bellies and a horizontally banded dorso-lateral colouration pattern. To the south of the Guadalquivir River pygmy newts as they are called, are small, with light coloured undersides and with a banded pattern also. These two groups enclose a third group in the southwest of the Iberian Peninsula, that is characterised by a reticulated dorso-lateral colouration pattern and light bellies. The northern group corresponds to T. marmoratus (Latreille, 1800) and to the south the species is T. pygmaeus (Wolterstorff, 1905), for which the central group has been described (T. pygmaeus lusitanicus Arntzen, 2023) as different from the southernmost nominotypical subspecies (fig. 1). When, in this paper, T. pygmaeus is written with quotation marks (‘T. pygmaeus’), this refers to the taxon before the here presented case for T. rudolfi sp. nov. as different from T. pygmaeus. For convenience of the reader the present-day knowledge is summarized in table 1.
The Iberian Peninsula with the approximate distributions of four taxa of large-bodied newts as described in table 1. Colour codes are: blue – Triturus marmoratus, dark red – T. pygmaeus pygmaeus, light red – T. p. lusitanicus and brown – T. rudolfi nov. sp., i.e., the newly described species from the wider Lisbon Peninsula. Letters indicate the capital cities Lisbon, Portugal (L) and Madrid, Spain (M) as well as Peniche (P) at the Atlantic coast. Major rivers partially coinciding with (sub)species borders are the Guadalquivir, the Vouga and the Tejo. The new species’ type locality Lagoa Seca near Valado dos Frades is indicated by a long arrow. The insert shows an adult male T. marmoratus.
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
animal drawing by bas blankevoortMaterial and methods
Fieldwork was carried out in France, Portugal and Spain under licenses as appropriate (see Acknowledgements). Newts were captured by dip netting or with funnel traps. The morphological characters measured are: snout-vent length up to and including the insertion of the hindlegs (SVl1), and up to and including the cloaca (SVl2, for descriptive purposes only), head length (Hl), head width (Hw), interlimb distance (ILd), forelimb length (FLl), third finger length (TFl), hindlimb length (HLl) and fourth toe length (FTl). Measurements were taken on live adults, with a plastic ruler in mm (SVl, ILd) or with plastic Vernier callipers at 0.1 mm precision (the other characters, for details, see Arntzen, 2018). Extremities were measured at the right side of the body. Because of a marked dimorphism (see Results) the sexes were treated separately. In salamanders, age and size at reproduction may vary from one population to the other, possibly as a direct response to conditions of the environment, a phenomenon especially prominent in pygmy newts (García-París et al., 1993; Díaz-Paniagua et al., 1996). To reduce the effect of overall size and to increase normality of the data, the standardized residuals of the regression of ln(character) to ln(SVl1) were used in multivariate analysis. To facilitate data interpretation and for comparability with published data, I also calculated body proportions from untransformed data (R_character = character/SVl1). Individuals were also inspected for the number of ‘links’ that they display, with most data taken from Arntzen (2024). Links are dorso-lateral colour pattern character states. They were counted as the number of uninterrupted connections following the green coloured surface at the lateral sides of the body, in between the insertion of the fore- and hindlegs, summed for the left and right side of the body (Nlinks). For a further explanation, and for illustrated examples and discussion of how the counting is done see Arntzen (2018). Although the morphological differentiation between T. marmoratus and ‘T. pygmaeus’ has already been analysed (Arntzen, 2018), data on the former species are included as a reference. Data for genetically admixed populations (see below) were excluded.
Tissues for molecular genetic analyses were larval or adult tail tips. Alternatively, recently deposited eggs (embryos) were raised in 5l buckets until hatching. dna was extracted from 95% ethanol preserved material under standard protocols with the KingfisherTM (Thermo Scientific) and DNeasy extraction kits (Qiagen, Valencia, CA, USA). Single nucleotide polymorphism (snp) data were gathered for 54 markers developed for the system (Lopez-Delgado et al., 2021), with data for 2622 individuals from 258 populations. Genotyping took place at the Institute of Biology Leiden snp line facility of the Institute of Biology, Leiden, using the Kompetitive Allele-Specific pcr system (kasp, lgc genomics, UK). A principal component analysis (pca) of the snp-data was performed with Adegenet (Jombart, 2008). In a later phase of the study, five populations were additionally studied to narrow the observation gap along the lower Tejo River. This material was studied for 14 of the most informative markers selected out of the original 54 (see supplementary table S1).
