Alytes obstetricans lusitanicus

urn: lsid: zoobank. org: act: 3 C 3 CD 577 - A 1 CE- 46 F 1 - 948 D- 1 AEA 16 F 53 B 1 D Identity First identified as a divergent mitochondrial lineage in the phylogeographic analyses of Fonseca et al. (2003) and Gonçalves e t al. (2007). It was retrieved in subsequent studies based on additional molecular markers and samples as lineage / haplogroup D or A. o. cf. boscai (e. g., Maia-Carvalho et al. 2014, 2018; Gonçalves et al. 2015; Dufresnes & Hernandez 2021; Ambu et al. 2023). Its range was inferred as western central Iberia, including central Portugal and western central Spain, where it was accordingly confounded with A. o. boscai given the distribution of this subspecies according to the historical literature (García-París & Martínez-Solano 2001). However, the type locality of A. o. boscai, regarded as “ Tuy en la provincia de Pontevedra ”, Spain (see García-París & Martínez-Solano 2001), corresponds to the lineage present in northwestern Iberia, including Spanish Galicia and northern Portugal (lineage / haplogroup C), to which the nomen boscai must be restricted (see also Dufresnes & Hernandez 2021). No taxonomic name is available for the western central Iberian lineage given as lineage / haplogroup D or A. o. cf. boscai (see e. g., Frost 2024), and we therefore describe it here as a new taxon, Alytes obstetricans lusitanicus ssp. nov.

Diagnosis A midwife toad from the subgenus Alytes, which becomes the fourth subspecies of A. obstetricans. According to phylogenomic analyses, A. o. lusitanicus ssp. nov. is the sister taxon of A. o. boscai, from which it diverged around the Plio-Pleistocene transition ca. 2.5 Mya (Ambu et al. 2023). It features 0.19 % of sequence divergence at ~ 282 kb of nuclear (RAD) loci from that subspecies. The mitochondrial diversity of A. o. lusitanicus ssp. nov. is counter-intuitive. The Spanish populations feature a “ ghost ” lineage different from the regular A. o. lusitanicus ssp. nov. mtDNA predominantly found in Portugal (Ambu 2024 b). Accordingly, the mtDNA of A. o. lusitanicus ssp. nov. differs from the mtDNA of A. o. boscai by 0.93 % (Portuguese lineage) or 0.99 % (Spanish ghost lineage) at 16 S, and by 5.2 % (Portuguese lineage) or 3.3 % (Spanish ghost lineage) at ND 4 (Table 1) – again noting that these mtDNA distances do not reflect the true divergence between taxa due to a past mitochondrial capture in A. o. boscai (Ambu et al. 2023). According to MOLD, the new subspecies can be distinguished from all other taxa from subgenus Alytes by the following diagnostic nucleotides in the ND 4 gene sequence: a “ C ” in the site 981, a “ T ” in the site 990, an “ A ” in the site 1,011 (Portuguese lineage); or an “ A ” in the site 1,062, a “ C ” in the site 1,179, a “ G ” in the site 1,365 (Spanish ghost lineage) (positions relative to the mitogenome of A. o. pertinax, accession AY 585337). Alytes o. lusitanicus ssp. nov. can also be distinguished from A. o. boscai by unique microsatellite genotypes (Maia-Carvalho et al. 2018) and private haplotypes at several nuclear introns, notably the C-myc gene (Gonçalves et al. 2015). Externally, these two subspecies partly differ in body shape based on a combination of morphological characters (Fig. 2), but none that are diagnostic or even significantly different when considered individually (Fig. 3; Table 2). The mating calls also appear to be similar (Fig. 2, 4; Table 3). The tadpoles have not been specifically studied and compared. In addition, A. o. lusitanicus ssp. nov. can be distinguished from more distantly related members of the subgenus Alytes based on molecular divergence at the loci mentioned above (notably at RAD and mitochondrial sequences; Table 1), but it is similar in terms of mating calls (Fig. 2; Table 3), morphology and size; the only exception is the shape of the head, which we found to be larger in A. o. lusitanicus ssp. nov. (Fig. 2; Table 2).

Distribution Mainly constrained between the Douro and Tagus rivers from the Atlantic coast to Sierra de Gredos (41.6 ° N to 39.0 ° N; 9.3 ° W to 4.9 ° W) (Fig. 8). The range encompasses the districts of Aveiro, Castelo Branco, Coimbra, Guarda, Leiria, Lisboa, Porto, Santarém and Viseu in central Portugal, as well as the provinces of Ávila, Cáceres, Salamanca and perhaps Zamora in western Spain. An isolated group of populations also subsists south of the Tagus River near the Spanish-Portuguese border, namely in the district of Portalegre (Portugal), as well as in the provinces of Badajoz and Cáceres (Spain). The altitudinal range spans from sea level to ~ 2000 m a. s. l. in the Gredos mountains. The northern boundary putatively follows the Douro River, which keeps A. o. lusitanicus ssp. nov. isolated from A. o. boscai, yet with traces of admixture that testify of occasional riverine dispersal even in the largest and brackish sections of the river (Ambu 2024 a). Better connected contact zones may be found in upstream sections, with A. o. boscai, and probably also with A. o. pertinax (provinces of Salamanca and Zamora in Spain). In the main eastern ranges (Sierra de Gredos), a mitochondrial ghost lineage segregates, and signatures of introgression (foreign nuclear alleles and mtDNA) suggest historical hybridization with A. o. pertinax, perhaps during the Late Pleistocene or the Holocene when these subspecies could have been in contact (Fig. 8). In this area, A. obstetricans is now mostly found in mountainous areas, and its altitudinal distribution is mediated by A. cisternasii that inhabits the lowlands (Lizana et al. 1988).

Etymology The nomen lusitanicus refers to the ancient Roman Province of Lusitania, which encompassed central and southern Portugal (south of the Douro River) and western central Spain (Extremadura, Castilla la Mancha and Castilla y León), thus broadly matching the distribution of the new taxon.

Holotype MNCN 50839 (Fig. 5), adult male collected by J. Ambu and C. Dufresnes on 6 July 2022, in an orchard situated alongside road AV-P- 711, 6.3 km from El Arenal, Ávila, Spain (40.2541495 N, 5.060717 W, 899 m a. s. l.). The specimen is curated at the herpetological collection of the Museo Nacional de Ciencias Naturales (MNCN). Its identity was genetically confirmed as the target lineage (“ lineage D ”, “ A. o. cf. boscai ”) using ddRAD-seq and 16 S analyses (Ambu 2024 a ‒ b).

Paratypes Nine specimens, eight curated at Museo Nacional de Ciencias Naturales (MNCN) and one curated at Muséum National d’Histoire Naturelle (MNHN), given as follows with body size (SVL) and locality of origin, and depicted in Fig. 6: MNCN 161 (34 mm) and MNCN 166 (36 mm) from Robleda, Salamanca, Spain; MNCN 315 (34 mm), MNCN 316 (31 mm) and MNCN 317 (31 mm) from Serra da Estrela, Portugal; MNCN 11369 (34 mm), MNCN 13133 (40 mm) and MNCN 13134 (36 mm) from San Martín del Pimpollar, Ávila, Spain; MNHN-RA- 1900.123 (36 mm) from Coimbra, Portugal.