Rana parvipalmata asturiensis, Dufresnes & Ambu & Galán & Sequeira & Viesca & Choda & Álvarez & Alard & Suchan & Künzel & Martínez-Solano & Vences & Nicieza, 2024

Dufresnes, Christophe, Ambu, Johanna, Galán, Pedro, Sequeira, Fernando, Viesca, Leticia, Choda, Magdalena, Álvarez, David, Alard, Bérénice, Suchan, Tomasz, Künzel, Sven, Martínez-Solano, Iñigo, Vences, Miguel & Nicieza, Alfredo, 2024, Delimiting phylogeographic diversity in the genomic era: application to an Iberian endemic frog, Zoological Journal of the Linnean Society 202 (1), pp. 1-15 : 10-12

Description of Rana parvipalmata asturiensis ssp.nov.

( Fig. 6 )

ZooBankregistration: urn:lsid:zoobank.org:act:89BF6DB4-36E4-4B3F-9735-D9D5D6F6D699 .

Identity: Both lineages T1 and T2 are currently assigned to the species Rana parvipalmata , a taxon originally described as Rana temporaria parvipalmata López-Seoane, 1885 . The original description ( López-Seoane 1885) reported this taxon from ‘the north-west of Spain, equally common from the level of the sea to an elevation of 1422 feet’, with the additional mention ‘ Near Pontevedra , Tui and Ferrol , it occurs conjointly with Rana iberica ’—all these explicitly mentioned localities are situated on the Galician coast. Although measurements of three specimens were given, no locality information for these was published and no type material is known to exist ( Frost 2023). Salvador (1974) proposed to restrict the type locality to ‘ La Coruña’ but did not designate a lectotype or a neotype. Given the localities and the diagnostic characters purported in the original description ( parvipalmata means ‘poorly webbed’), it is unambiguous that the nomen parvipalmata is to be assigned to lineage T1. For lineage T2, no taxonomic name is available (see e.g. Frost 2023), and we therefore here describe it as Rana parvipalmata asturiensis ssp. nov.

Diagnosis: Rana parvipalmata asturiensis is a member of the R. temporaria complex. It corresponds to phylogeographic lineage T2, distributed in Asturias and northern León (northwestern Spain), which has been recently attributed to the distinct species R. parvipalmata based on multilocus mitochondrial and nuclear DNA analyses ( Dufresnes et al. 2020b). Its closest relative is Rana parvipalmata parvipalmata , corresponding to phylogeographic lineage T1 distributed in nearby Galicia, from which it differs by 1.2% of divergence at mitochondrial genes (~4.4 kb spanning six genes), including 4.7% at cyt-b and 0.9% at 16S, and by 0.07% at nuclear RAD-seq markers conserved across distant Rana species (all data from Vences et al. 2017 and Dufresnes et al. 2020b). In the western parts of its range, R. p. asturiensis features mtDNA haplotypes derived from R. p. parvipalmata (cyt-b mitogroup T1b), following hybridization. As identified by MolD, R. p. asturiensis can be robustly identified by a combination of diagnostic nucleotides: ‘T’ in the site 666, ‘C’ in the site 714, ‘G’ in the site 732 for specimens carrying T1b mtDNA: ‘T’ in the site 627, ‘A’ in the site 715, ‘T’ in the site 825 for specimens carrying T2 mtDNA (positions relative to the full cyt-b gene of Rana temporaria ).

Externally, the new taxon generally resembles other common frogs of the R. temporaria complex. In western ranges, R. p. asturiensis can be confounded with R. p. parvipalmata , from which most individuals can be diagnosed by a combination of morphological characters ( Fig. 5 ; Supporting Information, Fig. S4 ), notably body size (larger in R. p. asturiensis, up to 80 mm), feet webbing (more extensive in R. p. asturiensis), femur and tibia lengths (respectively shorter and longer in R. p. asturiensis), and the metatarsal tubercle (larger in R. p. asturiensis). These differences alone are not fully diagnostic, however, and field identification should take into account distribution, and ideally rely on genetic barcoding. The mating call and tadpole of R. p. asturiensis have not been studied. Vences (1992) noted that Galician frogs (R. p. parvipalmata ) featured a lower number of pulses per call compared to populations from Central Europe ( R. temporaria ), but whether this pattern holds across the entire range of R. parvipalmata (i.e. including the new subspecies) is unknown. In eastern ranges, R. p. asturiensis can be confounded with the distinct species R. temporaria , from which it remains even more similar phenotypically. Finally, the new taxon shares its range (but rarely its habitats) with the Iberian frog Rana iberica but can be reliably distinguished based on a number of diagnostic criteria pertaining to size, shape, and coloration ( Dufresnes 2019).

