# A new subspecies of midwife toad (Anura, Alytidae, Alytes Wagler, 1829) supported by genomic taxonomy

Johanna Ambu, Íñigo Martínez-Solano, Christophe Dufresnes

<figure><img src="image_1.png" /></figure>

## To cite this version:

Johanna Ambu, Íñigo Martínez-Solano, Christophe Dufresnes. A new subspecies of midwife toad (Anura, Alytidae, Alytes Wagler, 1829) supported by genomic taxonomy. Alytes, 2024, 41 (1-4), pp.18-39. hal-04886694

HAL Id: hal-04886694

https://hal.science/hal-04886694v1

Submitted on 14 Jan 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# A new subspecies of midwife toad
*(Anura, Alytidae, *Alytes* Wagler, 1829)*
*supported by genomic taxonomy*

Johanna AMBU<fnref n="1" />, Íñigo MARTÍNEZ-SOLANO<fnref n="2" /> & Christophe DUFRESNES<fnref n="1" />,<fnref n="3" />,*

The mapping, delimiting and naming of biodiversity forge the links between academic research, conservation efforts and communication about wildlife. Midwife toads from the subgenus *Alytes* are a group of high conservation concern widely popular among European naturalists, but for which the taxonomy remains unsettled. Six phylogeographic lineages that diversified during the Pliocene and the Pleistocene epochs have been identified and delimited in two species (*A. obstetricans* and *A. almogavarii*), but only five subspecies are presently recognized (*A. o. obstetricans*, *A. o. pertinax* and *A. o. boscai*; *A. a. almogavarii* and *A. a. inigoi*). Accordingly, two distinct lineages found in northwestern and western Iberia are still regrouped under the same taxon *A. o. boscai*. Contrary to the discordant findings of earlier studies based on a few genes, phylogenomic analyses of thousands of nuclear markers have confirmed their independent evolution, estimated to exceed two million years. In this article, we detail molecular, morphological and behavioral variation in the subgenus *Alytes* to provide a taxonomic description for the previously unnamed western Iberian lineage. Like other taxa of this subgenus, the new taxon is supported by robust evidence for genetic divergence despite little external differentiation. It is designated as a subspecies of *A. obstetricans*, as per its phylogenetic placement and young evolutionary age, which compares to freely admixing *Alytes* subspecies. Combining genetic barcoding and distribution information, we provisionally define its range in central Portugal and western central Spain, and prompt to evaluate its potentially worrisome conservation status. Our study highlights how phylogeographic diversity can be acknowledged in zoological systematics, even when phenotypic differences are subtle, and illustrates the advantages of genomic approaches to overcome the

<footnote n="1">¹ Laboratory for Amphibian Systematics & Evolutionary Research, College of Biology & the Environment, Nanjing Forestry University, Nanjing, People's Republic of China.</footnote>

<footnote n="2">²Department of Biodiversity and Evolutionary Biology, Museo Nacional de Ciencias Naturales (MNCN-CSIC), c/ José Gutiérrez Abascal, 2, 28006 Madrid, Spain.</footnote>

<footnote n="3">³ Institut de Systématique, Evolution, Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE-PSL, Université des Antilles, Paris, France.</footnote>

<footnote>*Corresponding author: Christophe Dufresnes <christophe.dufresnes@hotmail.fr>.</footnote>
**limitations of single-gene analyses when implementing taxonomic revisions.**

**Keywords:** *Alytes obstetricans*; Iberian Peninsula; integrative taxonomy; species delimitation.

urn:lsid:zoobank.org:pub:8ADAF817-604B-41CC-A943-BCCAA32A71B5

INTRODUCTION

The mapping, delimitation and naming of biodiversity are crucial steps in biological sciences, as they directly affect the way populations are considered in research and conservation. Aggregating locality records to map distribution ranges, inferring red list status, and calculating diversity indices such as species richness, all depend on the taxonomic identity of populations (Vences *et al.* 2024). Molecular tools have become an essential resource for taxonomic revisions (“molecular taxonomy”); genetic data typically serve to establish the evolutionary divergence of taxa and to classify them as species or subspecies in the Linnean system (Padial *et al.* 2010).

Molecular taxonomists have essentially relied on DNA barcodes, i.e., genes bearing taxon-specific polymorphism, which are particularly useful to distinguish taxa in the absence of diagnostic external criteria (Hebert *et al.* 2003; Vences *et al.* 2005; Padial *et al.* 2010; Chenuil *et al.* 2019). Although the limitations of DNA barcodes such as mitochondrial DNA (mtDNA) genes are well-known (Rubinoff & Holland 2005), they remain insufficiently appreciated in the taxonomic context, and in effect the vast majority of new amphibian species described every year still consider the analysis of single loci such as 16S or COI as the main molecular evidence (Anonymous 2024). These analyses alone may be particularly insufficient in species complexes, i.e., widespread species that diversified into multiple intra- and interspecific evolutionary lineages. Phylogeographic studies in amphibians are emphasizing deep discordances between the patterns of genetic diversity retrieved from mitochondrial loci compared to the rest of the genome, which compromises species identification and delimitation based on mtDNA (Dufresnes & Jablonski 2022). For these reasons, it has been argued that taxonomic revisions should ideally incorporate genome-wide multilocus data obtained by high-throughput sequencing, thus extending molecular taxonomy to genomic taxonomy (Rubinoff *et al.* 2006; Dufresnes *et al.* 2023; Vences *et al.* 2024).

An emerging challenge in genomic taxonomy is to delimit species complexes without contributing to species-level taxonomic inflation, i.e., by oversplitting every lineage as a distinct species. As recently discussed (Dufresnes *et al.* 2023; Vences *et al.* 2024; and references therein), the high resolutive power of phylogenomic datasets may inevitably identify any structured population as a distinct lineage. It then becomes more appropriate to balance the delimitation of these lineages as species or subspecies based on evidence of reproductive isolation or panmixia, which can be assessed through the study of hybrid zones. The use of the subspecies category to designate phylogeographic lineages within a species might be particularly relevant to properly acknowledge and communicate about intraspecific diversity and attract conservation attention, yet without permanently reshuffling species lists.
This article focuses on midwife toads, genus *Alytes* Wagler, 1829¹. Midwife toads are western Mediterranean amphibians most famous for their remarkable parental care behavior, in which the male carries the eggs on its back until hatching (Lange *et al.* 2022). Widespread, essentially terrestrial, and accommodating to various habitats (Legros *et al.* 2015; Speybroeck *et al.* 2016; Dufresnes 2019), midwife toads are nevertheless a group of high conservation concern (Griffiths *et al.* 2008; Barrios *et al.* 2012). They face many threats such as habitat loss, global warming, as well as chytridiomycosis, a fungal disease that has been linked to the decline of many populations (Bosch *et al.* 2001, 2021).

