Research Article |
Corresponding author: Miguel Vences ( m.vences@tu-bs.de ) Academic editor: Raffael Ernst
© 2022 Miguel Vences, Jörn Köhler, Angelica Crottini, Michael Hofreiter, Carl R. Hutter, Louis du Preez, Michaela Preick, Andolalao Rakotoarison, Loïs Rancilhac, Achille P. Raselimanana, Gonçalo M. Rosa, Mark D. Scherz, Frank Glaw.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Vences M, Köhler J, Crottini A, Hofreiter M, Hutter CR, du Preez L, Preick M, Rakotoarison A, Rancilhac L, Raselimanana AP, Rosa GM, Scherz MD, Glaw F (2022) An integrative taxonomic revision and redefinition of Gephyromantis (Laurentomantis) malagasius based on archival DNA analysis reveals four new mantellid frog species from Madagascar. Vertebrate Zoology 72: 271-309. https://doi.org/10.3897/vz.72.e78830
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The subgenus Laurentomantis in the genus Gephyromantis contains some of the least known amphibian species of Madagascar. The six currently valid nominal species are rainforest frogs known from few individuals, hampering a full understanding of the species diversity of the clade. We assembled data on specimens collected during field surveys over the past 30 years and integrated analysis of mitochondrial and nuclear-encoded genes of 88 individuals, a comprehensive bioacoustic analysis, and morphological comparisons to delimit a minimum of nine species-level lineages in the subgenus. To clarify the identity of the species Gephyromantis malagasius, we applied a target-enrichment approach to a sample of the 110 year-old holotype of Microphryne malagasia Methuen and Hewitt, 1913 to assign this specimen to a lineage based on a mitochondrial DNA barcode. The holotype clustered unambiguously with specimens previously named G. ventrimaculatus. Consequently we propose to consider Trachymantis malagasia ventrimaculatus Angel, 1935 as a junior synonym of Gephyromantis malagasius. Due to this redefinition of G. malagasius, no scientific name is available for any of the four deep lineages of frogs previously subsumed under this name, all characterized by red color ventrally on the hindlimbs. These are here formally named as Gephyromantis fiharimpe sp. nov., G. matsilo sp. nov., G. oelkrugi sp. nov., and G. portonae sp. nov. The new species are distinguishable from each other by genetic divergences of >4% uncorrected pairwise distance in a fragment of the 16S rRNA marker and a combination of morphological and bioacoustic characters. Gephyromantis fiharimpe and G. matsilo occur, respectively, at mid-elevations and lower elevations along a wide stretch of Madagascar’s eastern rainforest band, while G. oelkrugi and G. portonae appear to be more range-restricted in parts of Madagascar’s North East and Northern Central East regions. Open taxonomic questions surround G. horridus, to which we here assign specimens from Montagne d’Ambre and the type locality Nosy Be; and G. ranjomavo, which contains genetically divergent populations from Marojejy, Tsaratanana, and Ampotsidy.
Amphibia, Anura, archival DNA, Mantellidae, new species, phylogeography
Among the hyperdiverse anuran fauna of Madagascar, with about 370 scientifically named species (
One such secretive group of Madagascar frogs is the subgenus Laurentomantis in the genus Gephyromantis. These frogs have long been so poorly known that the monograph of
Besides their apparent rareness, most species of the subgenus Laurentomantis are remarkable frogs by their very spiny and tubercular dorsal integument and have long been considered a separate genus (e.g.,
The present study provides a comprehensive molecular assessment of the frogs in the subgenus Laurentomantis, based on DNA sequences of all samples collected over the past 20 years. To clarify the taxonomic status of lineages currently included in G. malagasius, we applied targeted enrichment sequencing to genetically characterize the 110-year-old holotype of this species, which unexpectedly was found to be conspecific with G. ventrimaculatus; this surprising result indicates that all recently collected frogs assigned to G. malagasius instead belong to four species new to science. We herein formally name and characterize these new species, based on an integration of molecular, bioacoustic and morphological information.
This study is based on voucher specimens and call recordings collected during various field campaigns in Madagascar between 1994–2017, and tissue samples collected since the year 2000. Upon collection in the field, frogs were anesthetized and euthanized by immersion in tricaine methanesulfonate (MS222) or chlorobutanol solution. We removed tissue samples for molecular analysis and stored them separately in 1.5 ml vials with 95% ethanol. Vouchers were then fixed in 95% ethanol (or in 12.5% formalin), preserved in 70% ethanol, and deposited at the Museo Regionale di Scienze Naturali, Torino (
The holotype of Microphryne malagasia Methuen and Hewitt, 1913,
Morphometric measurements were taken by MV with an accuracy of 0.1 millimeter with a manual caliper. The measurements and abbreviations used are: snout–vent length (SVL); maximum head width (HW); head length from tip of snout to posterior edge of mouth opening (HL); horizontal tympanum diameter (TD); horizontal eye diameter (ED); 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, 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); and tibia length (TIBL). We report webbing formula according to
We recorded vocalizations in the field using different types of tape recorders (Tensai RCR-3222, Sony WM-D6C) with external microphones (Sennheiser Me-80, Vivanco EM 238), digital recorders (Tascam DR05, Edirol R-09, Marantz PMD 661 MkII, Olympus LS-10, Zoom H5) with built-in microphones or external microphones attached (Sennheiser K6+ME-66, ME-67, MKH-8060), and in one case extracted sound files from a published audio CD (
Two molecular dataset were assembled to examine the genetic variation and differentiation within the subgenus Laurentomantis:
(1) All available samples were DNA barcoded using a fragment of the mitochondrial 16S rRNA gene, which has previously been used as standard marker for Malagasy frogs (
(2) To understand the concordance between the variation in mitochondrial and nuclear-encoded genes, we amplified fragments of two nuclear-encoded genes: sacsin (SACS) and recombination-activating gene 1 (RAG1). For SACS, we applied the nested PCR approach of
We purified PCR products with Exonuclease I and Shrimp Alkaline Phosphatase digestion, and the purified products along with sequencing primers were shipped to LGC Genomics (Berlin) for sequencing on automated capillary sequencing instruments. The 16S fragment was sequenced with the forward PCR primer only, SACS and RAG1 were sequenced with the PCR primers in both directions and the two strands combined. Chromatograms were checked for base-calling errors and edited with CodonCode Aligner 6.0.2 (Codon Code Corporation, Dedham, MA, USA) and newly determined sequences submitted to GenBank (accession numbers OM885271–OM885340 and OM897144–OM897211).