A fragment of the mitochondrially encoded nadh dehydrogenase 4 gene (nd4) was amplified and sequenced with established primers following a set protocol (Wielstra and Arntzen, 2011; Wielstra et al., 2013). Sequences were aligned with the web version of Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo) under default settings and the result was exported in Nexus format. Sequences typical for ‘T. pygmaeus’ were identified as belonging to the ‘western’ (henceforth ‘southwestern’) and the ‘eastern’ haplotype group, based on a single differentiated nucleotide that corresponds to position 11241 in the full mitochondrial genome of Salamandra salamandra (Linnaeus, 1758) with Genbank accession number kx094971 (Mulder et al., 2016). ‘Triturus pygmaeus’ haplotypes found in pure or admixed populations of T. marmoratus and vice versa (as determined from snp and published nuclear genetic data (present paper, Arntzen, 2018) or, if so needed, inferred from the documented species distributions (fig. 1)) were omitted, because introgression, hybrid zone dynamics, population demographic analyses and species replacement are the topics of a forthcoming paper.
Blanket distribution maps composed of so-called Dirichlet cells (Matérn, 1979; Burrough et al., 2015) were obtained by spatial data interpolation of the extracted information with the ‘NearestPoint’ procedure in ilwis 3.8.6 (ilwis 2019). Characters of interest were analysed for spatial variation, with the focus on ‘T. pygmaeus’ populations along the northern edge of its range (see transect described below). The best fitting geographical clines were estimated with the Hybrid Zone Analysis under R (hzar) software (Derryberry et al., 2014), with a protocol used earlier (Arntzen et al., 2017). The reference point for longitudinal distance in this analysis is Entroncamento railway station at -8.478 E, 39.456 N, the putative centre of the hybrid zone (see below).
Finally, environmental data were extracted for Triturus newt populations with species identity known. Parameters available for selection were elevation and 19 climate variables obtained from the BioClim data base at 30 arc seconds spatial resolution (Fick and Hijmans, 2017). This data set was reduced to five largely uncorrelated variables (Spearman’s correlation coefficient |rho| < 0.7) following clustering with the upgma-procedure in Primer 7 (Clarke and Gorley, 2015). The number of 258 populations in three species groups was brought back to N = 232, due to positional overlap of neighbouring populations and because genetically admixed ones were excluded. For details on the parameter selection procedure, see supplementary fig. S1. Other statistical analyses were with spss 26 (ibm spss, 2021).
Results
A principal component analysis of snp data for 54 marker loci reveals a bimodal distribution of scores along the first pca-axis, composed of 821 T. marmoratus (31.3%) and 1689 ‘T. pygmaeus’ (64.4%). The latter cluster is subdivided in two groups along the second pca-axis that conform to T. rudolfi sp. nov. (see below) and T. pygmaeus. At the population level the corresponding numbers are 89 T. marmoratus, 154 ‘T. pygmaeus’ and 15 intermediate populations along the first axis and 123 T. pygmaeus and 26 T. rudolfi sp. nov. along the second axis with five intermediate populations (fig. 2). The spatial distribution of the genotypic clusters is geographically coherent, with T. marmoratus in the northern half of the Iberian Peninsula, the group of western pygmy newts (T. rudolfi sp. nov.) in the Lisbon Peninsula and along the adjacent Atlantic coast and the group of eastern pygmy newts (T. pygmaeus) inland and to the south (figs. 1 and 3). Genetically intermediate populations are all positioned at species range borders, so that it seems appropriate to interpret these as (rare) hybrid populations. The transition from T. marmoratus in the north to ‘T. pygmaeus’ in the south of the Iberian Peninsula is abrupt. The contact zone is parapatric with aspects of a mosaic, suggesting that species replacement has taken place under competitive exclusion, without extensive hybridization. The other main transition is from T. rudolfi sp. nov. in the west to T. pygmaeus in the southeast of the Iberian Peninsula. Analysis with hzar revealed a steep geographical cline for pca2 of the panel of snp markers (fig. 4). It has a central position at 0.3 km (95% confidence interval, ci -1.0–7.5 km) and a width of 35.2 km (ci 20.4–58.4 km). Details on the geographical clines are in supplementary table S2.
