Abstract
The herpetofauna of the Iberian Peninsula is relatively well-researched, yet detailed studies, at least in part relying on molecular genetic data, continue to reveal taxa new to science, mostly species and subspecies. Newts of the genus Triturus are one such group with undiscovered yet taxonomically relevant variation, as shown by the recent description of new (sub)species of pygmy newts (T. pygmaeus, T. rudolfi). The marbled newt, Triturus marmoratus, shows an equally deep and geographically coherent spatial-genetic diversification. It is here shown that a northern and a southern group are characterized by different mitochondrial dna profiles and are also differentiated in morphometry and colouration pattern. With no firm evidence for selection against intermediate genotypes, the southern group is described at the subspecies level, as T. marmoratus harmannis ssp. nov. The subspecies’ contact zone is situated at ca. 41.5 northern latitude and stretches from the Atlantic coast near Porto, Portugal to the northeast of Madrid, Spain.
Introduction
Biological diversity is not homogeneously distributed across the planet and outstandingly rich areas are known as biodiversity hotspots (Myers et al., 2000). The Mediterranean basin is one of those 25‒36 global biodiversity hotspots. Therein, the Iberian Peninsula contains more than 50% of the recognized European vertebrate and plant species and at ca. one-third of the total for plants and vertebrates, the endemicity rate is also high (Médail & Quézel, 1997; Williams et al., 2000). Among amphibians and reptiles, the Iberian Peninsula is by now relatively well-researched and well-documented, probably contributing to the documented pattern. I here delve into the morphological and genetic variation of western Mediterranean large-bodied newts (genus Triturus). The data so far resolved taxonomically relevant variation in pygmy newts, leading to the recognition of the species T. rudolfi Arntzen, 2024, as different from T. p. pygmaeus (Wolterstorff, 1905) and the subspecies T. p. lusitanicus Arntzen, 2023. However, within marbled newts, Triturus marmoratus (Latreille, 1800), two groups with coherent geographical ranges are also apparent (Kazilas et al., 2024). It is here investigated if the documented nuclear genetic differentiation of T. marmoratus has parallels in morphometry, colouration pattern and mitochondrial dna, to which the answer is affirmative. The data obtained so far suggest the presence of a wide transition area between the groups. With no evidence for selection against admixed genotypes, the new taxon is described at the subspecies level. Given that the type locality of T. marmoratus is situated at the very north of the species range (‘Paris’; Mertens and Müller, 1928) it is the southern taxon that requires recognition, for which the formal description (as Triturus marmoratus harmannis ssp. nov.) is in the Appendix.
Materials and methods
Fieldwork and morphological data
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 and released on the spot immediately after processing. Adults were measured and studied for their dorso-lateral colour pattern (Nlinks). Small amounts of tissue for molecular genetic analyses were taken as larval or adult tail tips and stored on 95% ethanol. Alternatively, recently deposited eggs (embryos) were raised in 5l buckets until hatching.
Measurements made were snout-vent length from the tip of the snout up to and including the insertion of the hind leg, or the posterior side of the cloaca (SVl1, SVl2), head length and head width (Hl, Hw), interlimb distance (ILd), fore- and hindlimb length (FLl, HLl) and third finger and fourth toe length (TFl, FTl). Measurements were taken in mm with a plastic ruler or on plasticized graph paper for SVl and ILd, and with plastic Vernier callipers at 0.1 mm precision for the other characters. Extremities were measured at the right side of the body. The measurement SVl2 was only used for descriptive purposes. To facilitate data interpretation and for comparability with published data, body proportions were calculated from untransformed data as character/SVl1. To further 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.
Nlinks is counted as the number of uninterrupted connections over the green coloured surface, from the position of the crest in males or the mid-dorsal line in females to the belly, over the region in between the insertions of the fore- and hindlegs, with numbers summed for the left and right side of the body (Arntzen, 2018). Analyses of morphological data were restricted to four groups of marbled newts (T. marmoratus) defined by two sexes and two inferred subspecies. Statistical analyses were done with spss 26 (ibm spss, 2019).