To obtain DNA sequences from the holotype of Gephyromantis malagasius, we applied targeted enrichment sequencing (
For library preparation, we used a single-stranded (ss-DNA) approach optimized for ancient and archival DNA (Gansauge et al. 2013,
The ss-DNA library of the G. malagasius holotype was then captured twice for the aforementioned target sequences using the Arbor Biosciences MyBaits kit (RNA-based in-solution sequence capture), with 14.5 μL of each indexed library in a 24 h reaction at a hybridisation temperature of 65°C, and following the MyBaits target enrichment protocol except reducing the bait volume to 2.75 μL and substituting the missing 2.75 μL in each reaction with nuclease-free water. After hybridization, the libraries were bound to streptavidin-coated magnetic beads, and the reactions washed and eluted according to the MyBaits kit protocol. We then performed PCR amplification in a reaction volume of 60 μL with the following PCR conditions: 120 s @ 95°C, then with an optimal cycle number determined using qPCR, 30 s@ 95°C, 45 s @ 60°C, 45 s @ 72°C, and final extension of 180 s @ 72°C. Amplifications were purified using a Min Elute PCR Purification Kit (Qiagen), with final elution in a total volume of 30 μL of 10 nM Tris-HCl, 0.05% TWEEN-20 solution (pH 8.0). This procedure was performed twice to increase target capture reactions success, as described in
After quality-trimming and adapter removal, all reads (duplicates not removed to keep information of read frequency) were compared against reference sequences of various Laurentomantis species using a custom script described in
We aligned the sequences for each locus individually in MEGA7 (
The 16S alignment was analyzed with a relatively simple (K2P) substitution model to avoid overparametrization for shallow branches, in a Maximum Likelihood analysis in MEGA 7 with NNI branch swapping, and 500 nonparametric bootstrap replicates to assess node support. We calculated uncorrected pairwise distances between 16S sequences using the program TaxI2, implemented in iTaxoTools (
The two nuclear-encoded genes (RAG1 and SACS) were analyzed separately from the mitochondrial gene and each other since our main interest was to understand concordance (or absence thereof) in the differentiation of unlinked genetic markers. We used a haplotype network visualization to graphically represent the relationship among alleles (haplotypes) of these genes. Haplotypes were estimated with the PHASE algorithm (
As in previous studies, we follow the general lineage concept (
The phylogenetic analysis of the 16S sequences of 88 individuals of Laurentomantis (plus one sequence of Gephyromantis (Vatomantis) webbi used as outgroup; total alignment length 508 nucleotides) revealed nine major clades separated by uncorrected pairwise distances >4%, which we consider as candidate species-level lineages (Fig.
Maximum Likelihood phylogenetic tree inferred from an alignment of 508 nucleotides of the mitochondrial 16S rRNA gene, in 88 individuals of Gephyromantis belonging to the subgenus Laurentomantis. A species of the subgenus Vatomantis (G. webbi) was used as the outgroup. Numbers at nodes are bootstrap values in percent (500 replicates; not shown if <50%). Colors correspond to main species-level lineages as discussed in the text.
The archival DNA analysis of the holotype of Microphryne malagasia yielded 3,481,149 raw sequence reads, of which 11,118 reads (0.32%) were retained after the filtering pipeline. These were used for downstream analysis for the sample, which led to a consensus sequence built with 10,497 reads. In the phylogenetic analysis, this consensus sequence clustered among specimens of the lineage commonly considered G. ventrimaculatus (e.g.,
The molecular evidence suggests that the name G. malagasius should take nomenclatural priority to refer to those frogs currently considered as G. ventrimaculatus. In order to assess whether the name Trachymantis malagasia ventrimaculatus Angel, 1935 represents a junior synonym of Microphryne malagasia Methuen and Hewitt, 1913, a comparison of the respective name-bearing types is necessary: (1) The lectotype of Trachymantis malagasia ventrimaculatus, examined previously by
As a consequence, no scientific name is available for those lineages that include the frogs with red ventral color on limbs that were assigned to Gephyromantis malagasius by
The red-legged individuals traditionally assigned to G. malagasius are placed by our analysis into four deep mitochondrial lineages, in this section provisionally named A–D. Lineage A (bootstrap support BS=94%) occurs at mainly low-elevation sites in Madagascar’s Southern and Northern Central East, from Ambohitsara (close to Ranomafana National Park) to Betampona and Befanjana Forest. Lineage B (BS=98%) occupies roughly the same region but appears to occur at mid-elevations, ranging from Ranomafana National Park to the Andasibe region and Anjozorobe. Lineage C (BS=95%) is only known from low elevations at the north-eastern localities Masoala and Makira. And finally, lineage D (BS=96%) is known from the Northern Central East, specifically from Sahafina and Betampona. These four lineages form a clade in our tree, which however did not receive strong bootstrap support. The smallest 16S uncorrected pairwise distances of 4.0–5.7% are found between lineages A and B, whereas the other comparisons among these four lineages yielded distances of 4.3–8.1%. Intra-lineage distances are up to 3% in lineage A, 1.8% in lineage B, 1.4% in lineage C, and 3.9% in lineage D.
Two further lineages correspond to the nominal species G. marokoroko and G. striatus and are confirmed by the analysis, with 100% and 99% bootstrap support, respectively. For G. marokoroko only individuals from the type locality (Vohidrazana) were available, and intra-lineage divergences are therefore negligible (<1%).
The final two species-level lineages revealed by our 16S tree correspond to a complex of enigmatic and poorly known taxa from northern Madagascar. They comprise specimens from Manarikoba forest in the Tsaratanana Massif that previously were assigned to G. horridus (
Analysis of the nuclear-encoded genes RAG1 (alignment length 475 nucleotides for 37 samples) and SACS (834 nucleotides for 30 samples) suggested genealogical concordance across unlinked markers in the differentiation of most lineages described in the previous paragraphs (no sequences of these two genes were available for G. morokoroko). The haplotype networks of both genes (Fig.
Maps showing locality records in Madagascar considered in this study, the majority of them based on molecular data. The left map shows records for G. marokoroko, G. horridus, G. malagasius, G. ranjamavo, and G. striatus in paler colors if they would benefit from further confirmation or taxonomic revision, as follows: pale yellow marks the type locality of G. ventrimaculatus (specimens not studied genetically) and records based on individuals phenotypically matching the types of ventrimaculatus (here considered to be a junior synonym of G. malagasius). Light green marks records assigned to G. striatus based on phenotype without genetic data. Light blue marks a record assigned to G. horridus tentatively due to the lack of genetic data from the type locality. Sky blue mark genetically divergent specimens here assigned to G. ranjomavo but in need of further study. The type locality of G. malagasius, Folohy, is here tentatively placed close to Toamasina, but its exact location is uncertain (north of Toamasina / east of Lake Alaotra, see species account).