Since the 1990s, the genus has been the focus of numerous molecular studies that contributed to taxonomic revisions. Midwife toads are presently divided into three subgenera and six species (see also Dubois *et al.* 2021; Frost 2024), namely the subgenus *Ammoryctis* Lataste, 1879b, with one species: *Alytes cisternasii* Boscá, 1879; the subgenus *Baleaphryne* Sanchíz & Adrover, 1979, with three species: *Alytes maurus* Pasteur & Bons, 1962, *Alytes muletensis* (Sanchíz & Adrover, 1979) and *Alytes dickhilleni* Arntzen & García-París, 1995; and the subgenus *Alytes* Wagler, 1829, with two species: *Alytes obstetricans* (Laurenti, 1768) and *Alytes almogavarii* Arntzen & García-París, 1995. The current arrangement is supported by recent phylogenomic and hybrid zone analyses (Dufresnes & Martínez-Solano 2020; Ambu *et al.* 2023), and is thus expected to remain stable.

The intraspecific taxonomy of the subgenus *Alytes* has also been regularly updated. Studies using allozymes (García-París 1995; Arntzen & García-París 1995), mitochondrial DNA (Fonseca *et al.* 2003; Martínez-Solano *et al.* 2004; Gonçalves *et al.* 2007, 2015), nuclear intron sequences (Maia-Carvalho *et al.* 2014; Gonçalves *et al.* 2015) and microsatellite markers (Maia-Carvalho *et al.* 2018; Lucati *et al.* 2022) unveiled multiple lineages that were progressively attributed to available nomina or described as new subspecies, i.e., *A. o. obstetricans* in extra-Iberian ranges (France, Switzerland, Belgium, Netherlands, Luxemburg and Germany; “lineage B” in Gonçalves *et al.* 2015), *A. o. boscai* Lataste, 1879c (western Iberia; “lineage C” and “lineage D”), *A. o. pertinax* García-París & Martínez-Solano, 2001 (eastern Iberia; “lineage A”), *A. a. almogavarii* Arntzen & García-París, 1995 (eastern Pyrenees; “lineage F”) and *A. a. inigoi* Dufresnes & Hernandez, 2021 (central Pyrenees; “lineage E”). This diversification is often referred to as the *A. obstetricans* species complex.

The five taxa currently recognized in the complex still under-estimate its diversity. The populations attributed to *A. o. boscai* correspond to two lineages, one distributed in northern ranges, namely northwestern Iberia, including Spanish Galicia and northern Portugal (“lineage C”), and one distributed in southern ranges, namely western central Iberia, including central Portugal and western central Spain (“lineage D”). The nomen *boscai* applies to the northern “lineage C” (see García-París & Martínez-Solano 2001) and the southern “lineage D” has remained unnamed, being provisionally referred to as “*A. o. cf. boscai*” (e.g., Dufresnes & Hernandez 2021; Ambu *et al.* 2023). However, previous studies reported conflicting patterns of lineage divergence and distribution, preventing a clear understanding of the evolution of the subgenus *Alytes* (reviewed by Dufresnes & Hernandez 2021). Microsatellite markers

<footnote n="1">¹ For the authorship and date of this nomen, see Dubois (1984, 1987, 1998) and (Dubois *et al.* 2021).</footnote>
retrieve the northern and southern populations of *A. o. boscai* as distinct genetic clusters (Maia-Carvalho *et al.* 2018), a multilocus intron-based phylogeny groups them together in a shallow clade (Maia-Carvalho *et al.* 2014), and the mitochondrial phylogeny does not retrieve them as sister lineages (Gonçalves *et al.* 2007, 2015).

Phylogenomic inferences based on double digest Restriction-Associated DNA sequencing (ddRAD-seq) recently clarified the diversity and evolution of midwife toads (Ambu *et al.* 2023; Fig. 1), in which the six candidate lineages of the subgenus *Alytes* were supported by thousands of nuclear loci. In particular, the analyses demonstrated that *A. o. boscai* sensu stricto and *A. o. cf. boscai* were sister lineages that diverged as early as the Plio-Pleistocene (2.5 My), after which the northern populations (*A. o. boscai* s. s.) experienced mitochondrial introgression from *A. o. obstetricans* (hence their distinctive placement in the mtDNA phylogeny). The delimitation workflow proposed for genomic taxonomy (Dufresnes *et al.* 2023) thus calls for the taxonomic naming of the southern populations, so they can be considered separately in biodiversity checklists and conservation assessments.

To provide a taxonomic description, here we summarize patterns of genetic, bioacoustic, morphological variation and distribution in the subgenus *Alytes*, based on data compiled in our associated research (Ambu *et al.* 2023; Ambu 2024a–b). Specifically, we screen for diagnostic characters, discuss evidence for species vs. subspecies distinction, and provide a distribution map for the new taxon.

