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https://doi.org/10.11646/zootaxa.5319.2.2
http://zoobank.org/urn:lsid:zoobank.org:pub:B0F1E485-5BF1-4B71-A59E-AA2CE354355F
178 Accepted by D. Blackburn: 27 Jun. 2023; published: 25 Jul. 2023
Article ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Zootaxa 5319 (2): 178–198
https://www.mapress.com/zt/
Copyright © 2023 Magnolia Press
Integrative revision of the Blommersia wittei complex, with description of a new
species of frog from western and north-western Madagascar
MIGUEL VENCES1,*, MALTE MULTZSCH1, JÖRN KÖHLER2, ANGELICA CROTTINI3,4,5, FRANCO
ANDREONE6, ANDOLALAO RAKOTOARISON7,8, MARK D. SCHERZ9 & FRANK GLAW10
1Zoologisches Institut, Technische Universität Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany
�
m.vences@tu-braunschweig.de; https://orcid.org/0000-0003-0747-0817
�
m.multzsch@tu-braunschweig.de; https://orcid.org/0000-0002-7189-0755
2Hessisches Landesmuseum Darmstadt, Friedensplatz 1, 64283 Darmstadt, Germany
�
joern.koehler@hlmd.de; https://orcid.org/0000-0002-5250-2542
3CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Campus de Vairão,
Universidade do Porto, 4485-661 Vairão, Portugal
�
acrottini@cibio.up.pt; https://orcid.org/0000-0002-8505-3050
4Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4099-002 Porto, Portugal
5BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairão, 4485-661 Vairão, Portugal
6Museo Regionale di Scienze Naturali, Via G. Giolitti, 36, 10123 Torino, Italy
�
franco.andreone@regione.piemonte.it; https://orcid.org/0000-0001-9809-5818
7Mention Zoologie et Biodiversité Animale, Université d’Antananarivo, BP 906, Antananarivo, 101 Madagascar
8School for International Training, VN 41A Bis Ankazolava Ambohitsoa, Antananarivo, 101 Madagascar
�
andomailaka@gmail.com; https://orcid.org/0000-0003-2620-440X
9Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, 2100, Copenhagen Ø, Denmark
�
mark.scherz@gmail.com; https://orcid.org/0000-0002-4613-7761
10Zoologische Staatssammlung München (ZSM-SNSB), Münchhausenstr. 21, 81247 München, Germany
�
glaw@snsb.de; https://orcid.org/0000-0003-4072-8111
*Corresponding author
Abstract
Frogs of the Blommersia wittei complex are widespread in western and northern Madagascar, and are one of two clades
of the family Mantellidae that have colonized the Comoran island of Mayotte. Based on a comprehensive set of DNA
sequences of the mitochondrial 16S rRNA gene and the nuclear-encoded RAG1 and SACS genes, integrated with
morphological and bioacoustic data, we here analyze the genetic differentiation of populations of this complex across
Madagascar. We confirm that a candidate species named B. sp. Ca5 in previous studies represents a genetically well-
defined evolutionary lineage distributed over much of western Madagascar, which we describe herein as Blommeria bara
sp. nov. based on its molecular and bioacoustic differentiation. Blommersia wittei occurs across northern Madagascar
but its type locality Ambanja, at the lower Sambirano river, is very close to the range of another, newly discovered
microendemic lineage that was only found at two sites along the upper Sambirano river (here named as candidate species
B. sp. Ca12). The B. wittei complex thus provides an example of a clade of closely related Malagasy frogs that contains
species widespread over hundreds of kilometers, as well as extreme microendemics. For a full resolution of this species
complex, more data need to be collected on the geographical contact among these two lineages, on the morphology and
bioacoustics of B. sp. Ca12, and on the north-eastern populations of B. wittei at Sambava, which are weakly differentiated
in mitochondrial genes but differ in bioacoustics and possibly in the extent of foot webbing.
Key words: Amphibia, Anura, Mantellidae, Blommersia bara sp. nov., microendemism, species delimitation
Introduction
Madagascar harbors a rich fauna of anuran amphibians, which in 2023 has exceeded 400 recognized and named
species (AmphibiaWeb 2023). The majority of these species are found in the rainforests of the eastern escarpment
and of the northern Sambirano region. This is in line with most other Madagascar-endemic vertebrate radiations
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whose species richness is strongly influenced by the colonization—or not—of these rainforests (Crottini et al.
2012). A number of species also occur in the more arid West and South-West of the island (Blommers-Schlösser
1979; Blommers-Schlösser & Blanc 1993; Glaw & Vences 2007), the majority of which live in small relict humid
forests such as in the canyons of the Isalo limestone massif (Mercurio et al. 2008; Cocca et al. 2018) or the
Tsingy de Bemaraha limestone karst (Bora et al. 2010). However, several species have adapted specifically to
arid environments, such as Scaphiophryne brevis, S. calcarata, S. obscura, Dyscophus insularis, or Laliostoma
labrosum, all of which are rather robust species that evade desiccation during dry episodes by burrowing into the
ground (Blommers-Schlösser & Blanc 1991; Glaw & Vences 2007; Pabijan et al. 2015; Scherz et al. 2021). Also,
some partly arboreal or scansorial species such as Heterixalus luteostriatus and H. tricolor, Boophis doulioti and B.
xerophilus, and a representative of the genus Blommersia, historically referred to as B. wittei (e.g., populations from
Ankarafantsika and Namoroka in Blommers-Schlösser & Blanc 1991), are widespread in relatively arid areas of
western Madagascar. More recently the latter frogs were assigned to a confirmed candidate species, B. sp. Ca5, due
to their strong genetic differentiation and apparent bioacoustic differentiation compared to B. wittei specimens from
areas near the species’ type locality Ambanja in the northern Sambirano region (Vieites et al. 2009).
Taking into account the latest species description (Vences et al. 2023), the genus Blommersia currently
contains 11 species from Madagascar, and two species from the Comoran island of Mayotte (AmphibiaWeb 2023).
Blommersia belongs to the family Mantellidae, an ecomorphologically diverse group endemic to Madagascar and
the Comoros (Glaw & Vences 2006), and consists of small to medium-sized semi-arboreal species that breed in
lentic, often temporary water bodies. Blommersia are morphologically relatively inconspicuous frogs, and the first
three species of the genus—including B. wittei—were described only in 1974 (Guibé 1974). Blommersia wittei
is a relatively common species at low elevations in northern Madagascar, where its calls are often heard in the
rainy season from temporary swamps. Molecular data place the species into a clade with B. sp. Ca5 from western
Madagascar, and with the Comoran species, B. nataliae and B. transmarina (Vieites et al. 2009, 2020; Wollenberg
et al. 2011; Vences et al. 2023).
In this study, we analyze the evolutionary relationships and taxonomy of frogs assigned to B. wittei and B. sp.
Ca5 based on combined evidence from DNA sequences, advertisements calls and morphology from a comprehensive
sampling across the ranges of these frogs.
Material and Methods
Voucher specimens and tissue samples were collected during field expeditions in Madagascar between 2000–2018.
Frogs were caught during nocturnal and diurnal searches, either opportunistically by catching animals on the ground
around swamps, or at night by locating calling males. Specimens were anesthetized by immersion in MS222 or
chlorobutanol solution, and subsequently euthanized with an overdose of the same substances. Tissue samples
for molecular analysis, typically parts of thigh muscles, were taken and stored in 1.5 ml vials with pure ethanol.
Voucher specimens were fixed in 95% ethanol and preserved in 70% ethanol, and deposited in the following
collections: Museo Regionale di Scienze Naturali, Torino, Italy (MRSN), Université d’Antananarivo, Département
de Biologie Animale (UADBA), Zoological Museum Amsterdam (ZMA; collections now in Naturalis, Leiden), and
Zoologische Staatssammlung München (ZSM). Additionally, type material from the Muséum national d’histoire
naturelle, Paris (MNHN) was studied. We use ACZC, ACP, DRV, FA, FAZC, FGZC, FGMV, ZCMV, MSZC, and
THC to refer to field numbers of A. Crottini, D. Vieites, F. Andreone, F. Glaw and M. Vences, M. D. Scherz, and
T. R. Fulgence, respectively. A full list of all field numbers and museum catalogue numbers, as well as sequences
and sequence accession numbers, is available as Supplementary Table 1 as Excel and tab-delimited table from the
Zenodo repository under DOI 10.5281/zenodo.8049142. Note that most vouchers deposited in UADBA have not
been catalogued in that collection and therefore have no final catalogue numbers yet. In addition several sequences
are from unvouchered tissue samples, held in the collection of A. Crottini (ACP series).