Mitochondrial and nuclear genetic data
dna was extracted from the preserved tissue samples under standard protocols with the KingfisherTM (Thermo Scientific) and DNeasy extraction kits (Qiagen, Valencia, CA, USA). A fragment of the mitochondrially encoded nadh dehydrogenase 4 gene (nd4) was amplified and sequenced with established primers, also following standard protocols (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 were trimmed to 600 bp to fit an earlier analysis and analysed by the minimum spanning network procedure (Bandelt et al., 1999) as supported in PopArt software (Leigh & Bryant, 2015) under default settings. This revealed the presence of six main haplotypes that each accommodate >2% of the data. A distance matrix was calculated in paup*4 (Swofford, 2003) for all individual sequences that were then allocated to the haplogroups H1–H6, in casu the representative main haplotype to which the uncorrected p-distances was smallest. For each population the dominant haplogroup (i.e., the one observed at highest frequency) was determined. It has been documented that the species border between marbled and pygmy newts is sharp, with limited hybridization and introgression (e.g., Arntzen, 2018). Yet, mtDNA haplotypes typical for one species were regularly found associated with the nuclear genome of the opposite species (Wielstra et al., 2013; Kazilas et al., 2024; present paper), but the analysis of interspecific cyto-nuclear discordances is left for a forthcoming article.
I reanalysed the nuclear gene capture data published by Kazilas et al. (2024), with the aims to estimate the geographical ranges of (four) taxa that were resolved as reciprocally monophyletic groups, and to scrutinize the intermediate status of several individuals that a Structure analysis had identified as mildly admixed. Analyses were done with HIest software (Fitzpatrick, 2012) and were restricted to markers with largely diagnostic properties for non-admixed reference samples. The panel contained 32 markers for within T. marmoratus, with a Cohen’s kappa (ĸ) measure of (dis)association as (1-ĸ) > 0.70 and 33 markers for T. pygmaeus and T. rudolfi with (1-ĸ) > 0.80.
Blanket distribution maps composed of so-called Dirichlet cells (Matérn, 1979; Burroughs et al., 2015) were obtained by the spatial interpolation of taxon and haplogroup information with the ‘NearestPoint’ procedure in ilwis 3.8.6 (ilwis, 2019), with a maximum extrapolation of ca. 90 km. The map derived from the gene capture data was used to allocate T. marmoratus populations not studied by Kazilas et al. (2024) to either the nominotypical subspecies, the new subspecies, or to the class of intergrades. The latter group was excluded from the morphological data analysis on account of its sparse representation.
Results
The gene capture data resolved two species of pygmy newts (T. rudolfi and T. pygmaeus) as well as two spatially coherent groups of marbled newts (T. marmoratus) (fig. 1). One individual pygmy newt standing out in the triangular HIest plot is allocated to T. rudolfi, in line with its origin from Serra de Sintra at the south of the species range. Another individual genetically akin to T. m. harmannis ssp. nov. has a coastal position and yet another three akin to T. m. marmoratus have an inland geographical position.
Classification and geographical distribution of European marbled and pygmy newts from a panel of 32–33 nuclear genetic markers (data from Kazilas et al., 2024). (A) HIests plot with ancestry and heterozygosity for within marbled newts (top panel, with Triturus m. marmoratus left and T. m. harmannis ssp. nov. to the right) and for pygmy newts (bottom panel, with T. rudolfi to the left and T. pygmaeus to the right). (B) Investigated Iberian populations shown by black dots with surrounding areas coloured as in A. Areas shown in white fall outside the documented range of the T. marmoratus species group and areas in shown grey are distant from a sampled locality. The open square symbol in the Lisbon Peninsula corresponds to the open round symbol in A.
Citation: Contributions to Zoology 93, 3 (2024) ; 10.1163/18759866-bja10060
Of all adult marbled newts with Nlink data, 167 individuals from 16 populations were allocated to T. m. marmoratus and 635 individuals from 36 populations were allocated to T. m. harmannis ssp. nov. Both subspecies show a wide range of values and have the same modal value (Nlinks = 0). However, character state distributions are different (Median Chi2 test, d.f. = 1, Chi = 33.2, P < 0.0001), with on average higher values for T. m. marmoratus (Ñlinks = 3.0) than for T. m. harmannis ssp. nov. (Ñlinks = 1.7) (fig. 2A). The difference between the averages (∆Ñlinks) is thus 1.3. For 28 well-sampled populations (N ≥ 10) a full separation among subspecies is achieved, with a cut-off point at Ñlinks = 2.3 (fig. 2B).