<figure><img src="image_3.png" /><figcaption>Figure 1. Phylogeny and distribution of midwife toads of the subgenus *Alytes* (*A. obstetricans* complex) based on genomic (ddRAD-seq) analyses (Ambu 2024a). The tree summarizes the evolution of the complex with the timing of events (in Mya), including the reticulate origin of *A. o. pertinax* (Ambu *et al.* 2023). The distribution map shows the subspecies ranges and the location of hybrid zones with ongoing gene flow (dash lines) (Ambu 2024a). The new subspecies is emphasized (credit: CD).</figcaption></figure>
METHODS

Genetics

Three types of loci were considered to assess the genetic divergence of the new
taxon compared to other lineages of the subgenus *Alytes*: (1) 278,267 bp of
concatenated RAD tag sequences used in phylogenomic analyses (taken from Ambu *et al.* 2023); (2) 44 haplotypes of the mitochondrial 16S rRNA gene (Ambu 2024b); (3) 107 haplotypes of the mitochondrial ND4 gene (Ambu 2024b). For each dataset, net pairwise sequence divergence was computed using MEGA X (Kumar *et al.* 2018). The range of the new taxon harbors two deeply-diverged mitochondrial lineages, a Portuguese one, and a Spanish one characterized as a “ghost” lineage, i.e., which does not correspond to any extant nuclear phylogeographic lineage (Ambu 2024b). These lineages were treated independently in the comparisons. For discussion, we also considered the ages of the most recent common ancestors between each pair of lineages, following the molecular clock analysis of Ambu *et al.* (2023).

For diagnostic purposes, we further screened for diagnostic positions that
distinguish the new taxon from other taxa at the hypervariable gene ND4 with MOLD
(Fedosov et al. 2022), as implemented in iTaxoTools (Vences et al. 2021), using the full
gene sequence of *A. o. pertinax* as reference (GenBank accession AY585337). The
analysis was not performed for 16S due to the lack of resolution of this marker.

Morphology

Variation in body shape was assessed based on the morphometric measurements
of 120 museum specimens from the Museo Nacional de Ciencias Naturales in Madrid
(MNCN), Muséum National d'Histoire Naturelle in Paris (MNHN), the Zoologisches
Forschungsmuseum Alexander Koenig in Bonn (ZFMK), and the National History
Museum in London (NHM). All measurements were obtained by Martínez-Gil et al.
(2022) and re-assigned to a lineage by Ambu (2024a). Here, we only considered
specimens that could be unambiguously attributed to one of the six lineages of the
subgenus *Alytes*. Eight characters were considered (details in Martínez-Gil et al. 2022):
snout-vent length (SVL); mouth length (ML); head width (HW); forelimb length (FLL);
femur length (FML); tibia length (TBL); foot length (FTL); and hindlimb length (HLL).

For standardization, we applied allometric size corrections with the *R* package *GroupStruct*, using the multispecies method to perform lineage-specific adjustments (Chan & Grismer 2022). To visualize the overall variation of body shape between lineages, we performed a Principal Component Analysis (PCA) using the *R* package *FactoMineR* (Lê *et al.* 2008). Differences between lineages were statistically assessed using a multivariate analysis of variance (MANOVA) with the *R* package *stats* (Anonymous 2023).

To screen for diagnostic characters potentially informative for field
identification, we also explored variation of simple body ratios at each character
separately (raw measurements divided by SVL) through boxplots (R package *ggplot2*;
Wickham 2016) and by testing for statistical differences with Tukey tests after verifying
normality and accounting for multiple testing (R package *stats*).
## Bioacoustics

Bioacoustic variation was assessed based on advertisement calls emitted by 119 individuals obtained from our own and publicly available recordings, as compiled by Ambu (2024a). Again, we only considered calls that could be unambiguously attributed to one of the six lineages of the subgenus *Alytes*. After identifying calls from single individuals, notes (on average 5 per individual) were isolated and analyzed in Raven Pro 1.6.1 to extract four parameters: the dominant frequency (DF) in kHz; the note duration (ND) in ms; the rising time (RT) in ms; and the pulse rate (PR) in ms-1.

As above, to assess differences between lineages, we performed a PCA and a MANOVA, visualized the variation of each variable separately using boxplots, and tested for statistical differences (Tukey tests). For the new taxon, we produced a spectrogram and oscillogram of a typical note using the R package *seewave* (Sueur *et al.* 2008).

## Distribution

To map the range of the new taxon, we considered 261 locality records from the scientific literature and public databases compiled by Ambu (2024a). In addition, we projected the distribution based on a niche model built from these records by Ambu (2024a), and reported the most contributing variables. The model was obtained using the Maxent methodology, as fully detailed in Ambu (2024a). All maps were drawn in QGIS 3.24.3

## Specimen collection

In July 2022, we searched for an adult specimen for the purpose of holotype designation in the Spanish range of the new taxon. A single individual was captured, and a buccal sample was taken and analyzed with ddRAD-seq and 16S to confirm its identification (Ambu 2024a-b). The specimen was euthanized and curated at MNCN. It was measured post-mortem by one of us (IMS) for an ad hoc set of morphological characters based on digital pictures. To this end, the specimen was positioned with a 90° angle to the camera, next to a scale (precision: 0.1mm), and pictures were analyzed with the software ImageJ (Schneider *et al.* 2012). Specimen collection was approved by permit AUES/CyL/222/2022 issued by the Junta de Castilla y León, Spain.