The taxonomic status of R. p. asturiensis is justified by the genuine evolutionary divergence and morphological differentiation from other R. parvipalmata populations, and the subspecies rank is justified by the recent Plio–Pleistocene origin, the weak sequence divergence at barcoding genes, and most importantly the extensive admixture with R. p. parvipalmata , suggestive of reproductive compatibility.

Holotype: MNCN 50655, adult male found by J. Ambu and C. Dufresnes on 14 March 2021 on a forest track c. 500 m northeast of Alto de la Fumarea , Picu Fario area , Asturias, Spain (43.4306403°, -5.5710408°, 549 m a.s.l.), and deposited at the Museo Nacional de Ciencias Naturales (MNCN) in Madrid, Spain. The specimen is depicted both live and post-mortem in Fig. 6 . Its identity was confirmed by mitochondrial (cyt-b) and nuclear (RAD-seq) analyses ( Figs 1–3 ).

Description of the holotype: Body size (SVL) of 64 mm for a wet weight of 24.1 g; head 16 mm wide: large protruding eyes (8 mm of diameter) spaced by 12 mm, with horizontal pupil; tympanum disk very distinctive and large (5 mm), located 3 mm behind the eyes; nostrils separated by 6 mm, roughly equidistant from the eye (5 mm) and the snout (6 mm); blunt snout with a rounded profile; dorsolateral lines distinguishable, spaced by 11 mm; vomerine teeth present, slightly pigmented, V-shaped and well separated; four fingers (Nos 1–4), including protruding thumbs (No. 1) with dark nuptial pads, and respective size (inner to outer) as follows: 3> 1> 4> 2; slender hind legs almost as long as the body, with femur 26 mm and tibia 33 mm, reaching the nostrils when projected on the head (‘leg test’); tarsus 30 mm with five elongated toes (up to 31 mm) with relatively extensive webbing, of formula I 1-1 II 1/2-1/2 III 2/3-2 IV 3-3 V; oval-shaped metatarsal tubercles (3 mm long by 2 mm high). Smooth skin; colourful dorsum with diffused dark blotches on a light-brown background; hints of creamy yellow on the flanks, groin, and armpits; predominantly white/light grey ventrum with faded dark spots and fine reddish speckles; throat greyish with dark reflections; iris yellow; temporal markings not pronounced (mostly brown, getting darker near the armpits); upper lip with reddish speckles on a light background, whiter near the tympanum and armpit. All observations and measurements were made post-mortem before fixation in ethanol.

Paratype: MNCN 50656, adult female found by A.G. Nicieza and A. Peláez on 12 March 2021 in a small pool near Lagunas de Valdecarrín , Puerto de Las Señales, León, Spain (43.07429°, -5.23967°) , and deposited at the Museo Nacional de Ciencias Naturales (MNCN) in Madrid, Spain .

Etymology: The new nomen, Rana parvipalmata asturiensis , makes reference to the region of Asturias where this taxon is mainly distributed. As vernacular names, we recommend using ‘Asturian common frog’ for R. p. asturiensis, and to restrict the name ‘Galician common frog’ to the subspecies R. p. parvipalmata . To avoid confusion, the species R. parvipalmata as a whole may be referred to as the ‘Western common frog’.

Diversity and distribution: Rana parvipalmata asturiensis is distributed in the western part of the Cantabrian mountains and coastal lowlands that mainly encompass the province of Asturias. It also mildly penetrates into the neighbouring provinces of Castilla y León (south) and Cantabria (southeast). In the west, the subspecies forms an extensive hybrid swarm with R. p. parvipalmata across Western Asturias, from about La Espina to the Eo River (at the border with Galicia). In the east, R. p. asturiensis meets and locally admixes with R. temporaria near the border between Asturias and Cantabria in the north, and near the border between Cantabria and Castilla y León in the south ( Montaña Palentina ). Populations of R. p. asturiensis are genetically structured due to isolation by distance ( Choda 2014, Dufresnes et al. 2020b), but they feature relatively homogenous patterns of genetic diversity at neutral markers ( Choda 2014, Cano et al. 2017, Dufresnes et al. 2020b). However, lowland and highland populations differ in genes related to metabolic function and immune response ( Cano et al. 2017).