Haplotype networks based on sequences of the nuclear-encoded genes RAG1 (475 nucleotides; 74 phased sequences corresponding to 37 individuals) and SACS (834 nucleotides; 60 phased sequences corresponding to 30 individuals). Colors correspond to sequences of individuals assigned to lineages based on mitochondrial DNA (Fig.
In this study we limit our morphological comparisons to those lineages for which we provide relevant novel taxonomic information compared to
The molecular data suggest a need to re-assess the identity of specimens from the Manarikoba Forest in the Tsaratanana Massif previously assigned to Gephyromantis horridus (
No photographs of the Manarikoba specimens in life were available, but we assign this population tentatively to G. ranjomavo based on the genetic similarity to the Ampotsidy individual and to the G. ranjomavo holotype. The two specimens of G. horridus from Nosy Be, from historical collections of the 19th century (Fig.
Specimens assigned to Gephyromantis horridus from Montagne d’Ambre National Park in life. A, B Adult female photographed in March 1994 (
The synonymy of G. ventrimaculatus with G. malagasius as supported by the genetic data from the holotype of G. malagasius is surprising. However, examination of the holotype of malagasius (Fig.
Individuals of lineages A, B, C and D (all previously assigned to G. malagasius) are characterized by reddish color on the posteriormost portion of the belly and the ventral side of the thighs (Figs
Advertisement calls in the subgenus Laurentomantis are multi-note calls, containing non-tonal notes. Calls are usually emitted in call series. The calls studied herein are all somewhat similar and simple in their structure, namely pulsatile or pulsed notes repeated at regular intervals, with only one exception where note repetition within calls is rather irregular. Despite the overall similar structure of advertisement calls in this species complex, detailed comparison of call parameters among the genetically identified clades reveals more or less pronounced differences in certain characters.
Among all calls analyzed, calls assignable to G. ranjomavo are longest in duration (1780–2526 ms) and differ from calls of G. horridus (call duration 543–618 ms) by notes consisting of a single pulse only (versus 2–4 pulses/note), low note repetition rate within calls (10.8–14.2 vs. 29.4–31.0 notes/second), and higher dominant frequency (2348–3204 vs. 1172–1369 Hz).
Calls of G. striatus contain very short single-pulse notes, but mainly differ from calls of G. ranjomavo by shorter call duration (439–1360 vs. 1780–2526 ms) and higher note repetition rate within calls (25.6–33.5 vs. 10.8–14.2 notes/second).
Calls of the recently described G. marokoroko contain multi-pulsed notes with clearly separated pulses. They are similar to calls of G. horridus in general character, but mainly differ by lower note repetition rate (18.2–19.6 vs. 29.4–31.0 notes/second) and higher dominant frequency (2916–3192 vs. 1172–1369 Hz).
Calls assignable to G. malagasius (previously reported under its synonym ventrimaculatus) contain multi-pulsed notes, with 2–10 pulses being partly fused and thus not always clearly distinguishable in oscillograms. Interestingly, calls from Vohiparara and Manombo generally agree in call duration, note duration and note structure, but differ considerably by a doubling number of notes per call and consequently much higher note repetition rate within calls of the Manombo population, which together with the sample from Befotaka Midongy forms a subclade within G. malagasius.
Among the calls assignable to the four unnamed lineages identified in the G. malagasius complex, calls of lineages A and C both contain multi-pulsed notes with a clear pulse structure, with the calls of lineage A containing a higher number of pulses per note (6–10 vs. 1–4) and exhibiting a lower note repetition rate within calls (20.8–21.6 vs. 43.5–54.1 notes/second) when compared to lineage C. Calls of lineage B from Andasibe, Ankeniheny and Vohidrazana contain single-pulse notes only and are unique among all calls analyzed in exhibiting rather variable note repetition rate within calls. Calls of lineage B from Ambatolahy (Ranomafana National Park) are generally in agreement with the latter, but differ slightly by exhibiting a regular note repetition within calls and more pronounced amplidute modulation. No call recordings are available from lineage D. For detailed call descriptions and comparison see species accounts and Table
Our results suggest the necessity of taxonomic changes in the subgenus Laurentomantis. We re-define G. malagasius by considering Trachymantis malagasia ventrimaculatus Angel, 1935 as a junior synonym of Microphryne malagasia Methuen and Hewitt, 1913. This leaves four genetic lineages (A–D) without scientific names. Of these, lineages A, B and C concordantly differed by a strong mitochondrial divergence (with uncorrected pairwise differences in the 16S gene > 4%), by a lack of haplotype sharing in two nuclear-encoded genes, and by temporal characteristics of advertisement calls. Only lineages B and D have limited haplotype sharing in one nuclear-encoded gene, but all four lineages have several morphological characters that enable their identification. This combined evidence suggests that lineages A, B, C and D represent biologically distinct species, which will be formally named in the respective species accounts in the following.