## RESULTS AND DISCUSSION

The diversification of the subgenus *Alytes* is primarily molecular (see also Ambu 2024a). The new taxon differs from *A. o. boscai* s. s. and other known taxa with comparable divergence to other subspecies of the complex at the three datasets used (Table 1).
<table><caption>Table 1. Pairwise sequence divergence (%) among nuclear (ddRAD-seq) and mitochondrial (16S, ND4) sequences. For 16S and ND4, we distinguish the two mitochondrial lineages found in the new taxon, which do not correspond to a comparable nuclear divergence (PO: Portugal; SP: Spain). 16S does not differentiate *A. o. obstetricans* from *A. o. pertinax*, so these taxa are lumped in the analysis.</caption><thead><tr><th></th><th>almogavarii</th><th>inigoi</th><th>boscai</th><th>lusitanicus ssp. nov.</th><th>obstetricans</th><th>pertinax</th></tr></thead><tbody><tr><td><strong>ddRAD-seq</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>almogavarii</td><td>—</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>inigoi</td><td>0.18 %</td><td>—</td><td></td><td></td><td></td><td></td></tr><tr><td>boscai</td><td>0.38 %</td><td>0.41 %</td><td>—</td><td></td><td></td><td></td></tr><tr><td><strong>lusitanicus ssp. nov.</strong></td><td>0.33 %</td><td>0.36 %</td><td>0.19 %</td><td>—</td><td></td><td></td></tr><tr><td>obstetricans</td><td>0.33 %</td><td>0.36 %</td><td>0.30 %</td><td>0.29 %</td><td>—</td><td></td></tr><tr><td>pertinax</td><td>0.24 %</td><td>0.28 %</td><td>0.25 %</td><td>0.22 %</td><td>0.14 %</td><td>—</td></tr><tr><td></td><td>almogavarii</td><td>inigoi</td><td>boscai</td><td>lusitanicus ssp. nov.</td><td>obstetricans/pertinax</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>PO</td><td>SP</td><td></td></tr><tr><td><strong>16S</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>almogavarii</td><td>—</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>inigoi</td><td>0.79 %</td><td>—</td><td></td><td></td><td></td><td></td></tr><tr><td>boscai</td><td>1.39 %</td><td>0.99 %</td><td>—</td><td></td><td></td><td></td></tr><tr><td><strong>lusitanicus ssp. nov. (PO)</strong></td><td>0.93 %</td><td>0.38 %</td><td>0.93 %</td><td>—</td><td></td><td></td></tr><tr><td><strong>lusitanicus ssp. nov. (SP)</strong></td><td>0.59 %</td><td>0.59 %</td><td>0.99 %</td><td>0.33 %</td><td>—</td><td></td></tr><tr><td>obstetricans/pertinax</td><td>1.12 %</td><td>0.76 %</td><td>0.21 %</td><td>0.71 %</td><td>0.76 %</td><td>—</td></tr><tr><td></td><td>almogavarii</td><td>inigoi</td><td>boscai</td><td>lusitanicus ssp. nov.</td><td>obstetricans</td><td>pertinax</td></tr><tr><td></td><td></td><td></td><td></td><td>PO</td><td>SP</td><td></td></tr><tr><td><strong>ND4</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>almogavarii</td><td>—</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>inigoi</td><td>4.39 %</td><td>—</td><td></td><td></td><td></td><td></td></tr><tr><td>boscai</td><td>3.86 %</td><td>4.07 %</td><td>—</td><td></td><td></td><td></td></tr><tr><td><strong>lusitanicus ssp. nov. (PO)</strong></td><td>5.51 %</td><td>4.14 %</td><td>5.21 %</td><td>—</td><td></td><td></td></tr><tr><td><strong>lusitanicus ssp. nov. (SP)</strong></td><td>3.64 %</td><td>2.51 %</td><td>3.29 %</td><td>4.42 %</td><td>—</td><td></td></tr><tr><td>obstetricans</td><td>3.73 %</td><td>3.20 %</td><td>1.54 %</td><td>4.73 %</td><td>2.44 %</td><td>—</td></tr><tr><td>pertinax</td><td>3.76 %</td><td>3.27 %</td><td>1.67 %</td><td>4.33 %</td><td>2.92 %</td><td>1.26 %</td></tr></tbody></table>

In contrast, none of the subspecies of the complex can be reliably distinguished without molecular data (Fig. 2–3). The morphological analysis reveals a significant effect of lineages on body shape (MANOVA's *F* = 3.4; *P* << 0.001). On the PCA, all lineages partly overlap, especially the highly variable *A. o. obstetricans* and *A. o. pertinax*, while *A. a. almogavarii* clusters with *A. a. inigoi* and *A. o. boscai* s. s. clusters with *A. o. cf. boscai* (Fig. 2). Tukey tests performed on body ratios revealed only a few significant (but non diagnostic) differences among the six lineages (Fig. 3; Table 2). The only significant specificity of *A. o. cf. boscai* was its proportionally larger head width (HW) compared to all other lineages, except *A. o. boscai* s. s. (Table 2). No body ratio significantly differed between these two sister lineages.
<figure><img src="image_4.png" /><figcaption>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.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>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.</figcaption></figure>
<table><caption>Table 2. Mean ± standard deviation and range (in brackets) for eight morphological characters; measures are given in mm; see Methods for abbreviations and data source.</caption><thead><tr><th></th><th>almogavarii</th><th>inigoi</th><th>lusitanicus<br>ssp. nov.</th><th>boscai</th><th>obstetricans</th><th>pertinax</th></tr></thead><tbody><tr><td>n</td><td>8</td><td>10</td><td>11</td><td>10</td><td>62</td><td>19</td></tr><tr><td>SVL</td><td>36.9 ± 3.9<br>(31.8-43.4)</td><td>38.8 ± 2.4<br>(34.7-43.0)</td><td>34.7 ± 4.9<br>(28.5-44.8)</td><td>37.7 ± 2.5<br>(33.0-40.1)</td><td>35.1 ± 4.8<br>(24.9-46.4)</td><td>37.2 ± 4.7<br>(26.0-43.9)</td></tr><tr><td>ML</td><td>14.7 ± 0.9<br>(13.4-16.6)</td><td>14.4 ± 1.0<br>(12.6-15.6)</td><td>13.7 ± 1.1<br>(11.8-16.5)</td><td>14.2 ± 0.6<br>(13.3-15.7)</td><td>13.1 ± 1.7<br>(9.7-16.2)</td><td>14.5 ± 1.8<br>(10.9-17.8)</td></tr><tr><td>HW</td><td>15.1 ± 1.2<br>(13.3-17.0)</td><td>15.5 ± 1.0<br>(13.8-16.9)</td><td>15.3 ± 1.3<br>(13.0-17.1)</td><td>15.7 ± 0.8<br>(14.4-16.6)</td><td>14.4 ± 2.0<br>(10.0-19.2)</td><td>15.2 ± 2.0<br>(10.6-17.6)</td></tr><tr><td>FLL</td><td>21.9 ± 1.2<br>(20.1-23.4)</td><td>20.9 ± 1.9<br>(18.6-25.3)</td><td>19.2 ± 2.6<br>(14.4-22.7)</td><td>19.9 ± 1.1<br>(17.9-21.7)</td><td>19.8 ± 2.8<br>(13.5-25.9)</td><td>20.1 ± 2.7<br>(14.4-23.3)</td></tr><tr><td>FML</td><td>16.4 ± 1.3<br>(15.2-18.6)</td><td>15.9 ± 1.9<br>(13.1-17.8)</td><td>14.3 ± 1.3<br>(11.4-15.6)</td><td>15.7 ± 1.21<br>(13.6-17.8)</td><td>15.2 ± 2.1<br>(10.3-20.1)</td><td>16.1 ± 2.3<br>(9.6-18.9)</td></tr><tr><td>TBL</td><td>16.6 ± 1.6<br>(14.3-18.9)</td><td>16.4 ± 1.0<br>(14.9-18.0)</td><td>14.6 ± 1.8<br>(11.6-17.5)</td><td>15.8 ± 1.1<br>(14.0-17.2)</td><td>15.1 ± 2.1<br>(10.0-20.3)</td><td>15.7 ± 2.3<br>(10.2-19.0)</td></tr><tr><td>FTL</td><td>24.6 ± 2.4<br>(21.2-28.6)</td><td>24.7 ± 2.0<br>(21.7-28.1)</td><td>21.9 ± 2.8<br>(17.7-25.7)</td><td>23.3 ± 1.4<br>(21.0-25.6)</td><td>22.8 ± 2.9<br>(16.2-27.7)</td><td>23.3 ± 3.3<br>(16.4-27.1)</td></tr><tr><td>HLL</td><td>52.2 ± 4.0<br>(46.8-59.7)</td><td>50.5 ± 3.5<br>(44.5-56.9)</td><td>46.0 ± 5.5<br>(36.3-53.8)</td><td>48.8 ± 2.5<br>(45.9-53.3)</td><td>47.6 ± 6.3<br>(30.3-61.5)</td><td>49.6 ± 6.8<br>(32.9-57.2)</td></tr></tbody></table>