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TABLE 1. Morphometric measurements (all in mm) and femoral gland conditions of adults of the Blommersia wittei species complex targeted in this study. For abbreviations of
measurements, see Materials and Methods; other abbreviations: HT, holotype; PT, paratype; F, female; M, male; FG, femoral gland. NA, not applicable; NM, not measured.
Catalogue
number
Field number Locality Status Sex SVL HW HL HTD HED END NSD NND FORL HAL HIL FOTL FOL TIBL FGL FGW
B. wittei
ZSM 51/2018 MSZC 404 Montagne d’Ambre PT F? 25.4 8.6 9.8 2.2 3.2 3.3 1.3 2.7 16.0 7.4 39.4 18.8 12.9 13.0 NA NA
ZSM 50/2018 MSZC 521 Montagne d’Ambre PT M 22.6 7.3 9.2 2.1 2.8 2.5 1.6 2.4 13.9 6.8 35.1 16.3 10.6 11.1 4.9 2.1
ZSM 384/2016 ZCMV 15039 Sambava PT F? 21.3 6.4 8.5 1.8 3.0 2.3 1.5 2.4 13.4 6.7 35.6 16.4 10.9 11.0 NA NA
ZSM 563/2000 FGMV
2000.378
Sambava PT M 24.7 7.5 9.1 2.4 2.8 2.3 1.5 2.4 14.6 7.3 40.3 19.1 13.1 12.4 4.3 2.3
ZSM 411/2000 FGMV
2000.121
Ambanja PT M 24.3 7.9 9.7 2.0 3.3 2.3 1.8 2.8 14.7 7.3 39.6 18.1 12.0 11.6 3.5 2.0
ZSM 405/2000 FGMV
2000.443
Benavony PT F 24.6 7.4 9.4 2.0 3.0 2.4 1.4 2.8 14.4 6.7 39.3 18.2 12.7 12.0 NA NA
ZSM 2229/2007 FGZC 1387 Forêt d’Ambre PT M 23.4 7.3 9.0 2.0 2.9 2.3 1.2 2.6 14.9 7.4 36.8 17.0 11.7 11.7 4.6 2.0
B. bara sp. nov. (B. sp. Ca5) PT
ZSM 31/2004 FGZC 57 Isalo HT M 18.5 5.8 7.1 1.7 2.7 2.2 1.4 2.3 11.5 5.3 28.6 14.0 9.4 9.0 3.8 1.9
ZSM 2284/2007 ZCMV 5801 Isalo PT M 20.5 6.3 7.7 2.0 2.8 2.1 1.4 2.0 12.6 6.2 NM 15.0 10.2 9.8 4.4 1.9
ZSM 22/2004 FGZC 34 Isalo PT M 18.2 5.6 7.5 1.8 2.3 2.0 1.1 2.0 12.1 5.4 30.4 13.9 9.4 9.0 3.8 1.9
ZSM 24/2004 FGZC 38 Isalo PT M 18.4 6.3 7.1 1.8 2.4 2.2 1.7 2.2 11.4 5.3 30.3 13.3 8.7 9.0 4.5 2.2
ZSM 2285/2007 ZCMV 5804 Isalo PT F 22.0 6.3 8.1 2.0 2.7 2.4 2.0 2.4 13.2 6.5 34.0 16.1 10.5 10.4 NA NA
ZSM 3222/2012 ZCMV 14143 Mariarano PT M 22.8 NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM
ZSM 247/2003 NA Kirindy PT M 23.3 7.0 9.0 1.7 3.4 2.0 1.9 2.4 14.4 7.0 34.4 16.6 11.6 10.7 4.9 2.6
ZSM 246/2003 NA Kirindy PT M 23.3 7.3 8.8 2.0 3.2 2.6 1.8 2.8 15.6 7.5 38.0 17.7 11.6 11.9 5.0 2.6
ZSM 52/2006 FGZC 779 Tsingy de Bemaraha PT M 23.3 7.6 8.8 2.1 3.3 2.2 1.6 2.7 14.9 7.4 38.3 17.3 11.7 11.3 4.8 2.5
ZSM 30/2006 FGZC 727 Tsingy de Bemaraha PT M 24.0 7.4 9.4 2.0 3.0 2.3 1.7 2.8 15.0 7.4 37.4 17.7 11.6 11.5 4.9 2.0
ZSM 13/2006 FGZC 691 Tsingy de Bemaraha PT F 26.4 7.7 9.7 2.2 3.3 2.5 1.8 2.5 16.3 7.8 40.3 19.2 13.4 12.6 NA NA
ZSM 706/2001 FGMV
2001.279
Ankarafantsika PT M 23.4 7.3 9.1 2.1 2.9 2.6 1.6 2.4 14.4 7.6 37.7 18.2 12.7 11.3 4.0 2.0
ZSM 2281/2007 ZCMV 5625 Ankarafantsika PT M 25.7 7.7 9.6 2.4 3.2 2.3 1.5 2.4 16.2 8.2 42.1 19.5 13.4 12.9 NA NA
ZSM 2320/2007 ZCMV 5621 Ankarafantsika PT M 25.6 7.4 9.7 1.9 3.3 2.4 1.5 2.5 15.9 7.6 39.7 18.4 12.7 12.4 5.3 2.6
ZSM 2283/2007 ZCMV 5643 Ankarafantsika PT F 24.8 7.9 9.2 2.0 3.1 2.5 1.6 2.4 15.7 8.1 41.6 18.4 12.6 12.9 NA NA
B. sp. Ca12 PT
ZSM 589/2001 FGMV
2001.34
Antsirasira PT M 23.2 7.0 8.9 2.0 3.0 2.8 1.7 2.4 14.7 6.9 38.9 18.1 12.2 12.0 3.4 2.0
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The following measurements were taken by MV using a manual caliper at an accuracy of 0.1 millimeter: snout-
vent length (SVL); maximum head width (HW); head length from posterior edge of snout opening in a diagonal
line to tip of snout (HL); horizontal tympanum diameter (HTD); horizontal eye diameter (HED); distance between
anterior edge of eye and nostril (END); distance between nostril and tip of snout (NSD); distance between both
nostrils (NND); forelimb length, from limb insertion to tip of longest finger (FORL); hand length, from the base
of the hand to the tip of the longest finger (HAL); hind limb length, from the cloaca to the tip of the longest toe
(HIL); foot length (FOL); foot length including tarsus (FOTL); tibia length (TIBL); femoral gland length (FGL);
and femoral gland width (FGW). Geographical regions within Madagascar are named according to Boumans et al.
(2007) and Brown et al. (2016).
We recorded anuran vocalizations in the field using different digital or analogue devices such as Sony WM-D6C
and Tensai RCR-3222 tape recorders with external microphones (Sennheiser Me-80, Vivanco EM 238), and Tascam
DR07, DR05, Marantz PMD 660 or Roland Edirol R-09 digital recorders, with built-in microphones (Tascam)
or accessorized with semi-directional microphones (Marantz and Roland). We obtained digital recordings at a
sampling rate of 44.1 kHz and 24-bit resolution and saved them as uncompressed files. Recordings were digitized or
resampled at 22.05 kHz and 32-bit resolution and computer-analyzed using the software CoolEdit Pro 2.0. Frequency
information was obtained through Fast Fourier Transformation (FFT; width 1024 points) with the Hanning window
function; audiospectrograms were drawn with the Blackman window function with 256 bands resolution. Temporal
characters were measured from oscillograms. Measured numerical call parameters were rounded and are provided
as range followed by mean ± standard deviation in parentheses. Terminology of call descriptions and methods for
call analyses follow those recommended by Köhler et al. (2017), using the call-centered terminological scheme.