Genetic differentiation in 258 marbled newt populations for 54 nuclear snps. Plotted are population averages for scores along the first and second principal component axis. The first axis explains 80.7% of the observed variation and separates Triturus marmoratus (blue symbols) from ‘T. pygmaeus’. The second axis explains 1.51% of the observed variation and separates western pygmy newts (T. rudolfi nov. sp., brown symbols) from southeastern pygmy newts (T. pygmaeus, red symbols). Note the unequal scales applied to the axes (for explanation see main text). Populations with intermediate positions are genetically admixed and presumed to contain hybrids between T. marmoratus and T. pygmaeus (14 grey symbols), T. marmoratus and T. rudolfi nov. sp. (one large, light grey symbol) and between T. pygmaeus and T. rudolfi nov. sp. (five open symbols).
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
(A) Genetically investigated populations of large-bodied newts from the western part of the Iberian Peninsula plotted with the nearest point method, with a maximum spatial extrapolation of ca. 25 km. Colour codes are: blue – T. marmoratus, red – T. pygmaeus and ochre – T. rudolfi sp. nov. Inferred hybrid populations of T. marmoratus and ‘T. pygmaeus’ are shown in grey (as in fig. 2). Note that populations from densely sampled areas do not always stand out separately. Five populations from along the lower Tejo area added a posteriori are shown by black dots. The boxed area in between 39.3-39.7 N encompasses the transect studied for spatial variation in morphometrics (35 populations), Nlinks (35 populations), mitochondrial dna (69 populations) and nuclear genetic composition (82 populations) with hzar software (for results see fig. 4). The nominal centre of the T. pygmaeus – T. rudolfi sp. nov. hybrid zone is at Entroncamento railway station (E, coordinates -8.478 E, 39.456 N). (B) Dirichlet tessellation of populations studied for variation in nuclear genetic composition, with colours like above. The yellow to red colour bar corresponds to the one shown in fig. 4. Distances in km are relative to Entroncamento railway station.
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
Geographical clines observed for ‘Triturus pygmaeus’ in a longitudinal transect across central Portugal (see fig. 3), with T. rudolfi nov. sp. in the west and T. pygmaeus in the east. The horizontal axis is distance in km, measured from the nominal centre of the contact zone at Entroncamento railway station. The vertical axes are from top to bottom, left column – pca2 for 54 snp markers and the frequency of the eastern mtDNA haplotype, and in the right-hand column – the number of links and body size (lnSVl1) of adult males and females. Solid dots represent populations and the grey areas represent the 95% credibility intervals. Note that the colour bar is applied to fig. 3B. The formal cline descriptions are in supplementary table S3.
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
A total of 1977 mitochondrial dna (mtDNA) sequences was split in two, about equally sized haplotype groups (N = 1638 when presumably introgressed haplotypes were excluded). The southwestern haplotype is dominant across the entire T. rudolfi sp. nov. and T. p. pygmaeus ranges as well as in the southern section of the T. p. lusitanicus range whereas the eastern haplotype is dominant in the northern section of the T. p. lusitanicus range. The spatial separation within T. pygmaeus is detailed in supplementary fig. S4. The haplotype distribution inside the transect is strongly bimodal. The geographical cline has a central position at 15.2 km (ci 12.9–16.1 km) and a width of 0.434 km (ci 0.048–8.659 km) (fig. 4).
Data on Nlinks were available for 2145 adult newts (N = 1953 when data for hybrid populations were excluded). Numbers of links are different for the (sub)species with low values for T. marmoratus and T. p. pygmaeus, intermediate values for T. rudolfi sp. nov. and high values for T. p. lusitanicus (fig. 5), with a statistically significant overall differentiation (Kruskal-Wallis test statistic 1177.3, P < 0.0001). Post-hoc comparisons of taxa show that pairwise comparisons are also significant (P < 0.0001), except for T. marmoratus and T. p. pygmaeus (P > 0.05). An analysis with logistic regression indicates that the optimal classification criterion for T. rudolfi sp. nov. versus T. pygmaeus lusitanicus is at Nlinks = 6.8. The fit of the classification, as expressed by the Area Under the Curve (auc) statistic is 0.851 ± 0.120 at the individual level and 0.929 ± 0.056 when data are averaged for populations. The geographical cline for Nlinks has a central position at -2.8 km (ci -15.5–18.0 km) and a width of 208.9 km (ci 20.4–425.0 km) (fig. 3).
Histogram for the number of links (Nlinks) observed in Iberian large-bodied newts, with low values for Triturus marmoratus (top panel), intermediate values for T. rudolfi sp. nov. (middle panel) and low to high values for T. pygmaeus (bottom panel). A distinction is made between T. p. pygmaeus from the Betic region (grey bars) and T. p. lusitanicus from the remainder of the species range (open bars) (see Arntzen, 2024). The optimal separation of T. rudolfi nov. sp. versus T. p. lusitanicus is achieved at Nlinks = 6.8, as shown by an interrupted line. To the right examples are shown of individuals with low and high link counts. Animals are facing left, with T. marmoratus from Gerês, northern Portugal at the top and T. p. lusitanicus from Sagres, southern Portugal at the bottom. Links are counted over the left and right side of the newts’ bodies, in between the insertion of the fore- and hind leg.