Call structure also significantly varied between lineages (MANOVA's *F* = 2.7; *P* < 0.001), but most lineages broadly overlap on the PCA (Fig. 2). The calls attributed to *A. o. boscai* s. s. and *A. o. cf. boscai* did not significantly differ at any bioacoustic variable (Fig. 4; Table 3). The only noticeable lineage-specific variation is the higher dominant frequency (DF) and pulse rate (PR) of *A. a. almogavarii* compared to some other lineages of the complex (Fig. 4; Table 3). In particular, our data confirmed the call similarities (frequency) between *A. a. almogavarii* and *A. o. pertinax* noted by earlier studies (Márquez & Bosch 1995).

The southern populations of *A. o. boscai* thus correspond to a unique phylogeographic lineage characterized by substantial genetic divergence but little external and vocal differentiation. Following the workflow of Dufresnes *et al.* (2023; see their Fig. 3), we advocate for the description of these populations as a new subspecies, based on the following elements.

The differentiation of *A. o. cf. boscai* from *A. o. boscai* s. s. is robustly supported by phylogenomics (Ambu *et al.* 2023), as well as analyses of mtDNA, introns and microsatellites (Dufresnes & Hernandez 2021). Their independent evolution throughout the Pleistocene is further illustrated by their contrasted histories of past secondary contact with other *A. obstetricans* taxa: the northern *A. o. boscai* s. s. captured a foreign mtDNA from *A. o. obstetricans* during the Late Pleistocene, while the southern *A. o. cf. boscai* retained its original mtDNA (Ambu *et al.* 2023).
<figure><img src="image_6.png" /><figcaption>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.</figcaption></figure>
<table><caption>Table 3. Mean ± standard deviation and range (in brackets) for four bioacoustic characters. See Methods for abbreviations and data source.</caption><thead><tr><th></th><th>almogavarii</th><th>inigoi</th><th>lusitanicus<br>ssp. nov.</th><th>boscai</th><th>obstetricans</th><th>pertinax</th></tr></thead><tbody><tr><td>n (notes)</td><td>37 (183)</td><td>5 (27)</td><td>11 (31)</td><td>15 (63)</td><td>43 (234)</td><td>8 (43)</td></tr><tr><td>DF (kHz)</td><td>1.419 ± 0.112<br>(1.206–1.636)</td><td>1.237 ± 0.104<br>(1.077–1.328)</td><td>1.332 ± 0.080<br>(1.219–1.464)</td><td>1.310 ± 0.076<br>(1.180–1.430)</td><td>1.322 ± 0.122<br>(1.120–1.636)</td><td>1.413 ± 0.130<br>(1.219–1.602)</td></tr><tr><td>ND (ms)</td><td>93 ± 15<br>(71–136)</td><td>104 ± 12<br>(94–124)</td><td>87 ± 14<br>(69–112)</td><td>85 ± 8<br>(73–101)</td><td>98 ± 18<br>(67–141)</td><td>80 ± 16<br>(62–108)</td></tr><tr><td>RT (ms)</td><td>9.3 ± 2.5<br>(4.0–16.0)</td><td>13.2 ± 5.5<br>(6.0–19.0)</td><td>10.5 ± 3.7<br>(6.0–18.0)</td><td>10.5 ± 3.4<br>(6.0–18.0)</td><td>9.3 ± 2.7<br>(3.0–17.0)</td><td>9.1 ± 3.0<br>(6.0–15.0)</td></tr><tr><td>PR (ms<sup>-1</sup>)</td><td>1.40 ± 0.12<br>(1.20–1.62)</td><td>1.22 ± 0.10<br>(1.07–1.31)</td><td>1.32 ± 0.07<br>(1.21–1.46)</td><td>1.30 ± 0.08<br>(1.17–1.42)</td><td>1.31 ± 0.12<br>(1.12–1.65)</td><td>1.40 ± 0.14<br>(1.19–1.60)</td></tr></tbody></table>

The taxonomic rank of phylogeographic lineages can be decided based on the extent of admixture in hybrid zones, or, when it is not possible, on their genetic divergence relative to confirmed species or subspecies (Dufresnes *et al.* 2023). The only sampled parapatric populations of *A. o. boscai* s. s. and *A. o. cf. boscai* (in coastal Portugal) are efficiently separated by the Douro estuary, and their genetic structure is thus not informative for species delimitation (Ambu 2024a). Nevertheless, because hybridizability and genetic divergence significantly correlate in the subgenus *Alytes* (Ambu 2024a) – as in anurans more generally (Dufresnes *et al.* 2021) –, we can predict where these lineages lie along the speciation continuum based on their evolutionary age and molecular distance. Specifically, the age of the split (~ 2.5 My) and the nuclear divergence (0.19 %) between *A. o. boscai* s. s. and *A. o. cf. boscai* are characteristic of other taxa confirmed as subspecies based on their levels of introgression across hybrid zones, e.g., *A. a. almogavarii* vs *A. a. inigoi*, *A. o. obstetricans* vs *A. o. pertinax* and *A. o. boscai* vs *A. o. pertinax* (Ambu 2024a).