All recordings were high-pass filtered at 600 Hz to remove unwanted low-frequency background noise. In all
cases, filtering was exclusively applied to frequencies outside the bandwidths of calls. For the purpose of easy
and immediate comparability, audiospectrograms and corresponding oscillograms of advertisement calls of all
populations are shown at a time scale of 600 ms.
DNA was extracted from ethanol-preserved tissue samples using a standard salt extraction protocol (Bruford
et al. 1992). We DNA barcoded available and not previously sequenced tissue samples assigned to B. wittei and B.
sp. Ca5 for a fragment of the mitochondrial 16S rRNA gene (16S) that spans about half of the gene at its 3’ terminal
portion and that has regularly been used for molecular taxonomy of Malagasy amphibians (e.g., Vieites et al. 2009),
including Blommersia (e.g., Glaw & Vences 2002; Vences et al. 2010; Pabijan et al. 2011; Glaw et al. 2019; Vieites
et al. 2020). The DNA fragment was PCR-amplified with primers 16SAL (5’-CGCCTGTTTATCAAAAACAT-
3’) and 16SBH-new: (5’-CCTGGATTACTCCGGTCTGA-3’), modified from Palumbi et al. (1991), with the
following cycling protocol: 94°C (90s), 33 x [94°C (45s), 55°C(45s), 72°C (90s)], 72°C (300s). The dataset
was complemented with sequences available from GenBank. We furthermore sequenced a subset of samples
for a fragment of the nuclear-encoded recombination activating gene 1 (RAG1), using primers Gephlut-RAG1-
F1 (5’-ATGGAGAGCCAACCCCTATC-3’) and Gephlut-RAG1-R1 (5’-KCCAGACTCGTTTCCTTCRC-
3’) with cycling protocol: 94°C (120s), 39 x [94°C (20s), 54°C (50s), 72°C (180s)], 72°C (600s) and used the
sequencing primer RAG1-Manti-Seq1 (5’-GCAAAGCCVTTTATTGAAACC-3’). Finally, a subset of samples
was sequenced for a fragment of sacsin (SACS). Amplifications of the SACS fragment were performed using a
nested approach (Shen et al. 2012) using external primers SACSF2 (5’-AAYATHACNAAYGCNTGYTAYAA-
3’) and SACSR2 (5’-GCRAARTGNCCRTTNACRTGRAA-3’) and internal primers SACSNF2 (5’-
TGYTAYAAYGAYTGYCCNTGGAT-3’) and SACSNR2 (5’-CKGTGRGGYTTYTTRTARTTRTG-3’) and with
cycling protocol for both PCRs as follows: 94°C (240s), 45 x [94°C (45s), 45°C (40s), 72°C (120s)], 72°C (600s).
PCR products were purified with Exonuclease I and Shrimp Alkaline Phosphatase digestion and sent to LGC
Genomics (Berlin) for sequencing on automated DNA sequencers. Chromatograms were checked and corrected for
obvious errors with CodonCode Aligner 3.7.1 (Codon Code Corporation, Dedham, MA, USA). All newly obtained
sequences were submitted to GenBank and are available under the following accession numbers: OR123906–
OR123961 and OR127037–OR127116).
Sequences were aligned using the Clustal algorithm implemented in MEGA7 (Kumar et al. 2016), and a
phylogeny inferred under the Maximum Likelihood (ML) optimality criterion, after selecting the most appropriate
substitution model based on the Bayesian Information Criterion. Node support was assessed with 500 bootstrap
replicates.
For graphically representing the relationships among alleles (haplotypes) of the RAG1 and SACS gene
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fragments, we used a network approach. Alleles (haplotypes) were inferred with the PHASE algorithm (Stephens et
al. 2001) implemented in the DnaSP software (Version 5.10.3; Librado & Rozas 2009), an ML tree inferred under
the Jukes-Cantor substitution model in MEGA7 (choosing this simple model to avoid over-parametrization), and
this tree and the respective alignment were used as input for Haploviewer (written by G. B. Ewing; http://www.
cibiv.at/~greg/haploviewer), a software that implements the methodological approach of Salzburger et al. (2011).
Pairwise genetic distances were calculated from the 16S alignment using TaxI2 (Vences et al. 2021). Species
partitions were inferred using ASAP (Puillandre et al. 2021) and subsequently we compared the favored ASAP
partition for concordance with evidence from RAG1 and SACS differentiation.
Results
Molecular differentiation and evolutionary relationships
The ML tree inferred from the alignment of the mitochondrial 16S fragment (alignment length 533 bp; 127 ingroup
samples; T92+G model) revealed several deep lineages within the B. wittei complex (Fig. 1). A lineage containing
samples assigned to B. wittei, including samples from the type locality Ambanja, was sister to a clade including the
two Comoran species, B. nataliae and B. transmarina, which are not further discussed herein. Samples in the B.
wittei lineage all originated from northern Madagascar (as defined by Brown et al. 2016). Two other mitochondrial
lineages split from nodes basal to this (wittei (nataliae, transmarina)) clade, one originating from a small area in
the Sambirano region, and the other from the West and South West of Madagascar (Fig. 2). We emphasize that this
16S tree is here mainly presented for the purpose of DNA barcoding, i.e., assigning samples to main mitochondrial
lineages. In fact, the short 16S fragment is not sufficient to reliably resolve deeper phylogenetic relationships among
the species involved (for a multi-gene tree with a hypothesis of the placement of the B. wittei complex within
Blommersia, see Vences et al. 2023).
The analysis with ASAP confirmed the existence of various species-level lineages in our data set. The species
partition with the best (lowest) ASAP score of 1.0 supported five ingroup partitions, corresponding to (1) B. nataliae,
(2) B. transmarina, (3) B. wittei (including the somewhat deviant samples from Sambava that will be discussed
below), (4) the microendemic Sambirano lineage for which we here coin the candidate species name Blommersia
sp. Ca12, and (5) the lineage from the West and South West, which corresponds to the previously named candidate
species B. sp. Ca5. The second-best species partition (ASAP score = 2.0) merged B. transmarina with B. wittei,
which is an unplausible result given the clear differences between these two species, and the third best partition
(ASAP score = 4.5) separated samples from Sambava from other B. wittei samples into an additional sixth ingroup
subset.
Considering the partition with five lineages, genetic distances between the main lineages were high. Uncorrected
pairwise distances in the 16S fragment were 5.0–8.2% between B. wittei and B. sp. Ca5, 5.5–7.0% between B. wittei
and B. sp. Ca12, and 4.9–6.4% between B. sp. Ca5 and Ca12. The Sambava specimens differed from other B. wittei
by a small divergence of 1.2%. The two Comoran species differed from the Malagasy lineages by a minimum of
4.6% (between B. transmarina and B. wittei), and by 5.1% from each other (a slightly inflated value because the B.
nataliae sequence is rather short, thus with overrepresentation of highly variable stretches).
The haplotype networks reconstructed from phased alleles of fragments of the nuclear-encoded genes for RAG1
(33 samples, 457 bp) and SACS (23 samples, 924 bp) revealed that each of the Malagasy mitochondrial lineages
(Comoran taxa were not included) formed independent phylogroups also from the nuclear perspective, in general
without haplotype sharing among lineages (Fig. 1), with the exception, in the RAG1 network, of the shared alleles
between B. wittei and B. sp. Ca12. Additionally, also in the RAG1 network, some B. wittei samples showed rather
divergent alleles that may represent sequencing errors or sequence contamination (two of the four sequences in this
deviant phylogroup were from individuals whose second allele was placed in the regular wittei phylogroup).
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FIGURE 1. Molecular differentiation in the Blommersia wittei complex. The panels show a maximum likelihood tree based on
533 bp of the mitochondrial 16S tree (on the right), numbers at nodes representing bootstrap percentages (500 replicates). The
two panels on the left show haplotype networks reconstructed from phased DNA sequences of fragments of the nuclear-encoded
SACS (23 samples, 924 bp) and RAG1 (33 samples, 475 bp) genes. Colors in the network correspond to assignment of samples
to main groups in the 16S tree.