Hemimantis horrida Boettger, 1880: 282
This species was described based on a juvenile specimen from Nosy Be, and a second specimen (an adult female) from the type locality has previously been reported by
Morphometric measurements (all in mm) of voucher specimens of Gephyromantis belonging to the subgenus Laurentomantis newly examined for this study and identified on the basis of DNA sequence data. See
Voucher | Field number | Status | Sex | Locality | SVL | HW | HL | ED | HTD | END | NSD | NND | FORL | HAL | HIL | FOTL | FOL | TIBL | FGL | FGW |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
G. horridus | ||||||||||||||||||||
|
MSTEMP 1 | M | Montagne d‘Ambre | 33.5 | 13.2 | 13.8 | 4.8 | 2.3 | 3.7 | 2.3 | 3.5 | 24.3 | 11.5 | 53.6 | 23.8 | 15.6 | 15.8 | 7.7 | 4.8 | |
G. ranjomavo | ||||||||||||||||||||
|
MSZC 0163 | M | Ampotsidy | 26.6 | 10.5 | 10.6 | 4.0 | 2.2 | 2.8 | 2.0 | 2.8 | 19.0 | 9.2 | 41.9 | 18.6 | 11.7 | 12.6 | 3.1 | 2.6 | |
G. malagasius (=ventrimaculatus) | ||||||||||||||||||||
|
ZCMV 5497 | M | Manombo | 27.0 | 10.7 | 11.4 | 4.4 | 1.8 | 3.3 | 1.8 | 2.2 | 19.0 | 8.6 | 44.1 | 18.9 | 12.0 | 14.0 | 5.8 | 2.4 | |
|
ZCMV 5486 | F | Manombo | 29.8 | 11.7 | 13.0 | 4.3 | 2.0 | 3.2 | 2.1 | 2.9 | 24.7 | 11.4 | 51.4 | 22.8 | 14.5 | 16.9 | NA | NA | |
|
ZCMV 3362 | M | Ranomafana | 23.6 | 9.9 | 10.0 | 3.7 | 2.0 | 3.2 | 1.7 | 2.4 | 19.0 | 8.8 | 40.8 | 17.2 | 11.2 | 12.7 | 5.4 | 2.0 | |
G. matsilo sp. nov. (lineage A) | ||||||||||||||||||||
|
ZCMV 7234 | HT | M | Ambatoroma, Befanjana | 21.9 | 8.8 | 8.9 | 3.4 | 1.7 | 2.7 | 2.0 | 2.2 | 17.0 | 8.0 | 37.1 | 16.1 | 10.8 | 12.2 | 5.1 | 2.0 |
|
ZCMV 7300 | PT | F | Babitanety, Befanjana | 24.9 | 9.6 | 10.9 | 4.0 | 2.0 | 2.8 | 1.8 | 2.6 | 18.6 | 8.5 | 42.1 | 17.8 | 11.1 | 13.1 | NA | NA |
|
ZCMV 90 | PT | M | Ambohitsara | 21.7 | 8.9 | 9.3 | 3.5 | 1.7 | 2.7 | 1.2 | 2.1 | 18.1 | 8.8 | 39.7 | 17.5 | 11.4 | 11.9 | 5.8 | 2.3 |
G. fiharimpe sp. nov. (lineage B) | ||||||||||||||||||||
|
FGZC 5181 | HT | M | Mandraka | 23.4 | 8.9 | 9.6 | 3.2 | 1.7 | 3.2 | 2.0 | 2.4 | 17.5 | 8.1 | 41.0 | 18.4 | 12.2 | 13.0 | 3.7 | 1.6 |
|
FGMV 2002.415 | PT | F? | Ranomafana | 25.8 | 9.2 | 10.7 | 3.4 | 1.9 | 2.7 | 1.7 | 2.0 | 18.5 | 9.2 | 43.5 | 19.3 | 12.5 | 13.3 | NA | NA |
G. oelkrugi sp. nov. (lineage C) | ||||||||||||||||||||
|
FGZC 5300 | PT | F? | Masoala | 21.1 | 8.4 | 8.7 | 3.3 | 1.8 | 2.8 | 1.5 | 2.3 | 14.8 | 7.1 | 32.6 | 14.5 | 9.6 | 10.1 | NA | NA |
|
NA | PT | M | Ambodivoangy | 21.6 | 8.7 | 8.9 | 3.7 | 2.2 | 2.1 | 1.2 | 2.6 | 16.9 | 17.8 | 38.9 | 16.7 | 11.0 | 12.0 | 3.9 | 1.6 |
|
FGZC 4220 | HT | M | Ambodivoangy | 21.9 | 8.4 | 9.0 | 3.0 | 2.3 | 3.1 | 1.2 | 2.4 | 15.0 | 6.8 | 33.8 | 14.8 | 9.9 | 11.0 | 3.2 | 2.0 |
G. portonae sp. nov. (lineage D) | ||||||||||||||||||||
|
ACZCV 0032 | HT | M | Betampona | 22.9 | 9.0 | 10.7 | 3.4 | 1.7 | 2.9 | 1.6 | 2.7 | 17.0 | 8.2 | 36.1 | 16.1 | 10.5 | 11.5 | 5.5 | 2.5 |
|
ACZCV 0223 | PT | F | Betampona | 26.1 | 9.8 | 10.5 | 4.0 | 1.9 | 3.0 | 1.3 | 2.8 | 19.3 | 9.5 | 45.2 | 20.3 | 12.5 | 13.6 | NA | NA |
|
ACZCV 0030 | PT | F | Betampona | 25.1 | 9.8 | 10.5 | 3.4 | 1.5 | 3.4 | 2.0 | 2.6 | 18.9 | 8.7 | 40.5 | 17.7 | 11.3 | 12.5 | NA | NA |
|
ACZCV 0025 | PT | M | Betampona | 22.4 | 8.3 | 9.1 | 3.1 | 1.8 | 2.5 | 1.9 | 2.4 | 19.3 | 9.0 | 41.2 | 17.7 | 12.1 | 12.6 | 5.1 | 2.4 |
Comparative numerical parameters of analyzed advertisement calls of frogs in the subgenus Laurentomantis. Values are provided as range followed by mean ± standard deviation in parentheses.