Phylogeographic diversity is an important component of Earth's biodiversity, even when it is not accompanied by external specificities perceptible by the human eye. In fact, phenotypic differences are not necessarily expected if species first diversify at the molecular level, which appears the hallmark of speciation in amphibians (Dufresnes *et al.* 2021; Dufresnes & Crochet 2022). Robust evidence for deep evolutionary divergence (as provided by genomic analyses) thus suffices to warrant a taxonomic distinction of the candidate lineages.

Hence, in the following we describe the southern lineage of *A. o. boscai* as a new subspecies of *A. obstetricans*.
*Alytes obstetricans lusitanicus ssp. nov.*

urn:lsid:zoobank.org:act:3C3CD577-A1CE-46F1-948D-1AEA16F53B1D

*Identity*

First identified as a divergent mitochondrial lineage in the phylogeographic analyses of Fonseca *et al.* (2003) and Gonçalves *et 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 2024b). 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 2024a-b).

<figure><img src="image_7.png" /><figcaption>MNCN 50839
*Alytes obstetricans lusitanicus*</figcaption></figure>

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

<figure><img src="image_8.png" /></figure>

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).
*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.

<figure><img src="image_9.png" /><figcaption>Figure 7. Spectrogram and oscillogram of a typical note of the advertisement call of *Alytes obstetricans lusitanicus* ssp. nov.</figcaption></figure>
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 2024a). 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).

<figure><img src="image_10.png" /><figcaption>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.</figcaption></figure>

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.
## 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, 1879a)<fnref n="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 2024a). 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 semi-temporary 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 2024b). 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.

<footnote n="2">² For the authorship and original reference of this nomen, see Frétey (2023: 7).</footnote>
ACKNOWLEDGEMENTS

This article is part of the PhD thesis of the first author and is published ahead of
the two chapters it refers to (Ambu 2024a-b), so the name *A. o. lusitanicus* ssp. nov.
becomes available and can be mentioned without ambiguities in the corresponding
upcoming publications. The authors are thankful to S. N. Litvinchuk, C. Caballero-Díaz,
G. Sánchez-Montes, A. G. Nicieza, G. Velo-Antón, H. Gonçalves, F. Martínez-Freiría,
H. Martínez-Gil, J. F. Beltrán, D. Donaire, A. Hernandez, T. Suchan, P.-A. Crochet, C.
Delmas, A. Trochet and B. Wielstra for their contributions to JA's PhD, to M. Calvo
and A. Sánchez Vialas (Herpetology Collection, Museo Nacional de Ciencias Naturales,
CSIC) for assistance in studying specimens under their care, to E. Ayllón, O. Cano, T.
García, C. Rozas and F. de Sá for assistance in locating and surveying populations of
the newly described taxon in Sierra de Gredos, and to the editor and reviewer for useful
comments on our article.

REFERENCES

Anonymous [R Core Team] (2023) *R: a language and environment for statistical computing*. Vienna, Austria (R Foundation for Statistical Computing). <https://www.R-project.org/>.

Ambu, J. (2024a) Genomic, phenotypic and environmental correlates of speciation in an iconic group of Mediterranean amphibians. *In*: Ambu, J., *A genomic view on the diversity and evolution of midwife toads*, PhD Thesis, Nanjing Forestry University: 52–75.

Ambu, J. (2024b) Genome-wide patterns of diversity in the European midwife toad complex: Phylogeographic and conservation prospects. *In*: Ambu, J., *A genomic view on the diversity and evolution of midwife toads*, PhD Thesis, Nanjing Forestry University: 76–94.

Ambu, J., Martínez-Solano, Í., Suchan, T., Hernandez, A., Wielstra, B., Crochet, P.-A. & Dufresnes, C. (2023) Genomic phylogeography illuminates deep cyto-nuclear discordances in midwife toads (*Alytes*). *Molecular Phylogenetics and Evolution*, **183** [107783]: 1–13.

Arntzen, J. W. & García-París, M. (1995) Morphological and allozyme studies of midwife toads (genus *Alytes*), including the description of two new taxa from Spain. *Contributions to Zoology*, **65** (1): 5–34.

Barrios, V., Olmeda, C., Ruiz, E., Ayres, C., Bentata, V., Böll, S., Borgula, A., Bosch, J., Bosman, W., Duguet, R., Goffart, P., Kordges, T., Schley, L., Schmidt, B., Teixeira, J., Uthleb, H. & Verbist, V. (2012) *Action Plan for the conservation of the common midwife toad* (Alytes obstetricans) in the *European Union*. European Commission, EU Species Action Plan, The N2K Group. 1–53.

Boscá, E. (1879) *Alytes cisternasii*, descripción de un nuevo batracio de la fauna española. *Anales de la Sociedad española de Historia natural*, Madrid, **8**: 217-227.

Bosch, J., Martínez-Solano, Í. & García-París, M. (2001) Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (*Alytes obstetricans*) in protected areas of central Spain. *Biological Conservation*, **97** (3): 331-337.

Bosch, B., Mora-Cabello de Alba, A., Marquínez, S., Price, S.J., Thumsová, B. & Bielby, J. (2021) Long-term monitoring of amphibian populations of a national park in Northern Spain reveals negative persisting effects of *Ranavirus*, but not *Batrachochytrium dendrobatidis*. *Frontiers in veterinary Science*, **8** [645491]: 1–9.

Boulenger, G. A. (1879) Étude sur les grenouilles rousses, *Ranae temporariae* et description d'espèces nouvelles ou méconnues. *Bulletin de la Société zoologique de France*, **4**: 158-193.