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
the imagery is reproduced from arntzen (2018)Body sizes as measured by SVl1 are large for T. marmoratus, medium for T. rudolfi sp. nov. and small for T. pygmaeus in both sexes (table 2) which is for the latter two species aptly illustrated by the geographical clines over the transect (fig. 4). The overall differentiation of species is significant (Independent samples Kruskal-Wallis test: males N = 714, test statistic 407.9, df = 2, P < 0.0001; females N = 876, test statistic 387.8, df = 2, P < 0.0001). Post-hoc tests show that pairwise species comparisons are also significant (P < 0.0001 in all combinations for both sexes). Analyses with logistic regression indicate that the optimal classification criterion for T. rudolfi sp. nov. versus T. pygmaeus lusitanicus is at SVl1 = 51.2 mm for males with auc = 0.671 ± 0.030 at the individual level and auc = 0.686 ± 0.107 when data are averaged for populations. For females the corresponding value is SVl1 = 57.3 mm, with auc = 0.680 ± 0.030 and auc = 0.743 ± 0.092, respectively. Geographical clines for body size have central positions of 65.7 km (ci 25.5–82.8) for males and 65.9 km (ci 22.9–89.2 km) for females. Cline widths are 56.4 km (ci 1.95–178.5 km) for males and 98.8 km (ci 47.7–246.2 km) for females (fig. 4).
Body proportions are different for the three species, except for R_FTl in both sexes (Kruskal-Wallis test, P > 0.05). Post-hoc comparisons suggest that relative extremity lengths (R_FLl, R_HLl) decrease in the order T. marmoratus – T. rudolfi sp. nov. – T. pygmaeus, that T. pygmaeus has a longer head (R_Hl) and T. marmoratus has a shorter body (R_ILd) compared to the other species (table 2). A principal component analysis on size-corrected morphometric data shows some differentiation among T. marmoratus and ‘T. pygmaeus’ as well as the near-complete overlap of scores for T. pygmaeus and T. rudolfi sp. nov., for males and for females (fig. 6). Percentages of variance explained are 41.4% and 15.2% for males and 36.4% and 17.4% for females, along the first and second axis, respectively. Measurements mostly contributing to the first axis concern the extremities, with loadings (L) 0.81<L<0.75 in males and 0.78<L<0.71 in females. Both head measurements and ILd (in females only) are more strongly contributing to the second axis, with 0.47<L<0.55 in males and 0.52<L<0.57 in females. The unprocessed morphometric data are available in supplementary table S3.
Results of a principal component analysis on size corrected morphometric data for three species of Iberian Triturus newts. The ellipses show the 95% confidence interval of the mean. Colours represent: blue – T. marmoratus, brown – T. rudolfi sp. nov., and red – T. pygmaeus. Top panel males and bottom panel females. Percentages of variance explained are indicated along the axes.
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
Non-parametric Kruskal-Wallis tests revealed significant ecological differentiation between the three species for each of the selected parameters (bio01, bio02, bio03, bio 12 and bio15; table 3). In post-hoc tests no significant differences were found for bio 15 (seasonality of precipitation) whereas for bio02 (mean diurnal temperature range) the species were all significantly different from one another. For bio01 (annual mean temperature), bio03 (isothermality) and bio12 (annual precipitation) climatic profiles were similar for T. marmoratus and T. rudolfi sp. nov., and significantly different for T. pygmaeus, suggesting that both former species share temperate, cooler and wetter environmental conditions, as different from T. pygmaeus.
The consistent genetic, morphological and ecological differentiation of western and eastern pygmy newt population groups warrants the description of a new taxon. The sharp genetic transition, in combination with a readily diagnosable differential morphology, particularly in colouration pattern and body size, justifies description at the species level. Because the type locality of ‘T. pygmaeus’ is the province of Cadiz, it is the western taxon that needs recognition. The formal description for T. rudolfi sp. nov. is in the Appendix, the taxonomic status at the species level is briefly discussed below and three individuals photographed alive are shown in Appendix Figure A2.