species | locality | call duration [ms] | note duration [ms] | notes per call | note repetition rate within call [notes/second] | pulses per note | dominant frequency [Hz] |
G. horridus | Montagne d’Ambre | 543–618 (585.1±27.4) | 6–12 (9.4±1.7) | 17–19 (18.1±0.8) | 29.4–31.0 (29.9±0.7) | 2–4 (3.0±0.5) | 1172–1369 (1264±71) |
G. ranjomavo | Manarikoba Forest | 1780–2526 (2237.9±255.6) | 10–19 (13.6±3.1) | 23–33 (29.0±3.2) | 10.8–14.2 (12.7±1.0) | 1 | 2348–3204 (2909±355) |
G. striatus | Marojejy | 439–1360 (848.5±319.1) | 3–7 (5.1±0.8) | 17–45 (29.8±8.4) | 25.6–33.5 (31.1±3.2) | 1 | 3768–4153 (3983±161) |
G. marokoroko | Vohidrazana | 557–1203 (968.7±357.7) | 10–26 (17.4±5.0) | 12–28 (21.0±8.2) | 18.2–19.6 (18.8±0.6) | 2–5 (3.3±0.9) | 2411–2476 (2444±28) |
G. malagasius | Vohiparara/ Ranomafana | 360–465 (428.4±37.2) | 8–17 (12.6±2.9) | 8–10 (9.6±0.8) | 19.8–21.1 (20.5±0.5) | 2–10 (6.2±2.1) | 2606–3516 (3127±254) |
G. malagasius | Manombo | 348–446 (386.3±34.5) | 9–16 (12.2±1.9) | 16–20 (17.5±1.6) | 42.7–45.5 (44.7±1.1) | 2–9 (5.5±1.9) | 2916–3192 (3041±107) |
G. matsilo sp. nov. (lineage A) | Betampona | 355–660 (552.3±88.0) | 13–21 (16.2±2.5) | 8–15 (12.6±1.9) | 20.8–21.6 (21.2±0.3) | 6–10 (6.9±1.1) | 3090–3434 (3200±116) |
G. fiharimpe sp. nov. (lineage B) | Ankeniheny | 609–935 (798.8±110.8) | 7–9 (7.8±0.8) | 11–19 (15.5±2.4) | 14.9–25.6 (18.3±3.9) | 1 | 3638–3723 (3676±23) |
G. fiharimpe sp. nov. (lineage B) | Andasibe | 840–1503 (1179.3±232.7) | 5–12 (7.6±2.2) | 26–43 (36.9±7.3) | 18.2–37.4 (25.5±6.5) | 1 | 3649–4078 (3796±157) |
G. fiharimpe sp. nov. (lineage B) | Vohidrazana | 950–1764 (1405.6±290.4) | 2–7 (4.3±1.7) | 26–52 (40.7±11.5) | 18.8–54.1 (32.1±13.1) | 1 | 3726–3913 (3781±68) |
G. fiharimpe sp. nov. (lineage B) | Ambatolahy/Ranomafana | 916–1162 (1073.9±86.0) | 4–7 (6.2±0.9) | 27–34 (30.6±2.3) | 25.6–28.0 (26.8±1.0) | 1 | 3305–3736 (3523±145) |
G. oelkrugi sp. nov. (lineage C) | Ambodivoangy | 279–504 (350.9±88.4) | 3–10 (6.8±1.3) | 13–25 (16.9±4.4) | 43.5–54.1 (46.5±4.0) | 1–4 (2.9±0.7) | 3266–3882 (3539±190) |
The advertisement call of specimen
Gephyromantis ranjomavo Glaw and Vences, 2011: 122
This species was previously known from two specimens (
Adult males of Gephyromantis ranjomavo in life. A Dorsolateral and B ventral view of the holotype,
We redescribe the advertisement call genetically assignable to Gephyromantis ranjomavo recorded on 17 February 1997 at Manarikoba Forest, Tsaratanana Strict Nature Reserve (air temperature 17.5°C;
Mantidactylus striatus Vences, Glaw, Andreone, Jesu and Schimmenti, 2002: 203
This species was described from lower elevations of the Marojejy Massif, and specimens from Tsararano and Masoala were assigned to the species based on morphology (
The advertisement call was recorded on 22 February 1995 at Marojejy National Park (air temperature 25.0°C;
Gephyromantis marokoroko Hutter, Andriampenomanana, Andrianasolo, Cobb, Razafindraibe, Abraham and Lambert, 2022: 486
A recently described species from the Northern Central East of Madagascar (
The advertisement call was recorded on 6 January 2016 at Vohidrazana (air temperature 20.4°C; call voucher
Microphryne malagasia Methuen and Hewitt, 1913b: 55
Trachymantis malagasia ventrimaculatus Angel, 1935: 205; syn. nov.
As discussed above, we redefine G. malagasius based on molecular data from the holotype as containing those frogs previously considered as G. ventrimaculatus (e.g.,
Specimens of Gephyromantis malagasius (previously considered as G. ventrimaculatus, herein considered to be a junior synonym of G. malagasius), in life. A, B Adult male from Ranomafana in dorsolateral and ventral view, photographed 2006, probably corresponding to voucher specimen
A, B Preserved holotype of Gephyromantis malagasius (originally named Microphryne malagasia; specimen
The advertisement call of G. malagasius recorded at Vohiparara (
A second recording from 23 February 2007 (Manombo; air temperature estimated at 23–25°C) consists of a series of pulsed notes and is emitted in series at regular intervals (Fig.
Audiospectrogram and corresponding oscillogram of one advertisement call of Gephyromantis malagasius recorded at Vohiparara. The oscillogram below shows a 100 ms section of the call figured above, showing two notes and their repective pulse structure. Recording band-pass filtered at 1500–7500 Hz.
Audiospectrogram and corresponding oscillogram of one advertisement call of Gephyromantis malagasius recorded at Manombo. The oscillogram below shows a 100 ms section of the call figured above, showing four notes and their repective pulse structure. Recording band-pass filtered at 1500–6200 Hz.
G. malagasius as redefined here is known based on genetically confirmed records from (1) the type locality Folohy, (2) Ranomafana, (3) Manombo, and (4) Befotaka-Midongy. Furthermore, morphologically identified specimens with the typical ventral pattern are known from (5) Andasibe, (6) Ambohitantely (based on phenotypically identified specimens collected by one of us [APR]; see Fig.
The occurrence of individuals morphologically corresponding to G. malagasius as redefined herein in Andasibe is supported by three records: one voucher specimen collected by Denis Vallan and reported in
The species epithet is derived from the Malagasy adjective matsilo (spiny) and refers to the spiny tubercles on the dorsum of this frog. The name is used as a noun in apposition.
A member of the subfamily Mantellinae based on the presence of intercalary elements between terminal and subterminal phalanges of fingers and toes (verified externally), and on the absence of nuptial pads and presence of femoral glands in males. Assigned to the subgenus Laurentomantis in the genus Gephyromantis based on the strongly tubercular dorsal skin, absence of foot webbing, completely connected lateral metatarsalia, and molecular phylogenetic relationships. The new species differs from all nominal species of the subgenus Laurentomantis as follows: From G. horridus by smaller body size (male SVL 21.7‒21.9 mm vs. 33.5 mm) and absence of a dorsal pattern of two blackish transverse patches (vs. presence); from G. ranjomavo by slightly smaller body size (male SVL 21.7‒21.9 mm vs. 23.5–28.1 mm), absence of light brown to orange-brown color covering limbs dorsally (vs. presence), and absence of a tibial gland (vs. presence); from G. striatus by absence of a vertebral stripe posteriorly on dorsum (vs. presence) and a more strongly tubercular dorsal skin; from G. malagasius (as redefined herein) by absence of a distinct bluish gray pattern on a dark venter (vs. presence); and from G. marokoroko by a more coarsely tubercular dorsal skin, presence of red color ventrally on limbs (vs. absence), absence of orange spots and vermiculations on dorsum (vs. presence), and absence of gray to whitish color on vocal sac (vs. presence). Also distinguished from G. horridus and G. striatus by longer notes in advertisement calls (13–21 vs. 3–12 ms). Furthermore, from all nominal Laurentomantis species distinguished by the presence of bright red color in the inguinal region and probably ventrally on limbs and posterior belly in life (see. Fig.