Chan, K. O. & Grismer, L. L. (2022) GroupStruct: an R package for allometric size correction. *Zootaxa*, **5124** (4): 471–482.

Chenuil, A., Cahill, A. E., Délémontey, N., Du Salliant, E. & Fanton, H. (2019) Problems and questions posed by cryptic species. A framework to guide future studies. *In*: Casetta, E., Marques da Silva, J. & Vecchi, D. (ed.), *From assessing to conserving bodiversity, History, philosophy and theory of the life sciences*, Berlin (Springer), **24**: 77–106.

Dubois, A. (1984) La nomenclature supragénérique des Amphibiens Anoures. *Mémoires du Muséum national d'Histoire naturelle*, (A), *Zoologie*, **131**: 1-64.
Dubois, A. (1987) Again on the nomenclature of frogs. *Alytes*, **6** (1–2): 27–55.

Dubois, A. (1998) List of European species of amphibians and reptiles: will we soon be reaching "stability"? *Amphibia-Reptilia*, **19**: 1–28.

Dubois, A., Ohler, A. & Pyron, R. A. (2021) New concepts and methods for phylogenetic taxonomy and nomenclature in zoology, exemplified by a new ranked cladonomy of recent amphibians (Lissamphibia). *Megataxa*, **5** (1): 1–738.

Dufresnes, C. (2019) *Amphibians of Europe, North Africa and the Middle East: a photographic guide.* London (Bloomsbury Publishing): 1–224.

Dufresnes, C., Brelsford, A., Jeffries, D. L., Mazepa, G., Suchan, T., Canestrelli, D., Nicieza, A., Fumagalli, L., Dubey, S., Martínez-Solano, Í., Litvinchuk, S. N., Vences, M., Perrin, N. & Crochet, P.-A. (2021) Mass of genes rather than master genes underlie the genomic architecture of amphibian speciation. *Proceedings of the national Academy of Sciences*, **118** (36) [e2103963118]: 1–10.

Dufresnes, C. & Crochet, P.-A. (2022) Sex chromosomes as supergenes of speciation: why amphibians defy the rules? *Philosophical Transactions of the royal Society*, (B), **377** (1856) [20210202]: 1–13.

Dufresnes, C. & Hernandez, A. (2021) Phylogeographic advances in midwife toads (*Alytes*) support the existence of a novel taxon endemic to the central Pyrenees. *Journal of zoological Systematics and evolutionary Research*, **59** (8): 2170–2179.

Dufresnes, C. & Jablonski, D. (2022) A genomics revolution in amphibian taxonomy. *Science*, **377** (6612): 1272.

Dufresnes, C. & Martínez-Solano, Í. (2020) Hybrid zone genomics supports candidate species in Iberian *Alytes obstetricans*. *Amphibia-Reptilia*, **41** (1): 105–112.

Dufresnes, C., Poyarkov, N. & Jablonski, D. (2023) Acknowledging more biodiversity without more species. *Proceedings of the national Academy of Sciences*, **120** (40) [e2302424120]: 1–9.

Fedosov, A., Achaz, G., Gontchar, A. & Puillandre, N. (2022) MOLD, a novel software to compile accurate and reliable DNA diagnoses for taxonomic descriptions. *Molecular Ecology Resources*, **22** (5): 2038–2053.

Fonseca, A., Arntzen, J. W., Crespo, E. G. & Ferrand, N. (2003) Regional differentiation in the common midwife toad (*Alytes obstetricans*) in Portugal: a picture from mitochondrial DNA. *Zeitschrift für Feldherpetologie*, **10**: 83–89.

Frétey, T. (2023) The publication dates of the printed issues of the *Bulletin de la Société zoologique de France* (1816 [*sic*, for 1876]–2015). *Bionomina*, **33**: 1–162.

Frost, D. R. (2024) *Amphibian Species of the World*. Version 6.2. New York, USA (American Museum of Natural History). < https://amphibiansoftheworld.amnh.org/index.php>. [Accessed on 11 February 2024].

García-París, M. (1995) Variabilidad genética y distribución geográfica de *Alytes obstetricans almogavarii* en España. *Revista española de Herpetología*, **9**: 133–138.

García-París, M. & Martínez-Solano, Í. (2001) Nuevo estatus taxonomico para las poblaciones ibero-mediterraneas de *Alytes obstetricans* (Anura: Discoglossidae). *Revista española de Herpetología*, **15**: 99-113.

Gonçalves, H., Maia-Carvalho, B., Sousa-Neves, T., García-París, M., Sequeira, F., Ferrand, N. & Martínez-Solano, Í. (2015). Multilocus phylogeography of the common midwife toad, *Alytes obstetricans* (Anura, Alytidae): contrasting patterns of lineage diversification and genetic structure in the Iberian refugium. *Molecular Phylogenetics and Evolution*, **93**: 363–379.

Gonçalves, H., Martínez-Solano, Í., Ferrand, N. & García-París, M. (2007) Conflicting phylogenetic signal of nuclear vs mitochondrial DNA markers in midwife toads (Anura, Discoglossidae, *Alytes*): deep coalescence or ancestral hybridization? *Molecular Phylogenetics and Evolution*, **44** (1): 494–500.

Griffiths, R. A., Garcia, G. & Oliver, J. (2008) Re-introduction of the Mallorcan midwife toad, Mallorca, Spain. *In: Global re-introduction perspectives: reintroduction case-studies from around the globe*, Abu Dhabi (IUCN/SSC Re-introduction Specialist Group): 54–57.

Hebert, P. D. N., Cywinska, A., Ball, S. L. & Jeremy, R. (2003) Biological identifications through DNA barcodes. *Proceedings of the royal Society*, (B), *Biological Sciences*, **270** (1512): 313–321.

Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. *Molecular Biology and Evolution*, **35** (6): 1547–1549.

Lange, L., Brischoux, F. & Lourdais, O. (2022) Benefits of paternal thermoregulation: male midwife toads select warmer temperature to shorten embryonic development. *Behavioral Ecology and Sociobiology*, **76** (48): 1–10.
Lataste, F. (1879a) [15 March] Société zoologique de France. Séance du 18 février 1879. *Revue internationale des Sciences*, **3** (3): 275-276.

