ArticlePDF Available

Natural history and conservation of the rediscovered Hula painted frog, Latonia nigriventer

Authors:
  • Ruppin Academic Center, Michmoret, Israel

Abstract and Figures

Dramatic global amphibian declines have recently led to an increased concern for many species of this animal class. The enigmatic Hula painted frog (Latonia nigriventer), the frst amphibian to be declared extinct but unexpectedly rediscov- ered in 2011, has remained one of the rarest and most poorly understood amphibians worldwide. Gathering basic biological information on this species, along with an understanding of its disease-related threats remains fundamental for developing risk assessments and conservation strategies. Our surveys in recent years confrmed that L. nigriventer is a localised species with elusive habits. The species appears to follow an opportunistic breeding phenology and has a tadpole morphology similar to its well-studied sister group Discoglossus. However, the adults’ extended annual presence in the aquatic habitat is a major dif- ference from species of Discoglossus. We detected the am- phibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), in northern Israel and on Hula painted frogs but did not observe any signs of chytridiomycosis in this species. Our preliminary data on aspects of the innate immunity of L. nigriventer suggest that the skin mucosome of this species contains antimicrobial peptides and a bacterial community differing from other syn- topic frogs (Pelophylax bedriagae). The combined knowledge of both natural history and innate immunity of L. nigriventer provides valuable insights to direct future research and conser- vation management of this critically endangered frog species.
Content may be subject to copyright.
Contributions to Zoology, 86 (1) 11-37 (2017)
Natural history and conservation of the rediscovered Hula painted frog, Latonia nigriventer
R.G. Bina Perl1, 2, 8, Sarig Gafny2, Yoram Malka3, Sharon Renan4, Douglas C. Woodhams5, Louise Rollins-Smith6, 7,
James D. Pask6, Molly C. Bletz1, Eli Geffen4, Miguel Vences1
1 Division of Evolutionary Biology, Zoological Institute, Braunschweig University of Technology, Mendelssohnstr. 4,
38106 Braunschweig, Germany
2 School of Marine Sciences, Ruppin Academic Center, Michmoret 40297, Israel
3 Israel Nature and Parks Authority, 3 Am Ve’Olamo Street, Jerusalem 95463, Israel
4 Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
5 Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, Massachusetts, 02125 USA
6 Departments of Pathology, Microbiology and Immunology and of Pediatrics, Vanderbilt University School of
Medicine, Nashville, Tennessee, 37232 USA
7 Department of Biological Sciences, Vanderbilt University, Nashville Tennessee, 37235 USA
8 E-mail: r.perl@tu-braunschweig.de
Key words: Amphibia, antimicrobial peptides, Anura, Batrachochytrium dendrobatidis, habitat, skin microbiota,
tadpole
Abstract
Dramatic global amphibian declines have recently led to an
increased concern for many species of this animal class. The
enigmatic Hula painted frog (Latonia nigriventer), the rst
amphibian to be declared extinct but unexpectedly rediscov-
ered in 2011, has remained one of the rarest and most poorly
understood amphibians worldwide. Gathering basic biological
information on this species, along with an understanding of its
disease-related threats remains fundamental for developing risk
assessments and conservation strategies. Our surveys in recent
years conrmed that L. nigriventer is a localised species with
elusive habits. The species appears to follow an opportunistic
breeding phenology and has a tadpole morphology similar to its
well-studied sister group Discoglossus. However, the adults’
extended annual presence in the aquatic habitat is a major dif-
ference from species of Discoglossus. We detected the am-
phibian chytrid fungus, Batrachochytrium dendrobatidis (Bd),
in northern Israel and on Hula painted frogs but did not observe
any signs of chytridiomycosis in this species. Our preliminary
data on aspects of the innate immunity of L. nigriventer suggest
that the skin mucosome of this species contains antimicrobial
peptides and a bacterial community differing from other syn-
topic frogs (Pelophylax bedriagae). The combined knowledge
of both natural history and innate immunity of L. nigriventer
provides valuable insights to direct future research and conser-
vation management of this critically endangered frog species.