Adult male in good state of preservation (Fig.
After twelve years in preservative, dorsal coloration of head and body uniformly dark brown, with darker crossbands on hind- and forelimbs. Posterodorsal surface of thigh with a small pigmentless patch near the knee joint, this area presumably corresponding to reddish color in life. Ventrally cream, with a rather faint and irregular brown marbling, which is more contrasted on the ventral surface of the hindlimbs.
Preserved holotype specimens of the four new Gephyromantis species in dorsal and ventral view. A, B G. matsilo sp. nov. (lineage A),
All photographed and examined specimens of G. matsilo lack a tibial gland. The male
The advertisement call (Fig.
Based on genetically verified records, the distribution area spans a south-north direction from: (1) Ambohitsara to (2) Betampona, (3) Ambodiriana, (4) two sites in Befanjana, and (5) Sahavontsira. The known elevational range of the species spans from near sea level (Ambodiriana, 53 m a.s.l.) to approximately 294 m a.s.l. (Ambohitsara). Very little is known on the natural history of this species. Despite intensive sampling, only three individuals of this species have been collected at Betampona, where the most commonly found Laurentomantis species is the lineage D. All individuals were collected in rainforest habitat.
The species epithet is derived from the Malagasy words fihary (gland) and fe (leg) which written together become fiharim-pe according to Malagasy grammar. The name makes reference to the tibial gland of the species, and is used as a noun in apposition.
A member of the subfamily Mantellinae based on the presence of intercalary elements between terminal and subterminal phalanges of fingers and toes (verified externally), and on the absence of nuptial pads and presence of femoral glands in males. Assigned to the subgenus Laurentomantis in the genus Gephyromantis based on the strongly tubercular dorsal skin, absence of foot webbing, completely connected lateral metatarsalia, and molecular phylogenetic relationships. The new species differs from all nominal species of the subgenus Laurentomantis as follows: From G. horridus by smaller body size (male SVL 22.0–24.0 mm vs. 33.5 mm), less expressed tubercles and ridges on the dorsum, and absence of a dorsal pattern of two blackish transverse patches (vs. presence); from G. ranjomavo by the absence of light brown to orange-brown color covering limbs dorsally (vs. presence), less expressed tubercles and ridges on the dorsum, and possibly slightly smaller body size (male SVL 22.0–24.0 mm vs. 23.5–28.1 mm); from G. striatus by absence of a vertebral stripe posteriorly on dorsum (vs. presence) and a more strongly tubercular dorsal skin; from G. malagasius (as redefined herein) by absence of a distinct bluish gray pattern on a dark venter (vs. presence), and less expressed tubercles and ridges on the dorsum; from G. marokoroko by a somewhat less tubercular dorsal skin, presence of red color ventrally on limbs (vs. absence), absence of orange spots and vermiculations on dorsum (vs. presence), and absence of gray to whitish color on vocal sac (vs. presence). Furthermore, differing from all the aforementioned species by the presence of light reddish color in the inguinal region and ventrally on limbs and posterior belly in life (vs. absence), and from all species except for G. ranjomavo by the presence of a tibial gland (vs. absence), and by a substantial genetic divergence (>6% uncorrected pairwise distance in the 16S gene).
According to the molecular phylogeny, G. fiharimpe is closely related to G. matsilo described above, and may be its sister species. It differs from G. matsilo by presence of a tibial gland (vs. absence), shorter note duration in advertisement calls (2–12 ms vs. 13–21 ms), a less tubercular dorsal skin, and an uncorrected genetic distance in the 16S gene of 4.0–5.7%.
For a distinction from the other new species described in the following (lineages C and D), see the diagnoses in the respective species accounts below.
Adult male in good state of preservation (Fig.
After five years in preservative, dorsal coloration of head and body uniformly dark brown, with darker crossbands on hind- and forelimbs. Posterodorsal surface of thigh largely pigmentless whitish/cream: this area in life was presumably reddish. Ventrally, throat, chest and anterior belly uniformly blackish brown, posteror belly fading into gray-cream. Ventral side of hindlimbs pigmentless cream, probably corresponding to reddish color in life.
A tibial gland is visible in all examined adult specimens, as well as in additional photographed individuals (
The advertisement call (Fig.
Specimens of Gephyromantis fiharimpe sp. nov. (lineage B) in life, in dorsolateral and ventral views. A, B Adult male, probably from An’Ala, photographed 1996. C–E Adult male from Andasibe, photographed 1994. F, G Adult male from Ranomafana, photographed 2003. Individuals not reliably attributable to a voucher specimen. Not to scale.
Audiospectrogram and corresponding oscillogram of one advertisement call of Gephyromantis fiharimpe sp. nov. recorded on 18 February 1994 at Ankeniheny. The oscillogram below shows a 100 ms section of the call figured above, showing two notes and their repective amplitude structure. Recording band-pass filtered at 1500–8000 Hz.
Additional calls recorded on 1 January 1994 at Andasibe (temperature unknown) generally agree in characteristics with those reported from Ankeniheny, including evident variation in note repetition rate within calls. The main differences compared to calls from Ankeniheny are longer call duration and higher number of notes per call. Numerical call parameters of 7 analyzed calls are as follows (range followed by mean ± standard deviation in parentheses): call duration 840–1503 ms (1179.3 ± 232.7 ms); note duration 5–12 ms (7.6 ± 2.2 ms); number of notes per call 26–43 (36.9 ± 7.3); note repetition rate within calls 18.2–37.4 notes/second (25.5 ± 6.5 notes/second); call repetition rate within call series approximately 25–27 calls/minute; dominant frequency 3649–4078 Hz (3796 ± 157 Hz); prevalent bandwidth 2500–4800 Hz.
Calls recorded on 12 January 2015 at Vohidrazana (air temperature 18.5°C; call voucher
Calls recorded on 13 February 2015 at Ambatolahy, Ranomafana National Park (air temperature 17.6°C; call voucher
Based on genetically verified records, the distribution area includes in a south-north direction the localities (1) Vevembe, (2) Ranomafana, (3) Mandraka, (4) Maromizaha, (5) Andasibe, (6) Ambatovy, (7) Vohidrazana, (8) Anjozorobe, and (9) Ambohitantely. Probably the species is also present at Ankeniheny. The known elevational range of the species spans from 580 m a.s.l. (Vevembe) to approximately 1500 m a.s.l. (Ambohitantely). The species apparently is restricted to rather intact rainforest habitat. Males call at night from perch heights of 5–50 cm in the low understory vegetation, not concentrated around water bodies.