Lataste, F. (1879b) [June] Sur un nouveau genre de batracien anoure d'Europe. *Comptes rendus hebdomadaires des Séances de l'Académie des Sciences, 88*: 983-985.

Lataste, F. (1879c) [15 December] Sur une nouvelle forme de batracien anoure d'Europe. *Revue internationale des Sciences, 4* (12): 543-545.

Laurenti, J. N. (1768) *Specimen medicum, exhibens synopsin Reptilium emendatam cum experimentiscirca venena et antidota Reptilium austriacorum*. Viennae (Joan. Thom. Nob. de Trattnern): i–ii + 1–215, pl. 1–5.

Lê, S., Josse, J. & Husson, F. (2008) FactoMineR: a package for multivariate analysis. *Journal of statistical Software, 25*(1): 1–18.

Legros, B., Puissauve, R., Massary, J.-C. de & Marty, V. (2015). Fiches d'information sur les espèces aquatiques protégées: Alyte accoucheur, *Alytes obstetricans* (Laurenti, 1768). Paris (Service du patrimoine naturel du MNHN & Onema): 1–4.

Linnaeus, C. (1758) *Systema Naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. Editio decima, reformata*. Tomus 1. Holmiae (Laurentii Salvii): [i–iv] + 1–824.

Lizana, M., Ciudad, M. J. & Pérez-Mellado, V. (1988) Distribución altitudinal de la herpetofauna en el macizo central de la Sierra de Gredos. *Revista española de Herpetología, 3*: 55-67.

Maia-Carvalho, B., Gonçalves, H., Ferrand, N. & Martínez-Solano, Í. (2014) Multilocus assessment of phylogenetic relationships in *Alytes* (Anura, Alytidae). *Molecular Phylogenetics and Evolution, 79*: 270-278.

Maia-Carvalho, B., Vale, C. G., Sequeira, F., Ferrand, N., Martínez-Solano, Í. & Gonçalves, H. (2018) The roles of allopatric fragmentation and niche divergence in intraspecific lineage diversification in the common midwife toad (*Alytes obstetricans*). *Journal of Biogeography, 45*(9): 2146-2158.

Márquez, R. & Bosch, J. (1995) Advertisement calls of the midwife toads *Alytes* (Amphibia, Anura, Discoglossidae) in continental Spain. *Journal of zoological Systematics and evolutionary Research, 33*(3-4): 185-192.

Martínez-Gil, H., Martínez-Freiría, F., Perera, A., Enriquez-Urzelai, U., Martínez-Solano, Í., Velo-Antón, G. & Kaliontzopoulou, A. (2022) Morphological diversification of Mediterranean anurans: the roles of evolutionary history and climate. *Biological Journal of the Linnean Society, 135*(3): 462-477.

Martínez-Solano, Í., Gonçalves, H. A., Arntzen, J. W. & García-París, M. (2004) Phylogenetic relationships and biogeography of midwife toads (Discoglossidae: *Alytes*). *Journal of Biogeography, 31*(4): 603-618.

Padial, J. M., Miralles, A., Riva, I. de la & Vences, M. (2010) The integrative future of taxonomy. *Frontiers in Zoology, 7*(16): 1-14.

Pasteur, G. & Bons, J. (1962) Note préliminaire sur *Alytes maurus*: gemellarité ou polytopisme? Remarques biogéographiques, génétiques et taxinomiques. *Bulletin de la Société zoologique de France, 87*: 71-79.

Pleguezuelos, J. M., Márquez, R. & Lizana, M. (2002) *Atlas y Libro Rojo de los anfibios y reptiles de España*. Madrid (Dirección General de Conservación de la Naturaleza-Asociación Herpetologica Española): 1-587.

Rubinoff, D., Cameron, S. & Will, K. (2006) A genomic perspective on the shortcomings of mitochondrial DNA for “barcoding” identification. *Journal of Heredity, 97*(6): 581-594.

Rubinoff, D. & Holland, B. S. (2005) Between two extremes: mitochondrial DNA is neither the panacea nor the nemesis of phylogenetic and taxonomic inferences. *Systematic Biology, 54*(4): 952-961.

Salvador, A., Pleguezuelos, J. M. & Reques, R. (2021) *Guía de los anfibios y reptiles de España*. Madrid (Asociación Herpetológica Española): 1-341.

Sanchíz, B. & Adrover, R. (1979) Anfibios fósiles del Pleistoceno de Mallorca. *Doñana, Acta Vertebrata, 4*: 5-25.

Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 years of image analysis. *Nature Methods, 9*: 671-675.

Speybroeck, J., Beukema, W., Bok, B. & Van Der Voort, J. (2016) *Field guide to the amphibians and reptiles of Britain and Europe*. London (Bloomsbury Publishing): 1-432.

Sueur J., Aubin T. & Simonis C. (2008) Seewave, a free modular tool for sound analysis and synthesis. *Bioacoustics, 18*: 213-226.
Vences, M., Thomas, M., Bonett, R. M. & Vieites, D. R. (2005) Deciphering amphibian diversity through DNA barcoding: chances and challenges. *Philosophical Transactions of the royal Society, (B)*, **Biological Sciences**, *360* (1462): 1859–1868.

Vences, M., Miralles, A., Brouillet, S., Ducasse, J., Fedosov, A., Kharchev, V., Kostadinov, I., Kumari, S., Patmanidis, S., Scherz, M. D. & Puillandre, N. (2021) iTaxoTools 0.1: kickstarting a specimen-based software toolkit for taxonomists. *Megataxa*, **6** (2): 77–92.

Vences, M., Miralles, A. & Dufresnes, C. (2024) Next-generation species delimitation and taxonomy: Implications for biogeography. *Journal of Biogeography*. <https://doi.org/10.1111/jbi.14807>.

Wagler, J. (1829) Descriptiones et icones Amphibiorum. *Isis von Oken, 1829*: 68–70.

Wickham, H. (2016) *ggplot2: elegant graphics for data analysis*. New York (Springer-Verlag): i–viii + 1–213.

*Submitted*: 8 March 2024.

*Accepted*: 8 April 2024.

*Published*: 13 May 2024.

*Corresponding Editor*: Daniel JABLONSKI