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FIGURE 2. Map of Madagascar showing the distribution records of the Blommersia wittei species complex. Only locations
confirmed by molecular data are shown.
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FIGURE 3. Specimens of Blommersia wittei in life. (A–C) Specimens UADBA-MSZC 451, ZSM 51/2018 (MSZC 404), ZSM
50/2018 (MSZC 521) from Montagne d’Ambre. (D) Specimen UADBA-FGZC 5576 from Nosy Be. (E,F) Male specimen from
Montagne d’Ambre (not clearly referable to a voucher specimen) photographed in 2003.
Morphological differentiation
Morphological examination of available specimens revealed no consistent differences among the main lineages
in the B. wittei complex (except for the distinct Comoran species that are not considered here). In contrast, a
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substantial polymorphism in various traits was apparent (Figs 3‒4). All specimens examined shared a separation of
the lateral metatarsalia by webbing (rather than connective tissue) which is a diagnostic trait of B. wittei and a few
other Blommersia species. Vomerine teeth were present in most specimens, but were sometimes weakly expressed
(e.g., in B. wittei from Montagne d’Ambre) and in some specimens (like the small-sized specimens of B. sp. Ca5
from Isalo) not recognizable. Femoral glands were distinct in many male specimens, but indistinct in the sole
male specimens available from Antsirasira and Sambava (Table 1). A very substantial difference was observed in
body size, especially within B. sp. Ca5 where specimens from Isalo (male SVL 18.5‒20.5 mm) were much smaller
than those from Ankarafantsika and Tsingy de Bemaraha (23.4‒25.7 mm) (Table 1). A thin middorsal line or a
broad middorsal stripe characterize some specimens, but otherwise color and pattern was inconspicuous and rather
uniform across specimens, populations and lineages.
FIGURE 4. Blommersia bara sp. nov. (= B. sp. Ca5) in life. (A,B): Male holotype ZSM 31/2004 (FGZC 47) from Isalo in
dorsolateral and ventral view. (C) Individual from Ankarafantsika displaying egg-guarding behavior. (D) Female paratype ZSM
13/2006 from Tsingy de Bemaraha. (E) Additional individual (not clearly referable to a voucher) from Tsingy de Bemaraha.
Bioacoustic differentiation
Advertisement calls within the Blommersia wittei complex all are characterized by consisting of a single short note
emitted in call series at more or less regular intervals (Figs 5‒7). Depending on motivation, call series might be of
different duration and contain differing numbers of calls (Vences et al. 2006). Although this character is shared by
all calls studied herein, there are some notable differences in note structure among the populations considered.
C
alls of populations here referred to as corresponding to B. wittei, including those from the type locality Ambanja,
differ distinctly in note structure from the other populations by notes composed of numerous pulses, mostly well
separated from each other and grouped in two pulse groups within each note, separated by a longer inter-pulse
interval (Fig. 5). In all cases, the first pulse group within a note exhibits less call energy than the second pulse group
of the same note. Although our call recordings of B. wittei differ markedly in recording quality, this character is
obvious in all B. wittei calls analyzed, except those from Sambava that are separately characterized below.
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FIGURE 5. Audiospectrograms and corresponding oscillograms of advertisement calls of Blommersia wittei from three different
localities (each showing several calls of a call series).
FIGURE 6. Audiospectrograms and corresponding oscillograms of advertisement calls of Blommersia bara sp. nov. (= B. sp.
Ca5) from two different localities (each showing several calls of a call series).
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FIGURE 7. Audiospectrograms and corresponding oscillograms of advertisement calls of Blommersia populations of
taxonomically uncertain status (each showing several calls of a call series). The recording from Antsirasira may refer to B. sp.
Ca12, but the recorded specimen has not been collected or genotyped. The call from Sambava is from a specimen of B. wittei
that belongs to a weakly divergent mitochondrial phylogroup and may also differ in some morphological features, but is here
assigned to B. wittei preliminarily.
Populations from western Madagascar, here referred to as B. sp. Ca5, emit advertisement calls with notes being
very short and barely structured with respect to amplitude modulation (Fig. 6). Calls from Isalo may contain 2–4
amplitude peaks, but are rather simple and homogeneous in structure. The same is true for calls from Ankarafantsika,
with sometimes only two recognizable amplitude peaks within notes and only slightly longer note duration when
compared to calls from Isalo. The differences among the calls of these two populations are rather small and would
not qualify for species-specific differences (see Köhler et al. 2017), given their geographic distance to each other.
Calls recorded from Antsirasira (no voucher specimen collected from the calling site; calls perhaps corresponding
to B. sp. Ca12) differ considerably from those of B. sp. Ca5 by a much more complex amplitude structure within
notes (Fig. 7), with each note containing several separated pulses (3–10 pulses/note), which can be partly fused.
Furthermore, note duration is considerably longer, when compared to calls from the dry west (34–66 versus 13–28
ms).
Calls from Sambava in the North-East (from a population here assigned to B. wittei) are rather similar when
compared to calls from Antsirasira (B. sp. Ca12), as these share a similar pulse pattern and note duration (Fig. 7).
However, pulses in Sambava calls were largely fused and the pulses in most notes are thus barely countable, and
dominant frequency seems to be distributed in a narrower frequency band.
In summary, the bioacoustic data are in general agreement with our results of molecular phylogenetic analyses
in supporting the distinctness of three Malagasy clades in this species complex, with advertisement calls showing
characteristic traits for each identified clade. Although call differences are slightly less pronounced among these
clades when compared to other groups in Blommersia (Vences et al. 2010; Pabijan et al. 2011), the bioacoustic
differences identified do support our taxonomic conclusions.
Taxonomic conclusions
The genetic data of mitochondrial and nuclear-encoded DNA sequences suggest the existence of various species
within the B. wittei complex. This is supported by deep mitochondrial divergences around 5% among the three
main Malagasy mitochondrial lineages, reduced or absent haplotype sharing between these lineages in two nuclear-
encoded gene fragments, bioacoustic differences between two of the lineages, and possibly by the phylogenetic
placement of the Comoran clade of two morphologically and ecologically distinct species, B. nataliae and B.
transmarina, nested among the Malagasy lineages.
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Completely resolving this complex of species is, however, hampered by uncertainties in both, assignment of
existing names and species delimitation. From a nomenclatural point of view, our data do not allow us to assign the
available name, B. wittei, with full confidence to one of the lineages, as no molecular data are available from the B.
wittei holotype. The closest occurrence of B. sp. Ca5, at Sahamalaza, is located at more than 70 km linear distance
from the B. wittei type locality Ambanja, and we therefore consider it as unlikely that B. sp. Ca5 would occur up to
Ambanja; its synonymy with B. wittei can therefore be excluded with sufficient probability. However, Ambanja is
located along the lower Sambirano river and in this general area two deep lineages occur: the widespread northern
lineage, which was found by us at Ambanja and nearby sites (Benavony, Nosy Be), and the deep microendemic
lineage here named Blommersia sp. Ca12 occurring somewhat further upstream. We here consider to be most likely
that the holotype and topotypical specimens of the paratype series are conspecific with the genetic lineage found by
us in Ambanja. We cannot, however, fully rule out that also B. sp. Ca12 occurs at Ambanja, and that the holotype of
B. wittei may belong to this lineage.
From the point of view of species delimitation, the status of the population from Sambava in the North East
remains unclarified. This lineage has unique haplotypes in the two nuclear genes and differs in advertisement call,
but exhibits only a weak mitochondrial divergence from typical B. wittei (ca. 1% 16S distance). Furthermore, one
sample purportedly from Sambava has a mitochondrial sequence similar to that of typical B. wittei. However,
this individual (voucher number FA m61, corresponding to the sequence with accession number AY848113 and
catalogued under MRSN A3785) was collected in 2000 (by MV) without assigning it a physical fieldnumber tag,
and we cannot exclude that the sample and/or voucher specimen has been incorrectly labelled as originating from
Sambava. New, more comprehensive collections are needed to understand if specimens with the mitochondrial
identity of typical B. wittei indeed occur in the Sambava region.