Discussion
The combined analysis of morphological and genetic data confirms that the Iberian Peninsula is inhabited by more than one species of Triturus newt, as is summarized in table 1 and fig. 1. The prime distinction is between Triturus marmoratus in the north and ‘T. pygmaeus’ in the south. The species border runs from Peniche at the Atlantic coast to the Madrid area, east of which both species cease to exist. Hybridization is rare and introgression is limited for most genetic markers (Espregueira Themudo and Arntzen, 2007a; Arntzen and Espregueira Themudo, 2008; Wielstra et al., 2013; Arntzen et al., 2014; Arntzen, 2018; Rancilhac et al., 2021; Gaczorek et al., 2023; Kazilas et al., 2023). The strongly parapatric and mosaic distribution has been interpreted as the signature of species replacement in northerly direction, most pronounced so along the Atlantic coast, by T. rudolfi sp. nov. at the expense of T. marmoratus (Espregueira Themudo and Arntzen, 2007b; Arntzen et al., 2021).
The southern half of the Iberian Peninsula accommodates more than one morphologically and genetically differentiated group of pygmy newts. Triturus p. pygmaeus from the Betic region differs from T. p. lusitanicus from north of the Guadalquivir by low link scores (fig. 5). Microsatellite genetic data revealed a narrow and steep transition between these subspecies, but information is limited to the Doñana National Park and the area of contact is small (Arntzen, 2024). For an intriguing hypothesis on how T. p. pygmaeus, along with presumably southern population groups of the salamander Pleurodeles waltl Michahelles, 1830 and the frog Pelophylax perezi (López Seoane, 1885) managed to colonize the southern stretches of Doñana National Park see Arntzen (2024). More work is needed to clarify the situation within Doñana National Park and across the Guadalquivir.
The Lisbon Peninsula and the Atlantic coast to the north are the territory of T. rudolfi sp. nov. The species meets up with T. marmoratus along the coastal dunes up to the river Vouga estuary (Arntzen et al., 2021). To the northeast T. rudolfi sp. nov. is wedged in between T. marmoratus and the Tejo River. To the east and southeast T. rudolfi sp. nov. and T. pygmaeus are probably widely separated by the Tejo River and the currently low-quality habitats brought about by agricultural land, with possibly some widespread introgression from T. rudolfi sp. nov. into T. pygmaeus (Kazilas et al., 2023). The area where the T. pygmaeus – T. rudolfi sp. nov. contact zone can most profitably be studied appears to be limited to a ca. 20 × 40 km stretch of land north of Entroncamento (fig. 3).
The geographical cline for mtDNA is sharper than the one for the nuclear genetic data and displaced to the east by 14.9 km (figure 4, supplementary table S2). Non-coincident and non-concordant clines in mtDNA versus nuDNA have generally been explained by differential introgression, for instance in scenarios where the demographic expansion of one species leads to range overlap and admixture in the advancing front, or when sex-biased dispersal promotes faster diffusion of the mitochondrial genome (Currat et al., 2008; Wielstra et al., 2017). The clines for morphology are substantially wider than for the genetic data, yet in approximately the same position for Nlinks and displaced to the east by ca. 65 km for body size, in both sexes. It therefore seems that a morphology-based estimate for the position of the species’ genetic transition is more readily, and considering the obtained auc-support values also more reliably, obtained from colouration pattern than from body size.
One point to be emphasized is that snp-marker development was geared up to the analysis of the T. marmoratus – ‘T. pygmaeus’ contact zone (Arntzen et al., 2021; Lopez-Delgado et al., 2021) and that the markers that are somewhat less informative for that purpose are those that perform best in distinguishing T. pygmaeus and T. rudolfi sp. nov. Accordingly, the separation of species achieved over the first (T. marmoratus – ‘T. pygmaeus’) and second axes of the principal component analysis (T. pygmaeus – T. rudolfi sp. nov.) (fig. 2) are not at the same scale so that inferences on the absolute level of differentiation would be invalid. Unbiased genetic data (Kazilas et al., 2023) resolve T. marmoratus and ‘T. pygmaeus’ as sister taxa, and T. rudolfi sp. nov. and T. pygmaeus as sister species within that clade. Under reference to an age of 24–16 Ma for the most recent ancestor in the genus (Steinfartz et al., 2007; Marjanović and Laurin, 2014), the date for these splits is estimated at ca. 5.0–3.3 Ma and 2.0–1.3 Ma, respectively. A phylogenetic analysis with a large panel of loci uncovered by restriction site-associated dna sequencing yields somewhat different relative branch lengths, resulting in the substantially higher estimates of ca. 8.0–5.3 Ma and 4.0–2.7 Ma, respectively (cf. Rancilhac et al., 2023), yet it has been argued that molecular phylogenetic inference requires careful attention to model assumptions, especially where it concerns the reconstruction of branch length (Leaché et al., 2015).