Histograms of Nlink values observed for adults of Triturus m. marmoratus (N = 167, shaded bars) and T. m. harmannis ssp. nov. (N = 635, open bars), with (A) all data and (B) averages for populations with a sample size ≥ 10. (C) Juvenile T. marmoratus from the subspecies transition area (Poço do Inferno near Valongo, Portugal). The photo aims to illustrate the species, and to discuss the usefulness of the character Nlinks for subspecies identification (details see text).
Citation: Contributions to Zoology 93, 3 (2024) ; 10.1163/18759866-bja10060
Of all populations with morphometric data, five populations were allocated to T. m. marmoratus (70 males and 80 females) and 46 were allocated to T. m. harmannis ssp. nov. (247 males and 338 females). Body size is smaller in T. m. harmannis ssp. nov. than in T. m. marmoratus. The relative sizes of extremities are shorter and head sizes larger in T. m. harmannis ssp. nov. than in T. m. marmoratus (table 1). A discriminant analysis of size corrected morphometric data retrieved a substantial differentiation across subspecies and sexes, more pertinent in females than in males (fig. 3). The first discriminant axis has high loadings (L) for the extremities (TFl, FLl, FTl and HLl, 0.48 < L < 0.82) and not the other characters (0.00 < L < 0.13) whereas the second axis has the highest loadings for Hl (L = 0.68) and Hw (L = 0.72).
Histogram of scores along the first axis for the discriminant analysis of size corrected morphometric data for Triturus marmoratus marmoratus (shaded bars) and T. m. harmannis ssp. nov. (open bars). This first discriminant axis is most strongly correlated with extremity lengths and not the other characters. Highest loadings on the second axis are for head length and head width (results not shown).
Citation: Contributions to Zoology 93, 3 (2024) ; 10.1163/18759866-bja10060
A total of 3324 nd4 sequences was obtained for altogether 278 populations of T. marmoratus, T. pygmaeus and T. rudolfi, increasing the data for Iberian Triturus species by up to two orders of magnitude. Sequences were dissimilar for pygmy versus marbled newts (fig. 4). Triturus pygmaeus and T. rudolfi fall in the same haplotype group (H6) that has a haplotype diversity π = 0.00088. Triturus marmoratus is characterized by five haplogroups (H1‒H5), with a haplotype diversity ranging from π = 0.00015 for H5 to π = 0.00269 for H3 (table 2). The haplogroups H1–H5 are not randomly distributed over T. m. marmoratus and T. m. harmannis ssp. nov. (Chi2 test, Chi = 729.23, d.f. = 4, P < 0.0001). Triturus m. marmoratus has mostly the H1 and H2 haplogroups whereas T. m. harmannis ssp. nov. carries mostly the H3–H5 haplogroups. The distribution of dominant haplogroups (fig. 5) is largely geographically coherent and coincides with subspecies ranges as estimated by the nuclear data (fig. 1). Five populations located in the coastal transition zone have H2 as the dominant haplogroup. For 64 T. marmoratus populations with an mtDNA sample size ≥ 10 the majority carried a single haplogroup (39 times, 61%). Co-occurring haplogroups were most often H3 with H4 (three times), H3 with H5 (two times) and H4 and H5 (18 times) whereas the co-occurrence of H1 and H2 was not observed.
Minimum spanning network for mitochondrial nd4 sequences retrieved from pygmy newts (Triturus rudolfi and T. pygmaeus) with a single haplogroup in red (H6), and marbled newts (T. marmoratus) with five haplogroups shown as H1 – green, H2 – light green, H3 – light blue, H4 – blue and H5 – purple. Satellite haplotypes are allocated to the nearest main haplotype, based on minimum uncorrected p-distances (details see text). Size of the circles corresponds to sample size (see legend). Internal and external branches have lengths of one or two substitutions, except for those marked with an ‘x’ that have 5‒7 substitutions.