The specific epithet is a patronym for Christopher Roland Oelkrug in recognition of his support for biodiversity research and nature conservation through the BIOPAT initiative.
A member of the subfamily Mantellinae based on the presence of intercalary elements between terminal and subterminal phalanges of fingers and toes (verified externally), and on the absence of nuptial pads and presence of femoral glands in males. Assigned to the subgenus Laurentomantis in the genus Gephyromantis based on the strongly tubercular dorsal skin, absence of foot webbing, completely connected lateral metatarsalia, and molecular phylogenetic relationships. The new species differs from all nominal species of the subgenus Laurentomantis as follows: From G. horridus by smaller body size (male SVL 21.6–21.9 mm vs. 33.5 mm), and absence of a dorsal pattern of two blackish transverse patches (vs. presence); from G. ranjomavo by the absence of light brown to orange-brown color covering limbs dorsally (vs. presence), a slightly smaller body size (male SVL 21.6–21.9 mm vs. 23.5–28.1 mm) and absence of a tibial gland (vs. presence); from G. striatus by absence of a vertebral stripe posteriorly on dorsum (vs. presence); from G. malagasius (as redefined herein) by absence of a distinct bluish gray pattern on a dark venter (vs. presence); from G. marokoroko by a more coarsely tubercular dorsal skin, presence of red color ventrally on limbs (vs. absence), absence of orange spots and vermiculations on dorsum (vs. presence), and absence of gray to whitish color on vocal sac (vs. presence). Furthermore, differing from all the aforementioned species by the presence of bright red color in the inguinal region and ventrally on limbs and on a small portion of posterior belly in life (vs. absence), and by a substantial genetic divergence (>6% uncorrected pairwise distance in the 16S gene).
According to the molecular phylogeny, G. oelkrugi is closely related to G. fiharimpe and G. matsilo described above. It differs from G. fiharimpe by the absence of a tibial gland (vs. presence), a more strongly tubercular dorsal skin, and brighter red ventral color that appears not to extend much on posterior belly (vs. less bright light red color extending onto posterior belly), probably by a faster note repetition rate in advertisement calls (43.5–54.1 vs. 14.9–37.4 notes/second in most recordings; but up to 54.1 in one recording of G. fiharimpe), and an uncorrected 16S genetic distance of 4.3–5.5%. The new species is morphologically most similar to G. matsilo but differs by a faster note repetition rate (43.5–54.1 vs. 20.8–21.6 notes/second) and a shorter note duration (3–10 ms vs. 13–21 ms) in advertisement calls, and an uncorrected 16S genetic distance of 4.3–5.5%.
For a distinction from the fourth new species described in the following (lineage D), see the diagnosis in the respective species account below.
Adult male in good state of preservation (Fig.
After eleven years in preservative, dorsal coloration of head and body uniformly dark brown, with darker crossbands on hind- and forelimbs. Posterodorsal surface of thigh with a large pigmentless patch in its distal part, this area presumably corresponding to reddish color in life. Ventrally, whitish, with some dark spotting along the lower lip, two symmetrical brown patches on chest, and brown pigment on distal part of thighs.
The coloration in life of several specimens is shown in Fig.
The advertisement call (Fig.
Specimens of Gephyromantis oelkrugi sp. nov. (lineage C) from the type locality, Ambodivoangy, in life. A, B, C Adult male (FGZC 4201) in frontal, dorsolateral and ventral view. D, E Adult male holotype (
Audiospectrogram and corresponding oscillogram of one advertisement call of the holotype of Gephyromantis oelkrugi sp. nov. (
Based on genetically verified records, the distribution area is restricted to (1) Ambodivoangy close to the Makira Reserve, and the Masoala Peninsula where it has been found at various sites, including (2) Andasin’i Governera, (3) Andranobe, (4) Ambatoledama, (5) Amparihy, (6) Antanamahalana, (7) Ambohitsitondroina, and (8) near hotel “Eco-Lodge chez Arol”. A species apparently restricted to lowland rainforest habitat. The holotype was found calling sitting on a twig of a shrub plant approximately 30 cm above the ground.
The specific epithet is a matronym for Ingrid Porton, our dear friend and colleague. Ingrid is a primatologist and Vice-Chair of Madagascar Fauna and Flora Group, and this honor is a recognition of her continuous support to the study of the unique biodiversity of Betampona Strict Natural Reserve, and her overall commitment to the conservation of Malagasy ecosystems.
A member of the subfamily Mantellinae based on the presence of intercalary elements between terminal and subterminal phalanges of fingers and toes (verified externally), and on the absence of nuptial pads and presence of femoral glands in males. Assigned to the subgenus Laurentomantis in the genus Gephyromantis based on the strongly tubercular dorsal skin, absence of foot webbing, completely connected lateral metatarsalia, and molecular phylogenetic relationships. The new species differs from all nominal species of the subgenus Laurentomantis as follows: From G. horridus by smaller body size (male SVL 22.4–22.9 mm vs. 33.5 mm), and absence of a dorsal pattern of two blackish transverse patches (vs. presence); from G. ranjomavo by the absence of light brown to orange-brown color covering limbs dorsally (vs. presence), a slightly smaller body size (male SVL 22.4–22.9 mm vs. 23.5–28.1 mm) and absence of a tibial gland (vs. presence); from G. striatus by absence of a vertebral stripe posteriorly on dorsum (vs. presence); from G. malagasius (as redefined herein) by absence of a distinct bluish gray pattern on a dark venter (vs. presence); from G. marokoroko by a more coarsely tubercular dorsal skin, presence of red color ventrally on limbs (vs. absence), absence of orange spots and vermiculations on dorsum (vs. presence), and absence of gray to whitish color on vocal sac (vs. presence). Furthermore, differing from all the aforementioned species (with the exeption of G. fiharimpe) by the presence of bright red color in the inguinal region and ventrally on limbs and on a small portion of posterior belly in life (vs. absence), and by a substantial genetic divergence (>6% uncorrected pairwise distance in the 16S gene).