The present paper recognizes a new species of pygmy newt, T. rudolfi sp. nov. from the wider Lisbon Peninsula as different from T. pygmaeus in the south of the Iberian Peninsula. Independent data revealed the long-lasting, reciprocal monophyly of independently evolving T. pygmaeus and T. rudolfi sp. nov. population groups (Kazilas et al., 2023), supporting species status under the phylogenetic (Eldredge and Cracraft, 1980) as well as evolutionary species concept (Wiley, 1978; de Queiroz, 2007). The newly gathered genetic data provide ample evidence for partial reproductive isolation within a narrow zone of intergradation, consistent with some selection against hybrids. These observations support species status under the biological species concept as well (Mayr, 1942). Triturus rudolfi sp. nov., while phylogenetically more closely related to T. pygmaeus than to T. marmoratus (Kazilas et al., 2023), deviates towards the latter species in terms of geographical position, climate conditions, overall size and colouration pattern.
Although Mertens and Wermuth (1960) only recognized T. marmoratus and the crested newt, T. cristatus (Laurenti, 1768), they had another five taxa listed (four subspecies and one ‘variety’) that are by now accepted as species, namely T. carnifex (Laurenti, 1768), T. dobrogicus (Kiritzescu, 1903), T. karelinii (Strauch, 1870), T. macedonicus (Karaman, 1922) and T. pygmaeus. Two crested newt species have since been described from the southeastern parts of the genus’ range (T. anatolicus Wielstra and Arntzen, 2016 and T. ivanbureschi Arntzen and Wielstra, 2013), to which it is now added T. rudolfi sp. nov. from Portugal, bringing the total to ten Triturus species.
The coastal zone of Portugal has for long gone unnoticed for a high level of amphibian diversity. However, with the uprise of molecular systematics, two endemic amphibian species, the newt Lissotriton maltzani (Boettger, 1879) and the frog Pelodytes atlanticus Díaz-Rodríguez, Gehara, Márquez, Vences, Gonçalves, Sequeira, Martínez-Solano and Tejedo, 2017 were resolved as different from L. boscai (Lataste, 1879) and P. ibericus Sánchez-Herráiz, Barbadillo, Machordom and Sanchiz, 2000 (Díaz-Rodríguez et al., 2017; Dufresnes et al., 2020; Sequeira et al., 2020). Whereas the former are morphologically cryptic taxa, T. rudolfi sp. nov. is readily diagnosable because the signal from nuclear and mitochondrial dna is paralleled by morphological differentiation, most prominently in the species’ dorso-lateral coloration pattern.
Editor: M. Laurin
Acknowledgements
I thank Onno Schaap and Notis Theodoropoulus for running the snp-line and Iñigo Martínez-Solano for constructive comments to an earlier version of the manuscript. Licenses to collect were provided as follows: France – Prefecture de la Mayenne, by permit number2003-A-2007; Spain – various provinces by permits numbers cn0010/12/aca, cn03/0085, cn04/0269, cn10/0030, dgmen/sen/avp_12_015_aut, dnp 27/2008, E.P-107/04 (mg), is/pa/epcyl/129/2012, sgyb/foa/afr/cfs and Doñana National Park with permit number 27/2008, and Portugal – Instituto da Conservação da Natureza, by letters dated 26/10/1998, 19/4/2000 and 19/3/2002 and by permit numbers 397/2007/capt, 102/2010/capt, 103/2010/capt and 107/2012/capt. I thank two anonymous reviewers for their helpful comments.
Data accessibility
Data on Triturus colouration patterns and the localities studied are available from a previous publication (Arntzen, 2024). The morphometric data are presented as supplementary table S3. The molecular genetic data here employed were collected for the purpose of a study on hybrid zone dynamics and species replacement that is in statu nascendi and are therefore not yet released.
Supplementary material
Supplementary material is available online at: https://doi.org/10.6084/m9.figshare.25158365
A three-dimensional model of the entire skeleton of the type specimen obtained with ct-scanning is available for inspection at https://doi.org/10.6084/m9.figshare.25264903, courtesy of Dr. Tijana Vučić.
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Appendix A1
Description of Triturus rudolfi sp. nov.
Descriptions were made from preserved material, except for the number of links that was determined in the field. Dorsal colouration invariably dark, without patterning clear enough to allow a detailed description.