Citation: Contributions to Zoology 93, 3 (2024) ; 10.1163/18759866-bja10060
The Iberian Peninsula with the ranges of Triturus pygmaeus in light red and T. rudolfi in brown (Arntzen, 2023, 2024). Populations of the counterpart species T. marmoratus are coloured according to the dominant mitochondrial haplogroups with colours as in the legend and fig. 4. The solid or interrupted black line shows the northern range of T. m. harmannis ssp. nov., as determined by the green, yellow and blue sections in fig. 1. Note that haplogroups 1 and 2 are associated with T. m. marmoratus and that haplogroups 3, 4 and 5 are associated with T. m. harmannis ssp. nov. Haplogroup 6 is associated with T. pygmaeus and T. rudolfi (for details see table 2). Areas shown in white fall outside the documented range of the T. marmoratus species group and areas shown in grey are distant from a sampled locality.
Citation: Contributions to Zoology 93, 3 (2024) ; 10.1163/18759866-bja10060
Discussion
The study of nuclear genetic variation in western European Triturus species revealed two reciprocally monophyletic and geographically coherent groups of individuals of similar evolutionary age of ca. 2.5 Ma (Arntzen, 2024; Kazilas et al., 2024;). One group is composed of the pygmy newts T. rudolfi and T. pygmaeus from the west and the south of the Iberian Peninsula (Arntzen, 2024; fig. 5). The other group is made up by marbled newts, T. marmoratus, from western-central Iberia and from northern Iberia and France (fig. 1). It is here shown that these marbled newt groups also possess different mtDNA haplotype profiles and have dissimilar morphologies. These results justify the recognition of a new taxon (see Appendix).
Even though both taxon-pairs are about equally deeply differentiated, the contact zones are narrow in pygmy newts (Arntzen, 2024) and wide in marbled newts (present paper). This observation is in line with the loose negative correlation of genetic differentiation and hybridization potential found in a wide range of organisms, including the genus Triturus (Jiggins & Mallet, 2000; Arntzen et al., 2014). Compared to the survey over 15 hybrid zones in frogs and toads (Dufresnes et al., 2021), the (sub)speciation events in Iberian Triturus are relatively recent, yet on par with the earlier events (also estimated at ca. 2.5 Ma) for the anuran subspecies Discoglossus g. galganoi Lanza, Nascetti, Capula and Bullini, 1986 and D. g. jeanneae Busack, 1986 that show a wide intergradation zone (ca. 140 km), and the species Alytes obstetricans (Laurenti, 1768) and Alytes almogavarii Arntzen and García-París, 1995, that engage in a narrow hybrid zone (ca. 18 km). In pygmy newts the steep transition zone (ca. 35 km; Arntzen, 2024) suggests selection against hybrids and consequently T. rudolfi was described as a species. Conversely, in the absence of evidence for selection against admixed genotypes in what is taken to be a wide transition zone, the southern taxon of the marbled newt is classified as a subspecies, namely T. m. harmannis ssp. nov.
The subspecies T. m. marmoratus and T. m. harmannis ssp. nov. are morphologically differentiated. The former taxon is of larger size than the latter one and has a relatively smaller head. The subspecies differ in the length of the extremities, that are longer in the former than in the latter taxon, be it that the difference is more pronounced in females than in males. While a strong sexual dimorphism in limb lengths is already documented for the genus Triturus (Malmgren & Tholessson, 1999), it is here shown that in T. marmoratus it aligns with subspecies differentiation (table 1, fig. 3).