According to the molecular phylogeny, G. portonae sp. nov. is related to G. fiharimpe, G. matsilo, and G. oelkrugi described above, but appears to be the genetically most divergent species of this complex, possibly representing the sister taxon of a clade composed by the other three species. It differs from G. fiharimpe by the absence of a tibial gland (vs. presence), a more strongly tubercular dorsal skin, and brighter red ventral color (vs. less bright light red color), and an uncorrected 16S genetic distance of 6.1–8.1%; from the sympatric G. matsilo by third toe distinctly longer than fifth (vs. of similar length or slightly longer) and an uncorrected 16S genetic distance of 5.9–7.3%; and from G. oelkrugi by a dorsal skin composed of mostly rather large and rounded tubercles (vs. equally prominent but smaller and more pointed tubercles), and an uncorrected 16S genetic distance of 5.1–7.5%.
Adult male in good state of preservation (Fig.
After eight years in preservative, dorsal coloration of head and body dark brown, washed with lighter brown color especially on the large tubercles. Darker brown crossbands on hind- and forelimbs. Ventral and posterodorsal surface of thigh with larger whitish/cream areas, which were presumably reddish in life. Ventrally, throat light brown to grayish with a few small light spots; chest brown; belly marbled with brown and light color.
The specimens examined and photographed all agree well with each other in external morphology. Several photographed individuals have very large and coarse dorsal tubercles, of less spiny appearance compared to G. oelkrugi and G. matsilo (see especially Fig.
Although the advertisement call has never been recorded and analysed, one male has been heard calling. Call sounded unmotivated with low amplitude notes.
Based on genetically verified records, the distribution area is restricted to (1) Sahafina and (2) Betampona. Calling males were observed in Betampona in a muddy bank close to slow running brooks, but the species is often encountered on the leaflitter in dense forest, along slopes and ridges of steep hills, far from water. The tadpole is unknown. A poorly known species apparently restricted to lowland rainforest habitat ranging from 200 m a.s.l. to approximately 500 m a.s.l. of elevation.
Frogs of the subgenus Laurentomantis are among the least known anurans in Madagascar. For instance, their reproductive biology is almost completely unknown: no report of their mating behavior has been published, no clutches have been found, and only for one species (G. malagasius, under the name G. ventrimaculatus) have tadpoles been described (
Although we assigned all known samples and specimens of Laurentomantis to species, several taxonomic enigmas still require revision. This mainly concerns G. horridus and G. ranjomavo where the populations assigned to G. ranjomavo present a substantial genetic divergence, possibly indicating a species complex (additional material and bioacoustic data from across the range of G. ranjomavo are needed to clarify this question). Conversely, since no fresh collections (and thus no male specimens and no molecular data) of G. horridus from its type locality Nosy Be are available, the identity of this species remains uncertain. Based on morphological similarity, we have followed previous studies (e.g.,
The molecular allocation of the holotype of Microphryne malagasia to the lineage previously considered as G. ventrimaculatus is perhaps the most suprising result obtained by DNA barcode fishing in historical types of Malagasy anurans (see:
According to the expanded data presented in this study, only two species of Laurentomantis have a distinct tibial gland: G. fiharimpe and G. ranjomavo. Given that for all species in the subgenus we have examined males emitting advertisement calls in the rainy season (thus sexually mature and reproductively active), and the presence or absence of these males could unambiguously be ascertained in all of them, it is obvious that absence of glands is not strictly linked to seasonal or ontogenetic effects. Considering the scarcity of material for some species, we cannot fully exclude individual, sexual, geographical or seasonal variation of this character, but it seems clear that it is genuinely absent in most species. Although our phylogenetic tree (Fig.
Despite a substantially improved understanding of the diversity of Laurentomantis, some species of this subgenus continue to be among the least collected frogs in Madagascar. For example, the recently described G. marokoroko (
Fieldwork in Betampona was funded by the Saint Louis Zoo’s Field Research for Conservation program (FRC# 16-09; FRC# 12-12) of the Wildcare Institute. Portuguese National Funds through FCT (Fundação para a Ciência e a Tecnologia) support the research project PTDC/BIA-EVL/31254/2017 and the 2020.00823.CEECIND/CP1601/CT0003 research contract for Angelica Crottini. This work was carried out in framework of collaboration accords among the author’s institutions, the Department of Animal Biology of the University of Antananarivo, and the Ministry of the Environment of the Republic of Madagascar. Various field expeditions were supported by the Volkswagen Foundation, Deutsche Forschungsgemeinschaft, and Deutscher Akademischer Austauschdienst. Sequencing of archival DNA was supported by a grant of the Deutsche Forschungsgemeinschaft (VE247/16-1 – HO 3492/6-1) in the framework of the Taxon-Omics priority program. Additionally, we thank the USA National Science Foundation (Graduate Research Fellowship award numbers 1540502, 1451148, 0907996 to CRH).
The authors have declared that no competing interests exist.
MV: Conceptualization, Investigation, Writing - Original draft. JK, Investigation, Writing – Original draft. FG, MDS, AC: Resources, Data Curation, Writing – Review and Editing. MP, MH, Methodology, Investigation, Writing – Review and Editing. CRH, LdP, AR, APR, GMR, Resources, Writing – Review and Editing.
We are grateful to numerous colleagues, students and guides who contributed to assembling the samples used in this study during field work, in particular F. Andreone, J. Forster, K. Glaw, T. Glaw, P.-S. Gehring, Georges, D.J. Harris, H. Lava, J. Nöel, M. Pabijan, D. Prötzel, M. Puente, L. Raharivololoniaina, L. Randriamanana, M. Randriamialisoa, J.E. Randrianirina, R.D. Randrianiaina, F.M. Ratsoavina, E. Rajeriarison, T. Rajoafiarison, D. Salvi, E. Scanarini, M. Thomas, J.H. Velo, and D.R. Vieites. M. Kondermann and G. Keunecke assisted with laboratory work. L. Mashinini granted access to the collection of the Transvaal Museum in Pretoria. S. Schweiger and G. Gassner (
Table S1
Data type: .docx
Explanation note: Summary of uncorrected pairwise distances between species in the subgenus Laurentomantis. For each pairwise between-species comparison, the minimum and maximum value from pairwise comparisons between individuals is reported. Cells marked in green report values of intra-specific distances (i.e., distances between individual sequences assigned to the same species, minimum and maximum). Yellow cells mark comparisons between lineages A–D, corresponding to the four new species described in this study.
Figure S1
Data type: .jpeg
Explanation note: Sections of the assembly of Illumina reads obtained by targeted capture from DNA extracted from the holotype of Microphryne malagasia (