Description of type specimen – preserved on ethanol in excellent storage condition at the Museo Nacional de Ciencias Naturales, Madrid, Spain under catalogue number 51784. Adult male in breeding condition with a well-developed crest and a large black cloaca. SVl1 58 mm, SVl2 68 mm, ILd 30 mm, Hw 11.6 mm, Hl 19.8 mm, FLl 23.7 mm, TFl 7.0 mm, HLl 24.5 mm and FTl 10.5 mm. Total length 125 mm. Relative finger lengths 4 < 1 < 2 < 3. Relative toe lengths 1 < 5 < 2 < 4 < 3. Nlinks left 2, right 2, total 4. Fourteen crème-white bands over the head and body part of the mid-dorsal crest. Grey ventral colouration with many large, irregular shaped dark dots and many evenly distributed white spots, denser in the central part than towards the gular and cloacal regions. Colouration of throat region continuous with that of the belly. Underside of legs with dark dots. See fig. A1.
Holotype of Triturus rudolfi sp. nov. at right (top) and left lateral view (middle), and in ventral view (bottom). Size bar is 1 cm. Stored at the Museo Nacional de Ciencias Naturales, Madrid, Spain under catalogue number 51784.
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
Triturus rudolfi sp. nov. from Serra de Sintra (top) and from the Colares and Janas region (middle and bottom).
Citation: Contributions to Zoology 93, 2 (2024) ; 10.1163/18759866-bja10057
photography r. malkmusDescription of paratypes – Preserved on ethanol in excellent storage condition at the Museo Nacional de Ciencias Naturales, Madrid, Spain under catalogue numbers 51785-54791, sequentially.
First paratype. Adult male in breeding condition with a well-developed crest and a large black cloaca. SVl1 43 mm, SVl2 52 mm, ILd 21 mm, Hw 9.5 mm, Hl 13.7 mm, FLl 19.1 mm, TFl 6.8 mm, HLl 19.9 mm and FTl 7.2 mm. Total length 90 mm. Relative finger lengths 1 = 4 < 2 < 3. Relative toe lengths 1 < 5 < 2 < 4 < 3. Nlinks left 4, right 4, total 8. Thirteen crème-white bands over the head and body part of the mid-dorsal crest. Continuous crème ventral colouration from the throat to the cloacal region and underside of fore- and hindlegs.
Second paratype. Adult male in breeding condition with a well-developed crest and a large black cloaca. SVl1 50 mm, SVl2 58 mm, ILd 26 mm, Hw 11.0 mm, Hl 15.1 mm, FLl 21.4 mm, TFl 7.6 mm, HLl 22.6 and FTl 8.4 mm. Total length 107 mm. Relative finger lengths 4 < 1 < 2 < 3. Relative toe lengths 1 < 5 < 2 < 4 < 3. Nlinks left 2, right 4, total 6. Fifteen crème-white bands over the head and body part of the mid-dorsal crest. Grey ventral colouration with many large, irregular shaped dark dots and many evenly distributed white spots. Throat with few dots and spots. Region towards cloaca and underside legs crème coloured.
Third paratype. Adult male in breeding condition with a well-developed crest and a large black cloaca. SVl1 53 mm, SVl2 61 mm, ILd 26 mm, Hw 11.2 mm, Hl 17.2 mm, FLl 23.2 mm, TFl 8.0 mm, HLl 24.4 and FTl 10.0 mm. Total length 110 mm. Relative finger lengths 1 < 4 < 2 < 3. Relative toe lengths 1 < 5 < 2 < 3 = 4. Nlinks left 0, right 4, total 4. Sixteen crème-white bands over the head and body part of the mid-dorsal crest. Light grey ventral colouration, with large, reticulated dark dots and white spots over half the surface. Colouration of throat continuous with belly. Crème colour towards cloaca. Underside hindlegs crème coloured with dark dots.
Fourth paratype. Adult female in breeding condition with ovarian eggs shining through. SVl1 66 mm, SVl2 72 mm, ILd 35 mm, Hw 12.6 mm, Hl 18.4 mm, FLl 23.6 mm, TFl 7.5 mm., HLl 23.1 and FTl 7.3 mm. Total length 134 mm. Relative finger lengths 1 < 4 < 2 < 3. Relative toe lengths 1 = 5 < 2 < 4 < 3. Nlinks left 2, right 3, total 5. Light grey ventral colouration with few small dark dots. Colouration of throat continuous with belly with small white spots. Crème coloured towards cloaca and underside hindlegs.