Colour and colouration pattern of western European Triturus species are highly variable and the character ‾N links captures just an aspect of the variation in marbled and pygmy newts (Arntzen, 2024). Moreover, the diagnostic power of ‾N links to identify geographically adjacent (sub)species varies as seen from the difference in the average number of links (∆ ‾N links) that is 2.9 for T. marmoratus – T. rudolfi, 7.3 for T. marmoratus – T. p. lusitanicus, 4.5 for T. rudolfi – T. p. lusitanicus and 7.4 for T. p. lusitanicus – T. p. pygmaeus (Arntzen, 2018, 2023, 2024). Accordingly, the weakest signal (∆ ‾N links = 1.3) is for the T. marmoratus subspecies here considered. Confounding factors are sample size (fig. 2AB) and the possibly wide zone of subspecies intergradation (fig. 1B). Ontogeny has also been implicated (Arntzen, 2023), but photographic material for T. pygmaeus lusitanicus from Guadalupe, Central Spain, shows that juveniles possess a colouration pattern already like that of the adults (Sergé Bogaerts, pers. comm., 8 November 2023). The juvenile T. marmoratus here shown here as example (fig. 2C) has no transversal green bands at either side of the body (Nlinks = 0) and is from inside the subspecies’ transition area and therewith remains of unknown subspecies affiliation. A remarkable feature for the group at large is that two southerly subspecies (T. marmoratus harmannis ssp. nov. and T. p. pygmaeus) are more striped (i.e., with lower Nlink values) than both northerly conspecifics (T. m. marmoratus and T. p. lusitanicus).
The observed mitochondrial dna variation is partitioned in five haplotype groups of which two associate with T. m. marmoratus (H1 and H2) and three with T. m. harmannis ssp. nov. (H3, H4 and H5), to which it may be noted that the haplogroups H1 and H2 are spatially partitioned whereas H3, H4 and H5 are frequently found together, yet separate out when the dominant haplogroup is considered (table 2, fig. 5). Because the mitochondrial and nuclear dna spatial distributions are largely concordant, i.e., with no widespread cyto-nuclear discordances for the subspecies, particular phenomena such as e.g., sex-dependent dispersal, adaptive introgression and hybrid zone movement (Ballard & Whitlock, 2004; Toews & Belsford, 2012) do not have to be invoked. The prime task at hand is the sequencing of the complete mtDNA molecule, to test the hypothesis that the H3, H4 and H5 haplogroups associated with the new subspecies are more closely related to one another than they are to H1 and H2.
Triturus from the Iberian Peninsula has now ‘triturated’ into five taxa with, from north to south T. m. marmoratus, T. m. harmannis ssp. nov., T. rudolfi, T. p. lusitanicus and T. p. pygmaeus. An overall similarity in spatial – temporal relationships within this monophyletic group and the continental European representatives of the genus Alytes can be discerned (García-París & Martínez-Solano, 2001; Ambu et al., 2023), in which the range of T. m. harmannis ssp. nov. coincides with that of Alytes obstetricans cf. boscai Lataste, 1879, as represented by the ‘green lineage’ of Maia‐Carvalho et al. (2018). While the Douro River basis may explain the local phylogeography of Alytes midwife toads (Maia‐Carvalho et al., 2018; Ambu et al., 2023) and was also implicated in the patterning of genetic variability in the golden-striped salamander, Chioglossa lusitanica Bocage, 1864 (Alexandrino et al., 2000), the correspondence is incomplete because T. m. harmannis ssp. nov. is found at either side of that river (Kalezic et al., 2023; fig. 1). Other recent additions to the amphibian checklist of western Iberia are Chioglossa lusitanica longipes Arntzen and Alexandrino, 2007 (Arntzen et al., 2007; Sequeira et al., 2022), Lissotriton maltzani (Boettger, 1879) (Sequeira et al., 2020), Pelodytes atlanticus Díaz-Rodríguez, Gehara, Márquez, Vences, Gonçalves, Sequeira, Martínez-Solano and Tejedo, 2017 (Díaz-Rodríguez et al., 2017), Rana parvipalmata Seoane, 1885, and R. p. asturiensis Dufresnes, Ambu, Galán, Sequeira, Viesca, Choda, Álvarez, Alard, Suchan, Künzel, Martínez-Solano, Vences and Nicieza, 2023 (Dufresnes et al., 2020, 2023). With these discoveries, the pinnacle of amphibian diversity in the Iberian Peninsula accentuates towards the Atlantic region, partly outside the Mediterranean biodiversity hotspot. Several of the above cases are, however, in need of a more thorough characterization of contact zones. This also applies to T. m. marmoratus – T. m. harmannis ssp. nov. Because their intergradation zone stretches over 2/3 of the width of the Iberian Peninsula (ca. 500 km), it should be relatively straightforward to find areas in which latitudinal transects can be studied in detail.
editor: r. vonk
Acknowledgements
Licenses for fieldwork were provided as follows: France – Prefecture de la Mayenne, by permit number 2003-A-2007; Spain – various provinces by permits numbers CN0010/12/aca, cn03/0085, cn04/0269, cn10/0030, dgmen/sen/avp_12_015_aut, 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 Bastian Reijnen and Notis Theodoropoulus for assistance with the mtDNA sequencing.