Natural history: Not specifically studied, but presumably similar to other lineages of the R. temporaria complex. In Asturias, common frogs are found across a variety of environments including wetlands, rivers, puddles and ditches in forest tracks, small retention dams, and any kind of low depth water bodies, such as mountain and cattle ponds. The breeding season extends from early autumn to early summer, starting progressively later with higher elevation ( Álvarez et al. 2012, Gutiérrez-Pesquera et al. 2022). The fine-scale spatial distribution may differ between highland areas, where breeding nuclei are larger but more isolated, and the more human-impacted mid-elevation and coastal areas, where breeding occurs in small nuclei in small and often anthropogenic water bodies.

Conservation: The Asturian common frog is abundant and widespread throughout its range, hence it is probably not threatened despite its narrow distribution (c. 10 000 km 2). The higher risks may be related to climate change and timber exploitation. Analyses of thermal tolerance and mechanistic niche modelling suggest a high vulnerability to climate warming, and suitability area projections point to an upward retreat of R. p. asturiensis populations, and extinction in most of the southern distribution ranges ( Enriquez-Urzelai et al. 2018, 2019a, b, 2020, Gutiérrez-Pesquera et al. 2022). Although forestry promotes suitable water bodies associated with forest tracks, eucalypt tree plantations may have a negative effect because eucalypt leachates are detrimental to the growth and development of larvae ( Burraco et al. 2018, Iglesias-Carrasco et al. 2022), and ultimately to metamorph survival (D. Álvarez and A.G. Nicieza unpublished data). Differences in functional genetic diversity along the altitudinal gradient ( Cano et al. 2017) suggest that conservation actions should be population specific.

Figures

Figure 1. Phylogeographic structure and diversity in R. parvipalmata based on RAD-seq analyses.The map (A) shows the location and numbering of sampled localities; the yellow shades outline the presumed distribution of the species.Barplots (B) and the PCA (C) display within-population expected heterozygosity He and between-sample genetic variation, both based on 812 unlinked SNPs. (D) The phylogenetic network shows genetic relationships among samples, based on ~155 kb sequences. Colour-coding follows a red-orange gradient that corresponds to the average genomic ancestries of samples/populations between the Galician R. p.parvipalmata T1 (red) and the Asturian R. p. asturiensis T2 (orange) lineages (Fig.2). Triangles and squares highlight the isolated southernmost and westernmost samples/populations. The star shows the type locality and the holotype of R. p.asturiensis.

Figure 2. Characterization of the hybrid zone between the Galician R. p.parvipalmata T1 (red) and the Asturian R. p. asturiensis T2 (orange) lineages.The map (A) and the STRUCTURE barplot (B) show population and individual ancestries, based on 812 unlinked SNPs. The cline plot (C) models the geographic transition by fitting a sigmoid function to the genome-average ancestry data (STRUCTURE Q) and to 17 unlinked individual SNPs fixed between the lineages.

Figure 3. Mitochondrial phylogeography of R. parvipalmata. Dots on the map (A) show the distribution of the three main cyt-b haplogroups identified on the phylogenetic network (B). The respective ranges of the two nuclear lineages are illustrated by shaded backgrounds (T1: red; T2: orange; dashed line:putative centre of the hybrid zone). Note how the T1b matrilines (pink dots) are found across T2 populations from western Asturias (orange area).

Figure 4. Comparison of body size in common frogs from northwestern Spain. (A) Violin plots showing variation in SVL among 1947 males of R. p.parvipalmata (T1, red), R. p. asturiensis (T2, orange), and R. temporaria from nearby Cantabria (T6, blue). (B) Neighbour-joining tree of pairwise size differences among these three lineages. Photo: T1 frog from La Garganta (credit: J. Ambu).

Figure 5. Comparison of body shape in common frogs from northwestern Spain. (A) PCA on 17 morphological characters (after allometric size correction) among 82 males of R. p.parvipalmata (T1, red circles), R. p.asturiensis (T2, orange circles), and R. temporaria from nearby Cantabria (T6, blue triangles); scatterplots show coordinates and circles show variable contributions on PC1–2 (top) and PC3–4 (bottom). (B) Neighbour-joining tree of multivariate pairwise differences among lineages.

Figure 6. The holotype of Rana parvipalmata asturiensis (MNCN 50655), depicted live (top) and post-mortem before ethanol preservation (bottom). Credit:C. Dufresnes.