Contents
Introduction ...................................................................................... 11
Material and methods ..................................................................... 14
Field surveys and sampling .................................................... 14
Adult and juvenile morphometrics and colour .................. 14
Tadpole description .................................................................. 14
Vocalisations ............................................................................. 15
Bacterial communities and Bd/Bsal screening ................. 15
Skin peptides collection and anti-Bd growth
inhibition assays ........................................................................ 16
Results ................................................................................................ 16
Range and habitat ..................................................................... 16
Activity patterns and movement ............................................ 17
Adult morphology ..................................................................... 17
Tadpole morphology ................................................................ 19
Reproduction ............................................................................. 20
Vocalisations ............................................................................. 22
Bd/Bsal screening, skin bacterial community,
and defensive skin peptides ................................................... 23
Discussion ........................................................................................ 24
Acknowledgements ........................................................................ 29
References ........................................................................................ 29
Appendices ....................................................................................... 34
Introduction
The Hula painted frog (Latonia nigriventer (Mendels-
sohn and Steinitz, 1943)) was rst discovered in the
eastern part of the Hula Valley in northern Israel on 22
March 1940 (Mendelssohn and Steinitz, 1943; Fig. 1
A–B) and appears to be strictly endemic to this area.
When the draining of the Lake Hula marshes in the
1950s caused the extinction or local disappearance of
several species of this area (Dimentman et al., 1992;
12 Bina Perl et al. – Natural history and conservation of the Hula painted frog
Goren and Ortal, 1999; Payne, 2012), the dramatic
ecosystem change most likely also caused the decline
of L. nigriventer. After the then last known individual
was collected in 1955 (Steinitz, 1955), the species was
the rst of 37 anuran species to be ofcially declared
extinct by the IUCN in 1996 (Baillie et al., 2010).
Almost 40 years after the drainage, the Hula Valley
was partly re-ooded and signs of ecological restora-
tion were observed (Kaplan et al., 1998; Cohen-Sha-
cham et al., 2011; Kaplan, 2012). Subsequently, in
No
vember 2011, a single postmetamorphic individual
of L. nigriventer was discovered in the Hula Nature
Reserve that protects the last remnant of the former
Lake Hula marshes. In the following two years, thir-
teen more postmetamorphic individuals were cap-
tured. All those recently discovered individuals were
observed within this same tiny patch of habitat (Biton
et al., 2013; SG, YM, EG personal observations).
Painted frogs (Discoglossus) belong to one of the
oldest anuran clades (Alytidae) which dates back to the
Jurassic. The Hula painted frog was originally described
in this genus and had been classied as such until 2013.
However, based on recent genetic and osteological anal-
yses the species was found to be a sister to a clade of all
remaining Discoglossus and was assigned to Latonia, a
genus of fossil giant frogs known from the Miocene
Fig. 1. Habitat of Latonia nigriventer.
A) Relief map of the Hula Valley (legend
shows surface elevation in meters);
B)
satellite image of the Hula Valley
showing the location of the Hula Nature
Reserve (indicated by the white circle);
C) water body close to the nding sites
within the Hula Nature Reserve; D) typ-
ical nding site within the Hula Nature
Reserve; ditch in Yesod HaMa’ala after
(E) and before (F) the vegetation was
trimmed by local authorities.
13Contributions to Zoology, 86 (1) – 2017
through Pleistocene but considered to be extinct since
(Biton et al., 2013). The fact that L. nigriventer is the
sole surviving species of an ancient clade, and thus
alone represents a high proportion of alytid phyloge-
netic diversity, calls for special attention to ensure its
survival. To date, almost nothing is known about the
natural history of this ancient, only recently rediscov-
ered frog that still ranges among the rarest amphibians
in the world. Apart from the brief tadpole and adult de-
scriptions published by Mendels sohn and Steinitz
(1943), the available information on its sister group Dis-
coglossus has remained the only reference to speculate
about the natural history of L. nigriventer.
Effective conservation of this unique species re-
quires precise knowledge on its distribution range, basic
ecology, and reproductive biology to (i) allow for a de-
nition of adequate conservation priorities targeting
aquatic and terrestrial landscape structures in the high-
ly transformed Hula Valley, and (ii) enable future in-situ
or ex-situ breeding initiatives. In addition, knowledge
about the presence of the chytridiomycete fungus, Ba-
trachochytrium dendrobatidis Long core, Pessier and
Nichols, 1999 (Bd), in the Hula Valley as well as on in-
nate skin defensive mechanisms of L. nigriventer
against this pathogen, e.g. mediated through benecial
skin bacteria or antimicrobial peptides (Colombo et al.,
Fig. 2. General appearance and colour
variants of Latonia nigriventer. Juvenile
individual (SVL 30.4 mm) in dorsal
(A),
ventral (B) and lateral (C) view;
adult female (SVL 100.5 mm) in dorsal
(D), ventral (E) and lateral (F) view;
adult male (SVL 98.2 mm) in dorsal
(G), ventral (H) and lateral (I) view;
J)
dark adult male (SVL 114.0 mm) dis-
playing almost no characteristic pattern
on dorsum; K) medium-sized juvenile
(SVL 43.0 mm) displaying a pale trans-
lucent venter; L) adult female (SVL
102.3 mm); M) smallest wild caught L.