As a taxonomic conclusion, we suggest that the western/north-western lineage (B. sp. Ca5) represents a
distinct species due to its genetic and bioacoustic divergence, and because its range is far enough from the B. wittei
type locality (Ambanja) to exclude that the B. wittei holotype belongs to this lineage. We also conclude that the
microendemic Sambirano lineage (B. sp. Ca12) very probably represents a distinct species which, however, we
cannot formally name and describe at this time due to the lack of voucher specimens available for examination, the
absence of the molecular identification of the B. wittei holotype, and the closeness of its range to the type locality
of B. wittei. Finally, we do not formally name the Sambava population (neither as species, subspecies nor candidate
species) due to its low mitochondrial divergence and uncertainty about its co-occurrence with typical B. wittei.
Species accounts
Blommersia wittei (Guibé, 1974)
Figures 3, 8
Identity and type material. Clarifying the taxonomy of the B. wittei complex requires first ascertaining the identity
of B. wittei sensu stricto. The species was described by Guibé (1974) as Mantidactylus wittei, and type specimens
are deposited in the Paris museum (acronym MNHN, in earlier times written MNHNP), with the following verbatim
information in the original description: “Holotype : no 1953-60 MNHN Paris. Environs d’Ambanja. J. Guibé (11-
1951). Paratypes : nos 1953-60 A à 1953-60 L MNHN Paris. Même provenance. Nos A.682 à A.684 : Ampijora,
station forestière à 45 km de Marovaoy (Ouest). Nos 1973-944 à 1973-951 MNHN Paris : forêt d’Ankarafantsika.
Ch. P. Blanc (11-1973).”
Due to some imprecise information in the handwritten MNHN catalogue and extensive re-labelling of several
of these specimens, there has been confusion on the type series in subsequent publications and in the current digital
MNHN catalogue. Frost (2023) correctly states that the holotype is MNHNP 1953.60, by original designation,
but merges two separate sites in the type locality account: “‘Environs d’Ambanja’, Forest Ankarafantsika,
Madagascar.”
In contrast, the digital type catalogue of the MNHN (downloaded from gbif.org in 2022) as well as the MNHN
online catalogue (https://science.mnhn.fr/institution/mnhn/collection/ra/; accessed 11 February 2023) state that the
holotype originated from “Vallée de la Tsiribihina” and furthermore provide the following list of paratypes: MNHN
1973.944–1973.951 (8 specimens) from Ankarafantsika, and 1993.682–1993.684 (3 specimens) from Ampijoroa.
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FIGURE 8. Preserved name-bearing type specimens of Blommersia wittei and Blommersia bara sp. nov. in dorsal and ventral
views. Photographs of the B. wittei holotype by RECOLNAT (ANR-11-INBS-0004) / Antoine Fraysse, available from https://
science.mnhn.fr/institution/mnhn/collection/ra/item/1953.60.
Vences et al. (2010) provided measurements of part of the type series of B. wittei, purportedly all from Ambanja:
the holotype MNHN 1953.60, and the following paratypes: MNHN 1991.2529–1991.2533 (5 specimens; previously
labelled MNHN 1953.60 A to E) and MNHN 1991.2536–1991.2539 (4 specimens, previously labelled MNHN
1953.60 H to K).
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After inspecting the original (handwritten) MNHN catalogues (partly available from https://science.mnhn.fr/
catalogues; accessed 11 February 2023) we conclude that:
(1) The holotype of Mantidactylus wittei Guibé, 1974 (Fig. 8) unambiguously is the specimen MNHN 1953.60
for which morphometric measurements were provided by Vences et al. (2010) and whose locality, according to
the original description and original catalogue entry is “Environs d’Ambanja”, with the addition in the catalogue:
“Cacaoyéres” (= cacao trees, thus indicating the specimen was collected in a cacao plantation).
(2) A series of 11 paratypes (originally labelled MNHN 1953.60 A to 1953 K, currently re-labelled as MNHN
1991.2529–1991.2539) originates from the same locality as the holotype.
(3) A series of 8 paratypes (MNHN 1973.944–1973.951) originate from Ankarafantsika,
(4) A series of 3 paratypes (MNHN 1993.682–1993.684) originate from Ampijoroa, which represents a forestry
station (currently local headquarters of Madagascar National Parks) within the Ankarafantsika forest.
As explained in the Taxonomic conclusion account above, based on the provenance of the holotype from
Ambanja, we continue assigning the name B. wittei to the genetic lineage occurring across much of northern
Madagascar and collected by us at Ambanja and nearby localities. The paratype series is however mixed: specimens
from Ankarafantsika (MNHN 1973.944–1973.951) and Ampijoroa (MNHN 1993.682–1993.684) are here assigned
to B. sp. Ca5. Only the 11 paratypes from the type locality are likely conspecific with the holotype.
Morphology. Measurements of the type series of B. wittei have been published by Vences et al. (2010) and
measurements of additional specimens by Pabijan et al. (2011). Measurements of further specimens are included in
Table 1 herein. A full set of measurements including those from the previous publications is given as Supplementary
Table 2 (available from the Zenodo repository under DOI 10.5281/zenodo.8049142). Based on these data, male
SVL is 21.0–27.0 mm and female SVL is 20.7–25.0 mm. In the specimens examined for the present study (listed in
Table 1), vomerine teeth are usually recognizable (clearly visible in specimens from the type locality Ambanja) but
weakly expressed in several specimens from Montagne d’Ambre. In ZSM 563/2000 from Sambava, vomerine teeth
are weakly recognizable, and this specimen apparently has more developed webbing than many other individuals
examined.
Vocalizations. Advertisement calls recorded in February 1991 at the type locality Ambanja (recording temperature
not taken) consist of a single short pulsed note repeated in call series at regular intervals and very fast succession
(Fig. 5). Recording quality is poor and the detailed call structure is probably partly masked by background noises.
However, each call (= note) seems to contain two pulse groups which are separated from each other, with the first
pulse group being of lower amplitude. Pulse structure in not very obvious in the recording and most pulses appear
basally fused. Maximum call energy is distributed towards the middle of the call’s duration. Numerical parameters
of 16 analyzed calls of one male are as follows: call duration (= note duration) 62–77 ms (66.8 ± 5.3 ms); inter-call
intervals within regular call series 15–31 ms (18.6 ± 5.6 ms); pulses/note 9–18 (13.8 ± 3.3); duration of regular call
series 1275 ms (n = 1); call rate within call series approximately 740 calls/minute; dominant frequency 4694–4886
Hz (4797 ± 82 Hz); second frequency peak around 2500 Hz; prevalent bandwidth 1800–6000 Hz.
Advertisement calls recorded on 7 February 1992 from north of Andoany, Nosy Be (air temperature 25°C),
consist of a single short pulsed note repeated in short call series at regular intervals and very fast succession (Fig. 5).
Each call (= note) exhibits two pulse groups which are clearly separated from each other, with the first pulse group
being of lower amplitude. Pulses are partly fused, but countable. Maximum call energy is distributed in the middle
of the call, namely the beginning of the second pulse group. Numerical parameters of 10 analyzed calls of one male
are as follows: call duration (= note duration) 65–83 ms (74.8 ± 6.3 ms); inter-call intervals within regular call series
16–32 ms (21.5 ± 5.6 ms); pulses/note 14–19 (16.3 ± 1.9); duration of regular call series 490 and 500 ms (n = 2); call
rate within call series approximately 640 calls/minute; dominant frequency 4823–5240 Hz (5033 ± 163 Hz); second
frequency peak around 2500 Hz; prevalent bandwidth 2000–8200 Hz.
Advertisement calls recorded on 15 March 1994 on Montagne d’Ambre (air temperature 22°C), consist of a
single short, distinctly pulsed note repeated in call series at regular intervals and very fast succession (Fig. 5). Each
call (= note) exhibits two pulse groups which are clearly separated from each other, with the first pulse group being
of lower amplitude. Pulses are barely fused and rather distinctly separated. Maximum call energy is distributed in
the middle of the call, namely the beginning of the second pulse group. Numerical parameters of 19 analyzed calls
of one individual are as follows: call duration (= note duration) 59–73 ms (65.4 ± 4.2 ms); inter-call intervals within
regular call series 22–39 ms (29.6 ± 7.1 ms); pulses/note 7–11 (8.9 ± 1.3); duration of regular call series 1821 ms
(n = 1); call rate within call series approximately 600 calls/minute; dominant frequency 4489–4597 Hz (4542 ± 41
Hz); prevalent bandwidth 1800–7500 Hz.
