Bioacoustics of Alytes obstetricans

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 16S, and by 5.2 % (Portuguese lineage) or 3.3 % (Spanish ghost lineage) at ND4 ( 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 ND4 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 AY585337 ). 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 ).

Conservation Despite a relatively large range and high number of populations, the subspecies A. o. lusitanicus ssp. nov. may be threatened by pathogens causing viral (Ranaviruses) and fungal (chytrid) infections, to which other subspecies of A. obstetricans are sensitive (Bates et al. 2018; Bosch et al. 2001 , 2021 ); by the early desiccation of semitemporary water bodies used for reproduction such as stream puddles, as a side effect of global warming and aridification; by habitat loss following landscape developments in the most anthropic areas (e.g., the Portuguese coast) – but noting that midwife toads can accommodate to peri-urban and even urban habitats; and by invasive predatory species such as exotic fishes and crayfish introduced in water bodies for ornamental or recreative reasons. Populations previously assigned to “ A. o. boscai ” are considered as Near Threatened in the Spanish Red List ( Pleguezuelos et al. 2002 ). This assessment combines A. o. boscai s. s. from northwestern Spain with A. o. lusitanicus ssp. nov. in central Spain , so the actual situation of A. o. lusitanicus ssp. nov. might be more worrisome, especially since the Spanish populations of A. o. lusitanicus ssp. nov. appear genetically impoverished compared to the main Portuguese range ( Ambu 2024 b ). In Portugal , A. obstetricans is listed as Least Concern in the national red list, which considers A. o. boscai and A. o. lusitanicus ssp. nov. together. The recognition of A. o. lusitanicus ssp. nov. as a distinct subspecies thus calls to evaluate its conservation status separately, for which our preliminary range map ( Fig. 8 ) offers a first-hand resource.