Fifth paratype. Adult female in breeding condition with ovarian eggs shining through. SVl1 57 mm, SVl2 63 mm, ILd 29 mm, Hw 11.3 mm, Hl 15.4 mm, FLl 21.9 mm, TFl 7.7 mm, HLl 22.0 and FTl 8.0 mm. Total length 120 mm. Relative finger lengths 1 = 4 < 2 < 3. Relative toe lengths 5 < 1 < 2 < 3 = 4. Nlinks left 0, right 1, total 1. Grey ventral colouration with few small dark dots. Colouration of throat continuous with belly. Crème coloured towards cloaca and underside hindlegs.
Sixth paratype. Adult female in breeding condition with ovarian eggs shining through. SVl1 63 mm, SVl2 68 mm, ILd 32 mm, Hw 11.8 mm, Hl 18.5 mm, FLl 22.0 mm, TFl 7.6 mm, HLl 21.0 and FTl 7.2 mm (measured at left side, because toe at right side is either malformed or regenerating). Total length 127 mm. Relative finger lengths 1 < 4 < 2 < 3. Relative toe lengths 1 < 5 < 2 < 3 = 4 (left side). Nlinks left 1, right 1, total 2. Upper side of tail damaged, possibly a bite mark. Grey ventral colouration with medium number of dark dots and few white spots. Colouration of throat continuous with belly. Crème coloured towards cloaca and underside hindlegs.
Seventh paratype. Adult female in breeding condition with ovarian eggs shining through. SVl1 60 mm, SVl2 66 mm, ILd 32 mm, Hw 11.7 mm, Hl 17.2 mm, FLl 22.5 mm, TFl 7.6 mm, HLl 21.2 and FTl 7.8mm. Total length 121 mm. Relative finger lengths 1 < 4 < 2 < 3. Relative toe lengths 1 = 5 < 2 < 3 < 4. Nlinks left 0, right 2, total 2. Light grey ventral colouration, with few small dark dots. Colouration of throat region similar to that of belly, but lighter and with small white spots. Crème coloured towards cloaca and on underside hind legs.
Other material from the type locality – zma.rena.9332 (N = 1, leg. J. W. Arntzen) and zma.rena.19271 (N = 4, leg., J. W. Arntzen and E. Froufe).
Locality and date of collecting – Lagoa Seca, Valado dos Frades near Nazaré, Portugal at 39.596 northern latitude and 9.012 western longitude. Elevation 27 m a.s.l. Date of collecting 20 March 2013, leg. J. W. Arntzen. Date of deposition at the Museo Nacional de Ciencias Naturales 16 August 2023.
Diagnostic features – the newly recognized species is most closely related to, yet morphologically distinguishable from T. pygmaeus, on account of a lower number of green coloured dorso-lateral transversal bands (‘links’). Body size larger than in T. pygmaeus. Significant genetic differences for population groups at either side of the cline near Entroncamento were found at 34 out of 54 investigated nuclear markers, as well as for mitochondrial dna. Body size smaller than in T. marmoratus, with a higher number of links and with a light rather than dark coloured underside.
Derivatio nominis – the species name is chosen in honour of Mr. Rudolf Malkmus, in recognition of his contribution to the knowledge of the Portuguese herpetofauna. Mr. Malkmus placed the Portuguese herpetofauna on the map, not just as a figure of speech, but also literally (Malkmus, 2004).
Suggested vernacular name – Malkmus’ pygmy newt or Lisbon pygmy newt. I further advocate to restrict the name ‘marbled newt’ to T. marmoratus and to use ‘pygmy newts’ for T. pygmaeus and T. rudolfi sp. nov. (see also table 1).
Distribution – the Lisbon Peninsula in Portugal, reaching northward along the Atlantic Ocean up to the river Vouga estuary. Approximate range borders: bounded by T. marmoratus in the north at ca. 39.5 N, separated from T. pygmaeus in the east by the Tejo River and in the northeast by a narrow hybrid zone that is wedged in between T. marmoratus territory and the river Tejo.
Conservation status – vulnerable, on account of the small range probably more so than other Iberian Triturus species.
Nomenclatorial act – the electronic ‘on-line early’ version of this article is considered a published work according to the International Code of Zoological Nomenclature. The new name has also been registered in ZooBank (http://zoobank.org/) where it can be accessed under http://zoobank.org/urn:lsid:zoobank.org:pub: D6D62940-6B0E-42E5-8212-383D28DC3D6A.