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Advertisement calls recorded from specimen ZSM 50/2018 (MSZC 521) on 25 December 2017 on Montagne
d’Ambre (air temperature ca 19°C), consist of a single short, distinctly pulsed note repeated in call series at regular
intervals and very fast succession (Fig. 5). Each call (= note) exhibits clearly separated pulses. Intervals between
pulses are somewhat irregular, in some calls resulting in two pulse groups separated by a larger interval. Maximum
call energy is evident in the middle of the call. Numerical parameters of 16 analyzed calls of one male are as follows:
call duration (= note duration) 68–88 ms (78.2 ± 5.7 ms); inter-call intervals within regular call series 28–50 ms
(41.4 ± 4.2 ms); pulses/note 7–11 (9.0 ± 1.3); duration of regular call series 1590 ms (n = 1); call rate within call
series approximately 500 calls/minute; dominant frequency 5066–5232 Hz (5137 ± 80 Hz); prevalent bandwidth
2000–10000 Hz.
Advertisement calls recorded on 20 March 2000 in Sambava (air temperature 24.7°C), here tentatively allocated
to B. wittei, consist of a single, short, pulsed note repeated in call series at regular intervals within series. Calls (=
notes) exhibit a pulsed structure, but most pulses are largely fused, resulting in an irregular pulse pattern and varying
number of countable pulses (Fig. 7). Maximum call energy is distributed among the first two thirds of the call’s
duration. Numerical parameters of 22 analyzed calls of one male are as follows: call duration (= note duration) 27–
59 ms (39.4 ± 11.8 ms); inter-call intervals within regular call series 54–76 ms (67.9 ± 7.7 ms); pulses/note 2–6 (4.1
± 1.0); duration of regular call series 2209 ms (n = 1); call rate within call series approximately 600 calls/minute;
dominant frequency 5316–5598 Hz (5440 ± 145 Hz); second frequency peak around 2600 Hz; prevalent bandwidth
2100–8600 Hz.
Blommersia bara sp. nov.
Figures 4, 8
Identity. This species was considered Mantidactylus wittei by Guibé (1974) and Blommers-Schlösser & Blanc
(1991) for specimens from Ankarafantsika, and called Mantidactylus cf. wittei by Glos (2003), Blommersia sp. aff.
wittei “Isalo” by Glaw & Vences (2007), Blommersia sp. aff. wittei by Mercurio et al. (2008), Blommersia sp. (aff.
wittei) by Bora et al. (2010), Blommersia sp. 5 by Vieites et al. (2009) and Wollenberg et al. (2011), Blommersia sp.
Ca5 by Perl et al. (2014), Penny et al. (2016) and Glaw et al. (2019), Blommersia sp. Ca05 by Penny et al. (2017),
and Blommersia sp. aff. wittei Ca05 “Isalo” by Cocca et al. (2018).
Holotype. ZSM 31/2004 (field number FGZC 47), an adult male, collected by F. Glaw, M. Puente, M. Thomas
& R. Randrianiaina at a stream near Ranohira, Isalo Massif (22.5856°S, 45.3997°E, 813 a.s.l.), southwestern
Madagascar, on 21 January 2004.
Paratypes. A total of 30 specimens, all from western Madagascar. ZSM 22/2004 (FGZC 34) and ZSM 24/2004
(FGZC 38) two adult males with the same collecting data as the holotype; ZSM 706/2001 (FGMV 2001.279),
adult male, collected by M. Vences, D.R. Vieites, G. Garcia, V. Raherisoa, & A. Rasoamamonjinirina at Ampijoroa,
Ankarafantsika National Park (approximately 16.3°S, 46.82°E) on 24 February 2001; ZSM 246/2003 and ZSM
247/2003, two adult males, collected by J. Glos at Kirindy Forest (site “G2”) in January 2001; ZSM 13/2006 (FGZC
691), ZSM 30/2006 (FGZC 727), ZSM 52/2006 (FGZC 779), and ZSM 68/2006 (FGZC 809), two adult females
and two adult males, collected by F. Glaw, J. Köhler, P. Bora & H. Enting at Tsingy de Bemaraha National Park,
Andranopasazy, “Camp 1” (18.7086°S, 44.7189°E, 146 m a.s.l.), on 17, 19, and 24 March 2006, respectively; ZSM
2284/2007 (ZCMV 5801) and ZSM 2285/2007 (ZCMV 5804), one adult male and one adult female, collected by
M. Vences and collaborators at Isalo National Park, on 17 February 2007; ZSM 2281/2007 (ZCMV 5625), ZSM
2282/2007 (ZCMV 5626), ZSM 2283/2007 (ZCMV 5643), and ZSM 2320/2007 (ZCMV 5621), collected by L.
du Preez, C. Weldon & L. Raharivololoniaina at Ankarafantsika, on 9 February 2007; ZSM 3222/2012 (ZCMV
14143), adult male collected by A. Rakotoarison, J. Erens & E. Rajeriarison at Mariarano (15.4978°S, 46.6943°E,
11 m a.s.l.), on 28 December 2012; MRSN A2957 and A2958 (FAZC 11807–11808), two males, collected by F.
Andreone, G. Aprea & V. Mercurio in the Isalo Massif, Andohasahenina (22.8333°S, 45.1880°E, 876 m a.s.l.) on
15 January 2004; MRSN A5349 (FAZC 12550), female, and MRSN A5350 (FAZC 12551), male, collected by F.
Andreone, F. Mattioli & V. Mercurio in the Isalo Massif, Zahavola (22.6215°S, 45.3587°E, 881 m a.s.l.), on 17
November 2004; MRSN A5353 and A5354 (FAZC 12968 and 12970), two males, collected by F. Andreone, F.
Mattioli & V. Mercurio in the Isalo Massif, Sakamalio (22.4348°S, 45.2552°E, 726 m a.s.l.) on 16 December 2004;
MRSN A5351 (FAZC 12591), female, collected by F. Andreone, F. Mattioli & V. Mercurio in the Isalo Massif,
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Andriamanero (22.3672°S, 45.3920°E, 663 m a.s.l.), on 20 November 2004; as well as seven specimens deposited
in the UADBA collection (uncatalogued but accessible via field tags): UADBA-ZCMV 5620, 5641, 5642, 5644,
four specimens collected by L. du Preez, C. Weldon & L. Raharivololoniaina at Ankarafantsika, on 9 February 2007;
and UADBA-ZCMV 5800, 5802, 5803, three specimens collected by collected by M. Vences and collaborators at
Isalo National Park, on 17 February 2007.
Diagnosis. A species of the genus Blommersia in the subfamily Mantellinae of the family Mantellidae based
on presence of intercalary elements between penultimate and ultimate phalanges of fingers and toes (verified by
external examination), occurrence in Madagascar, relatively small body size (male SVL < 27 mm), presence of
femoral glands in males and absence of femoral gland rudiments in females, head distinctly longer than wide, and
molecular phylogenetic relationships.
From other species of Blommersia, the new species is mainly distinguished as follows: from B. angolafa by the
presence of vomerine teeth in many individuals (vs. absence) and a totally different color pattern without whitish
spots on the flanks and on the finger- and toe-tips (vs. presence); from B. dejongi by a femoral gland placed centrally
on the thigh (vs. distally); from B. blommersae by having lateral metatarsalia separated by webbing (vs. connected
by dense tissue); from B. domerguei by having lateral metatarsalia separated by webbing (vs. connected by dense
tissue), by the presence of vomerine teeth in many individuals (vs. absence), a larger body size (SVL >20 mm vs.