Data availability
Many data used in this study are available from published works ‒ for morphology and localities see Arntzen (2023, 2024) and for molecular data see Wielstra et al. (2013) and Kazilas et al. (2024). New mtDNA sequences 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.
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Appendix
Description of Triturus marmoratus harmannis ssp. nov.
Descriptions were made from preserved material, except for the Nlinks character state that was determined in the field. Dorsal colouration invariably dark, yet with the green colouration pattern discernible. Morphological data retrieved for a series of live animals are in Table A1.
Description of type specimen – preserved on ethanol in excellent storage condition at the ‘Museo Nacional de Ciencias Naturales’, Madrid, Spain under catalogue number mncn 51792. Adult male in breeding condition with a well-developed crest and a large back cloaca. Thirteen crème-white bands over the head and body part of the crest. SVl1 64.5 mm, SVl2 73.5 mm, ILd 32.5 mm, FLl 28.3 mm, TFl 8.5 mm, HLl 28.1 mm, FTl 11.6 mm, Hw 14.1 mm and Hl 19.7 mm. Total length 136.5 mm. Relative finger lengths 1<4<2<3. Relative toe lengths 1<5<2<4<3. Nlinks left 0, right 0, total 0. Solid dark grey ventral colouration with many more or less evenly distributed white spots, denser in the middle part than towards the gular and cloacal regions. Throat region light grey with many small white dots. Underside of the legs and cloacal region with light and dark regions. The holotype is shown in fig. A1. 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.25358890, courtesy of Dr. Tijana Vučić’.
Holotype of Triturus marmoratus harmannis ssp. nov. at right and ventral view. Size bar is 1 cm. Stored at the Museo Nacional de Ciencias Naturales, Madrid, Spain under catalogue number 51792.
Citation: Contributions to Zoology 93, 3 (2024) ; 10.1163/18759866-bja10060
Description of paratypes – Preserved on ethanol in excellent storage condition at the ‘Museo Nacional de Ciencias Naturales’, Madrid, Spain.
First paratype mncn 51793. Adult male in breeding condition with a well-developed crest and a large back cloaca. Seventeen crème-white bands over the head and body part of the crest. SVl1 65.0 mm, SVl2 73.5 mm, ILd 34.0 mm, FLl 26.2 mm, TFl 9.3 mm, HLl 27.0 mm, FTl 11.5 mm, Hw 13.6 mm and Hl 19.9 mm. Total length 135 mm. Relative finger lengths 1=4<2<3. Relative toe lengths 1<5<2<4<3. Nlinks left 0, right 1, total 1. Grey ventral colouration with many dark dots. Many white spots mostly positioned on the undotted sections. Colour of the throat continuous with that of the belly. Underside of the legs and cloacal region with light and dark regions.
Second paratype mncn 51794. Adult male in breeding condition with a low crest and a large back cloaca. Eleven crème-white bands over the head and body part of the low crest. SVl1 56.5 mm, SVl2 64.0 mm, ILd 28.5 mm, FLl 23.4 mm, TFl 7.7 mm, HLl 23.6 mm, FTl 9.6 mm, Hw 12.6 mm and Hl 17.7 mm. Total length 118 mm. Tail tip regenerating. Relative finger lengths 1<4<2<3. Relative toe lengths 1<5<2<4<3. Nlinks left 1, right 2, total 3. Solid black ventral colouration with many, evenly distributed small white spots. Throat colouration continuous with belly with equally dense but larger white spots. Underside of the legs and cloacal region light with few dark regions.
