Alytes obstetricans lusitanicus, Ambu & Martínez-Solano & Dufresnes, 2024

Ambu, Johanna, Martínez-Solano, Íñigo & Dufresnes, Christophe, 2024, A new subspecies of midwife toad (Anura, Alytidae, Alytes Wagler, 1829) supported by genomic taxonomy, Alytes 41 (1 - 4), pp. 18-39 : 30-35

Alytes obstetricans lusitanicus ssp. nov.

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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 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 ).

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 16S analyses ( Ambu 2024 a ‒b).

Description of the holotype

Small-sized body (33.9 mm of SVL); oval-shaped head (14.7 mm wide); blunt, rounded snout, with nostrils small and round; eyes large (3.6 mm of diameter) with an interorbital space flat (10 mm), wider than the internarial space (3.4 mm); tympanum well defined, round, and large (2.8 mm); tympanum annulus distinct; vomerine teeth present, well separated; three prominent metacarpal tubercles; four fingers with a relative length of I <IV <II <III (5.9 mm for the longest); hind limbs relatively short, tibia slightly shorter (13.4 mm) than the thigh (15.7 mm), and twice longer than the tarsus (6.7 mm); five toes moderately long with rounded tips and relative length as I <V <II <III <IV (9.0 mm for the longest); webbing present, thin; metatarsal tubercle barely distinct. Dorsum and ventrum slightly granulated; thin parotids almost indistinct from the dorsolateral ridge. Color live: dorsum, flanks and upper side of limbs with a pale grey background, brownish and greenish blotches, and irregular orange warts along the dorsolateral ridges, dorsum and eyelids; ventral surface whitish, slightly translucent, with darker pectoral and abdominal areas.

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 .

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.

Advertisement call

Single short high-pitched “whistling” note emitted at regular intervals, as in other midwife toads, and with characteristics like other taxa of the subgenus Alytes ( Fig. 2 ; Table 3). From the analyzed recordings, on average, notes last 87 ms, starting by a rising time of 10.5 ms, and with a dominant frequency of 1.33 kHz (corresponding to a pulse rate of 1.32 ms-1). A spectrogram is provided in Fig. 7 .

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).

Natural history

Not specifically studied, but presumably like other A. obstetricans subspecies (see e.g., Pleguezuelos et al. 2002; Speybroeck et al. 2016; Dufresnes 2019; Salvador et al. 2021). The taxon inhabits various habitats with permanent water bodies for larval development, such as tanks, small ponds, streams, notably those retaining small water puddles during the driest months. Tadpoles are frequently found alongside other Iberian amphibians such as Rana iberica Boulenger, 1879 , Lissotriton boscai ( Lataste, 1879 a) 2 or Salamandra salamandra ( Linnaeus, 1758) . These habitats are found in relatively humid parts of central Portugal and western central Spain. The niche model of A. o. lusitanicus ssp. nov. (projected in Fig. 8 ) is mostly built from precipitation variables, including precipitation of the coldest quarter (Bio19, 43.2 %), precipitation of the driest month (Bio14, 12.9 %), and precipitation seasonality (Bio15, 6.9 %), in addition to global land cover (7.8 %) ( Ambu 2024 a). The model might however be biased towards the conditions found in the Portuguese ranges, where many records exist in hilly landscapes of low to middle elevations, compared to the Spanish ranges, where A. o. lusitanicus is more localized and scattered at higher altitudes; the suitability scores are accordingly lower for the Spanish populations ( Fig. 8 ).

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.

Figures

Figure 2. PCAs on (size-corrected) morphological and bioacoustic data in the subgenus Alytes (Ambu 2024a). Each dot corresponds to an individual. Hulls show the parameter space occupied by each taxon on the first two axes. Diagram circles show variable contributions. See Methods for abbreviations and data sources.

Figure 3. Variation of SVL and body ratios at individual morphological characters in the subgenus Alytes. Top bars show significant pairwise comparisons in the Tukey tests (P <0.05). See Methods for abbreviations and data sources.

Figure 4. Variation of bioacoustic variables in the subgenus Alytes. Top bars show significant pairwise comparisons in the Tukey tests (P <0.05). See Methods for abbreviations and data sources.

Figure 5. The holotype MNCN 50839 of Alytes obstetricans lusitanicus ssp. nov., depicted live (credit: CD) and post-mortem (credit: A. Sánchez Vialas).

Figure 6. The paratypes of Alytes obstetricans lusitanicus ssp. nov. First row: MNCN 11369, MNCN 13133 and MNCN 13134; second row: MNCN 315, MNCN 316 and MNCN 317; third row: MNHN-RA- 1900.123 (inset), MNCN 161 and MNCN 166. Credit: IMS (MNCN) and CD (MNHN).

Figure 7. Spectrogram and oscillogram of a typical note of the advertisement call of Alytes obstetricans lusitanicus ssp. nov.

Figure 8. (A) Distribution map for A. o. lusitanicus ssp. nov., distinguishing occurrence records and genetically-barcoded populations. The type locality is noted with a green star. The distribution of the mtDNA ghost lineage is shown. The potential presence of A. o. pertinax in the northeastern ranges (where data is lacking) is emphasized by orange dash lines. (B) Projected distribution of A. o. lusitanicus ssp. nov. under current conditions. See Methods for abbreviations and data sources.