<18 mm), and absence of a distinct pattern with three dark brown bands on a copper-brown dorsum (vs. presence);
from B. dupreezi by having lateral metatarsalia separated by webbing (vs. connected by dense tissue) and absence
of distinct black lateral stripe (vs. presence); from B. galani by presence of vomerine teeth in many individuals
(vs. absence); from B. grandisonae by presence of vomerine teeth in many individuals (vs. absence) and a largely
different color pattern (but see discussion in Vences et al. 2023 on the identity of B. grandisonae); from B. kely
by having lateral metatarsalia separated by webbing (vs. connected by dense tissue), presence of vomerine teeth
in many individuals (vs. absence), and larger body size (SVL >20 mm vs. <17 mm); from B. nataliae by ovoid
femoral glands in small distance to each other (vs. more rounded, distant glands) and absence of a dark face mask
(vs. presence); from B. sarotra by having lateral metatarsalia separated by webbing (vs. connected by dense tissue);
from B. transmarina by a narrower head (male HW/SVL ratio 0.29‒0.34 vs. 0.34–0.36) and somewhat shorter
hands (male HAL/SVL ratio 0.29–0.33 vs. 0.30–0.35); from B. variabilis by having lateral metatarsalia separated
by webbing (vs. connected by dense tissue in most individuals) and probably a smaller distance between femoral
gland. Furthermore, differentiated from all these species by differences in advertisement calls and genetic distances
>5% in the analyzed mitochondrial 16S rRNA gene fragment of these two species. It differs from B. wittei, however,
in a substantial divergence of mitochondrial genes (5.0‒8.2% uncorrected pairwise distance in 16S) and lack of
haplotype sharing in RAG1 and SACS, and in advertisement calls lacking distinct pulses vs. being clearly pulsed in
B. wittei (except in the Sambava population whose taxonomic status is in need of revision).
Description of the holotype. Adult male specimen with distinct femoral glands, in a good state of preservation
(Fig. 8). Tongue removed as tissue samples for molecular analysis. SVL 18.5 mm, for further measurements see
Table 1. Body slender; head longer than wide, of same width as body; snout rounded in lateral view, obtusely pointed
in dorsal and ventral views; nostrils directed laterally, protuberant, nearer to snout tip than to eye; canthus rostralis
rounded; loreal region slightly concave; tympanum distinct, round, its diameter 63% of eye diameter; supratympanic
fold distinct, curved above tympanum where it follows the tympanum outline; tongue absent, its shape therefore
not ascertainable; vomerine teeth not visible; choanae small, round, located toward the front of the palate; maxillary
teeth present. Arms slender, subarticular tubercles present, single; fingers without webbing; relative length of fingers
1<2<4<3, finger discs enlarged, nuptial pads absent. Hindlimbs slender; tibiotarsal articulation reaches center of eye
when the hind limb is adpressed along the body; lateral metatarsalia entirely separated by webbing; comparatively
small inner and much smaller outer metatarsal tubercles, which are present but not very distinct; webbing formula
1(traces), 2i(traces), 2e(1), 3i(2.5), 3e(1.75), 4i(2.75), 4e(3), 5(1.75); relative length of toes 1<2<5<3<4. Skin on the
upper surface smooth, without folds or ridges. Ventral skin smooth to slightly shagreened. Femoral glands distinct
and prominent.
After 19 years in preservative (Fig. 8), the dorsum is rather uniformly brownish, with a weakly recognizable
dark brownish inverted chevron, a faint small dark transverse bar on the forehead anterior to the eyes, and a very
weakly recognizable dark interorbital bar. A dark stripe runs from the nostril to the eye and continues broader
posterior to the eye, encompassing the entire tympanic region underneath the supratympanic fold. Underneath these
brown elements, a weakly expressed light frenal stripe is visible from the snout tip to the forelimb insertions. Fore-
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and hindlimbs dorsally with distinct and sharply delimited brown crossbars (three crossbars on thigh and four on
shank). Ventral side uniformly beige (unpigmented), throat being a bit lighter than chest, belly and limbs. In life,
color was very similar but somewhat more contrasted (Fig. 4A); whitish color was present on the throat; femoral
glands granules were somewhat yellowish (Fig. 4B).
Variation. ZSM 52/2006 exhibits a broad middorsal light band. Vomerine teeth are clearly visible in paratype
ZSM 2281/2007 from Ankarafantsika, also recognizable (although weakly expressed) in the paratypes from
Bemaraha. We observed substantial differences in body size among populations, with individuals from the type
locality Isalo being distinctly smaller than those from other sites. According to available data (Supplementary Table
2; available at DOI 10.5281/zenodo.8049142) male SVL across all populations ranges between 18.2–25.7 mm and
female SVL between 22.0–26.4 mm.
Etymology. Named after the Bara people, the ethnic group living in the area of Madagascar that includes the
type locality of the new species, the Isalo Massif. The name is used as a noun in apposition.
Natural history. A relatively common species in Madagascar, living in areas of a certain humidity such as
streams or swamps, including areas of secondary vegetation. During the rainy season, males call during day and
night from the low vegetation of these water bodies. In Isalo this species is commonly found on the ground of gallery
forests (e.g., Andriamanero, Sakamalio), along temporary rivers and in Pandanus swamps in the open savannah
(e.g., Ilakaka, Zahavola). Tadpoles develop in temporary ponds and individuals of this species have been observed
in egg-guarding behaviour both in Ankarafantsika (Fig. 4C) and Isalo (Mercurio et al. (2008). In the Sahamalaza
Peninsula, this species seems to be abundant and it is found along streams and ponds in intact forested areas as well
as in paddy fields in cleared areas. In Bemaraha, specimens were found in the leaf litter. In Kirindy, Glos (2003)
found the species in the largest out of 200 ponds studied, and observed males calling at night from a bush at the
water edge between 1–4 m above ground.
Vocalizations: Advertisement calls recorded on 29 January 1994 at Isalo (air temperature 21.5°C) consist of a
single short pulsatile note repeated in call series at rather regular intervals within series (Fig. 6). Each call exhibits
some irregular amplitude modulation, sometimes with 2–4 peaks recognizable within each call, with the second
peak usually having the highest energy. Numerical parameters of 23 analyzed calls of one male are as follows: call
duration (= note duration) 13–18 ms (15.2 ± 1.6 ms); inter-call intervals within regular call series 69–78 ms (74.8 ±
3.0 ms); duration of regular call series 2034 ms (n = 1); call rate within call series approximately 670 calls/minute;
dominant frequency 5813–6115 Hz (5968 ± 119 Hz); second dominant frequency peak around 3000 Hz; prevalent
bandwidth 2600–9500 Hz.
Advertisement calls recorded on 24 February 2001 at Ankarafantsika (air temperature 28°C) consist of a single
short pulsatile note repeated in call series at regular intervals within series (Fig. 6). In some calls (= notes) two
barely separated peaks of amplitude are recognizable, but call energy is distributed rather equally along the first two
thirds of the call’s duration, then dropping rapidly towards its end. Numerical parameters of 18 analyzed calls of
one male are as follows: call duration (= note duration) 19–28 ms (23.2 ± 2.5 ms); inter-call intervals within regular
call series 94–107 ms (97.3 ± 4.8 ms); duration of regular call series 2078 ms (n = 1); call rate within call series
approximately 500 calls/minute; dominant frequency 4694–4758 Hz (4735 ± 27 Hz); second frequency peak around
2370 Hz; prevalent bandwidth 1900–9700 Hz.
Distribution. Known from Isalo in the South-West up to Sahamalaza Peninsula in the North-West (see Fig. 2 for
genetically confirmed sites). The precise contact zone with B. wittei has not yet been identified but can be expected
to be located between Sahamalaza and Ambanja.
Blommersia sp. Ca12
Identity. This candidate species name refers to a new lineage discovered in the course of this study and found only
at two sites along the Sambirano river (Maevatanana and Antsirasira).
Morphology. Unknown. One voucher specimen from Antsirasira (ZSM 589/2001; see Table 1) could not be
genotyped as no tissue sample for molecular analysis was taken in the field from this specimen. This specimen
is an adult male with rather indistinct femoral glands and weakly expressed vomerine teeth. Photos in life are not
available.