Third paratype mncn 51795. Adult female in breeding condition with ovaries shining through. SVl1 64.0 mm, SVl2 70.0 mm, ILd 33.0 mm, FLl 25.1 mm, TFl 8.3 mm, HLl 23.7 mm, FTl 7.6 mm, Hw 14.5 mm and Hl 19.6 mm. Total length 140.5 mm. Relative finger lengths 1<4<2<3. Relative toe lengths 1=5<2<4<3. Nlinks left 2, right 2, total 4. Solid, medium-grey ventral colouration with few, evenly distributed small white spots. Throat coloration continuous with that of the belly. Underside of the legs and cloacal region solid light grey.
Fourth paratype mncn 51796. Adult female in breeding condition with ovaries shining through. SVl1 73.0 mm, SVl2 79.5 mm, ILd 39.5 mm, FLl 28.1 mm, TFl 9.3 mm, HLl 26.4 mm, FTl 8.8 mm, Hw 16.5 mm and Hl 21.7 mm. Total length 149 mm. Relative finger lengths 1<4<2<3. Relative toe lengths 1<5<2<4<3. Nlinks left 0, right 0, total 0. Dark grey ventral colouration with few small dark dots and few white spots. Colouration of throat continuous with belly. Underside of the legs and cloacal region light with few dark regions.
Fifth paratype mncn 51797. Adult female in breeding condition with ovaries shining through. SVl1 71.0 mm, SVl2 77.5 mm, ILd 37.5 mm, FLl 27.4 mm, TFl 9.1 mm, HLl 26.0 mm, FTl 7.2 mm, Hw 15.4 mm and Hl 22.2 mm. Total length 150.5 mm. Relative finger lengths 1=4<2<3. Relative toe lengths 1=5<2<4<3. Nlinks left 1, right 2, total 3. Light grey ventral colouration with large dark dots and few white spots towards the flanks. Colouration of throat continuous with belly. Underside of the legs and cloacal region light with few dark regions.
Sixth paratype mncn 51798. Adult female in breeding condition with ovaries shining through. SVl1 72.0 mm, SVl2 78.5 mm, ILd 38.5 mm, FLl 28.0 mm, TFl 9.0 mm, HLl 27.2 mm, FTl 8.7 mm, Hw 16.1 mm and Hl 21.1 mm. Total length 146 mm. Relative finger lengths 1=4<2<3. Relative toe lengths 1<5<2<3=4. Nlinks left 2, right 2, total 4. Light grey ventral colouration with medium number of large dark dots and few white spots. Colouration of throat continuous with belly. Underside of the legs and cloacal region light with few dark regions.
Locality and date of collecting – Arrochela, near Madeirã, Portugal at 39.9386 northern latitude and 8.1025 western longitude. Elevation 396 m a.s.l. Date of collecting 20 March 2013, leg. J. W. Arntzen. Date of deposition at mncn 16 August 2023.
Diagnostic features – the newly recognized subspecies is closely related to and morphometrically similar to T. m. marmoratus, yet at the population level characterized by a lower number of green coloured dorso-lateral transversal bands (‘links’). In comparison with T. m. marmoratus, it has a small body size, significantly shorter extremities in females and a relatively big head in both sexes. The distinctiveness of the subspecies is supported by nuclear and mitochondrial dna data.
Derivatio nominis – the subspecies name is chosen to commemorate the Dutch couple Harm and Annie (or Ann) Walen, who lived from 1926‒2005 (Mr. H. C. Walen) and 1928‒ 2018 (Mrs. A. A. van Silfhout). After Harm’s retirement from his taxidermist job at the Zoological Museum in Amsterdam, the twosome eventually landed in Nisa, Portugal where they constructed their own ‘quinta’ and felt enormously at place. Without ‘harmann’s’ hospitality and moral support, my extensive fieldwork in Portugal and adjacent Spain late last century would hardly have been sustainable. The new subspecies’ name refers to a lifelong couple and only indirectly to individual people so that, matrimony being gender neutral, the third declension is used.
Suggested vernacular name – Harmann’s marbled newt, or central Iberian marbled newt Distribution – central-western Iberia. The southern range border is determined by a sharp, parapatric, yet mosaic range border with two pygmy newt species, namely T. rudolfi in the west and T. pygmaeus in the centre of the Iberian Peninsula. The northern edge of the range is positioned at ca. 41.5 N. The transition area with the nominotypical subspecies may be wide.
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:6DE32B0B-FB92-4F45-80BB-15EBC49DD23E.