Vocalizations: Advertisement calls were recorded on 31 January 2001 at Antsirasira (air temperature 26°C), but
INTEGRATIVE REVISION OF THE BLOMMERSIA WITTEI COMPLEX Zootaxa 5319 (2) © 2023 Magnolia Press · 195
call vouchers were not collected and calls are therefore not assignable to B. sp. Ca12 with full reliability. These calls
consist of a single, short, pulsed note repeated in call series at slightly irregular intervals within series (Fig. 7). Calls
(= notes) generally exhibit a pulse structure, although some pulses are not clearly separated but rather largely fused,
resulting in an irregular pulse pattern and varying number of countable pulses. Numerical parameters of 10 analyzed
calls of one male are as follows: call duration (= note duration) 34–66 ms (47.8 ± 10.8 ms); inter-call intervals within
regular call series 48–72 ms (61.4 ± 10.2 ms); pulses/note 3–10 (6.9 ± 2.3); duration of regular call series 1434 and
995 ms (n = 2); call rate within call series varied between 500–580 calls/minute; dominant frequency 5316–5598 Hz
(5440 ± 145 Hz); second frequency peak around 2850 Hz; prevalent bandwidth 2200–9000 Hz.
Discussion
The initial goal of this study was to clarify the taxonomic status of the candidate species Blommersia sp. Ca5 which
had been identified over 15 years ago based on its bioacoustic and strong genetic divergence from B. wittei (Glaw
& Vences 2007; Vieites et al. 2009). Unexpectedly, however, the taxonomy within the B. wittei-complex turned out
to be more convoluted than initially thought. We found the new species, herein formally named B. bara, occurring
not only in the West (Isalo, Makay and Bemaraha) but also in several localities in the North West of Madagascar,
including Ankarafantsika, which previously was thought to be inhabited by B. wittei (Glaw & Vences 2007) and
which is the origin of several paratypes of that species. We found B. wittei to be somewhat genetically heterogeneous,
with the population from the North East (Sambava) differing by a pairwise 16S distance of 1.2%, and distinct also
in bioacoustics (less pulsatile call structure) from other populations. Most importantly, however, we discovered
a previously unnoticed deep genetic lineage in the Sambirano valley, here named B. sp. Ca12, that differed both
by deep genetic divergence in 16S (up to 7% uncorrected genetic distance to B. wittei) and by very reduced allele
sharing in nuclear genes even from neighboring populations of B. wittei. Taken together, the biogeographic pattern
in the B. wittei complex likely reflects the complexity of speciation mechanisms in the Malagasy herpetofauna
(Brown et al. 2014): with a widespread species in the West and North West geographical regions (B. bara), a
widespread species in the Sambirano, North and North East regions containing at least one deep genetic lineage
(B. wittei), two species from the Comoran island of Mayotte that likely speciated in sympatry on this rather small
island (Glaw et al. 2019; Vieites et al. 2020), and one microendemic species only known from lowlands along the
Sambirano river (B. sp. Ca12).
Western Madagascar is characterized by relatively arid and highly seasonal climate (Jury 2022). Such conditions
could be hypothesized to be unsuitable for small, partially arboreal frogs which therefore would be restricted to relict
areas of more humid conditions e.g., in isolated massifs or in river gallery forests. However, our analysis revealed
low and poorly structured mitochondrial divergence in B. bara across a wide range from Isalo to Sahamalaza, at
an aerial distance of about 1000 km. The 16S distances between the main phylogroups of B. bara were at 1.6% or
below, which is only slightly more than the distances observed among populations of the larger-sized co-distributed
species Laliostoma labrosum (up to 1.1%; Pabijan et al. 2015), but considerably more than in Boophis doulioti
(<1%; Vences & Glaw 2002). Interestingly, despite this relatively weak genetic differentiation among B. bara
phylogroups, a quite substantial size difference was observed between B. bara individuals from Isalo vs. other sites.
No obvious reasons for these size differences are apparent, but they align with the situation in at least one other
frog species (Boophis obscurus) where Isalo populations are characterized by a distinctly smaller body size than
those occurring in rainforest (Glaw et al. 2010). To understand whether these are genetically fixed differences in
maximum size, or effects of e.g. timing of reproduction, speed of larval development, or resource availability, more
in-depth studies of the natural history of these populations is necessary. If, for instance, in one population specimens
metamorphose later or at smaller sizes at the end of the wet season than in other populations, these specimens will
have less time for growing until the onset of the subsequent reproductive season and thus reproduce at smaller sizes
(Scherz et al. 2023).
According to our revision, Blommersia wittei occurs in the Sambirano region, in the North, and North East
regions of Madagascar, but its populations show some differentiation across its range. For example, specimens from
Montagne d’Ambre appear to have more weakly expressed vomerine teeth than those from Ambanja, and especially,
specimens from Sambava differ by bioacoustics, a mitochondrial genetic divergence of 1.2%, and lack of haplotype
sharing in SACS and RAG1. The sole voucher specimen from this site also has poorly expressed femoral glands
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196 · Zootaxa 5319 (2) © 2023 Magnolia Press
and more extended webbing than voucher specimens from other sites. These differences may indicate taxonomic
distinctness of these populations, but current data are insufficient for final conclusions: (i) the observed mitochondrial
divergence is distinctly below the 3% used as a yardstick by Vieites et al. (2009) to define candidate species, and (ii)
there is no call voucher that could reliably link bioacoustics, genetics and morphology of the Sambava population.
Future material from Sambava and a fine-scale sampling of possible contact zones, e.g., northwards towards
Montagne d’Ambre, and eastwards towards the Sambirano region, are necessary to comprehensively understand
the level of differentiation within B. wittei.
The species status of Blommersia bara is obvious due to the concordance of genetic and bioacoustic divergence
to its morphologically and phylogenetically closest congener on Madagascar, B. wittei. The diagnostic advertisement
call structure of the new species appears to be constant across its range (verified for Isalo and Ankarafantsika). It
is striking that the newly discovered candidate species B. sp. Ca12, has only been found at two sites, the small
villages of Antsirasira and Maevatanana, both located in the lower Sambirano valley, whereas at two sites closer
to the Sambirano river mouth, Benavony and Ambanja, we found B. wittei. Antsirasira is also the only known site
from which the miniaturized species Wakea madinika is known (Vences et al. 2002), a deep mantellid lineage sister
to the genus Mantella and superficially similar to Blommersia. This calls for further exploration of this area, which
consists largely of anthropogenically transformed habitat covered with cocoa plantations, but may still harbor other
undetected microendemic species.
Acknowledgments
We acknowledge the help and assistance received from numerous colleagues, students and guides during a large
number of field expeditions in Madagascar, carried out between 2000–2018, in particular G. Aprea, P. Bora, H.
Enting, P. Eusebio Bergó, J. Glos, E. Z. Lattenkamp, F. Mattioli, V. Mercurio, J. and C. Patton, S. Penny, M. Puente,
L. Raharivololoniaina, J.E. Randrianirina, E. Rajeriarison, T. Rajoafiarison, S. M. Rakotomalala, L.M.S. Rakotozafy,
R.-D. Randrianiaina, T.J. Razafindrabe, G. M. Rosa, M. Thomas, and D. R. Vieites. Our research was carried out
in the framework of collaboration accords between the Zoological Institute of TU Braunschweig, the Zoologische
Staatssammlung München, the Mention Zoologie et Biodiversité Animale of the Université d’Antananarivo, and
the Ministere de l’Environnement, des Eaux et des Forêts of the Republic of Madagascar. We are grateful to the
Malagasy authorities and to Madagascar National Parks for research, collection, and export permits, and to MICET/
ICTE for logistic support. Portuguese National Funds through FCT (Fundação para a Ciência e a Tecnologia)
support the research contract to AC [2020.00823.CEECIND/CP1601/CT0003].
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Supplementary Materials. The following supporting information can be downloaded at the DOI landing page of
this paper.
SUPPLEMENTARY TABLE 1. GenBank accession numbers, voucher numbers and localities of all sequences
used in the molecular analysis.
SUPPLEMENTARY TABLE 2. Measurements of all species of Blommersia.