nigriventer individual (SVL 16.2 mm);
N) housed L. nigriventer individual
(SVL 103 mm, female) only displaying
the rostral portion of the head while the
rest of the body is submerged; O) adult
female (SVL 81 mm) carrying a trans-
mi tte r.
14 Bina Perl et al. – Natural history and conservation of the Hula painted frog
2015), are a valuable piece of information for an inte-
grated risk assessment of this species.
Here we have compiled observations and data on
multiple aspects of the natural history of L. nigriven-
ter. We also provide the rst evidence for the presence
of Bd in northern Israel as well as preliminary infor-
mation regarding the innate immune defences and
skin microbial community of L. nigriventer, to aid
fu
ture conservation management of this critically en-
dangered species.
Material and methods
Field surveys and sampling
We carried out eight eld surveys between November
2013 and September 2015 at numerous amphibian hab-
itats over ca. 177 km2 in the Hula Valley. Daytime sur-
veys for L. nigriventer involved searching terrestrial
habitats as well as various water bodies where we used
dip nets to search for adults, tadpoles and egg clutches.
Night surveys only involved visual inspection of water
bodies and their banks. To minimise disturbance, sites
were usually not inspected more than twice a week.
Equipment and shoes were either completely dried
or
disinfected with Virkon S or 10% bleach solution
between locations (i.e. those not connected by water-
ways) following Johnson et al. (2003). Detailed coor-
dinates of conrmed locations are not published to
avoid disturbance and collecting, but have been com-
municated to the Israel Nature and Parks Authority.
Metamorphosed individuals were captured with
gloved hands, photographed in dorsal and ventral
views and morphometric measurements were taken.
We collected tissue and buccal swabs for assessing ge-
netic variation, and took skin and cloacal swabs for
exploring the microbial communities of L. nigriventer
and other local amphibians. Skin swabs were also used
for Bd screening (Hyatt et al., 20 07). Before swabbing,
frogs were rinsed with sterilised distilled water to re-
move transient bacteria (Culp et al., 2007; Lauer et al.,
2007; Rebollar et al., 2014). Water volume was adjust-
ed based on SVL (50–150 ml). The skin of each indi-
vidual was swabbed dorsally and ventrally using two
separate swabs with each side receiving 10 strokes.
Tissue samples and buccal swabs were directly
stored in 95–99% ethanol, while skin swabs were im-
mediately placed on ice and transferred to freezer
storage (–20 °C) within 8 hours. All metamorphosed
individuals were released back to their collection site
after examination. None were sacriced but dead indi-
viduals (e.g. due to predation, road kills) were pre-
served in ethanol. Several tadpoles died shortly after
capture and were xed in 70% ethanol and later pre-
served in 70% ethanol or 5% formalin.
External transmitters (SOPR-2038; Wildlife Mate-
rials International, Inc.) were tted to seven large (> 70
mm) individuals with an elastic waistband. Specimens
were kept in a terrarium for up to three days to ensure
that the waistband was not causing undue harm. Re-
leased individuals were tracked with a TRX-16 receiv-
er and 3-element folding antenna (Wildlife Materials
International, Inc.). Tracking was completed twice a day
for up to 18 days, but individuals were only visually
inspected every second or third day in order to mini-
mise disturbance (Fig. 2 O).
Adult and juvenile morphometrics and colour
In adult and juvenile frogs the following measure-
ments were taken with a calliper to the nearest 0.1 mm,
and weight was taken with a digital scale or Pesola
spring scale (precision: 0.1 g): horizontal eye diameter
(ED), horizontal eye neck fold to snout (FS), hand
length (HAL), head width at eyes (HW), interorbital
distance (IOD), length of elbow to nger tip (LE), tip
of characteristic colour patch on forehead to snout
(PS), snout-vent length (SVL), tarsal length (TSL),
weight (W), webbing formula is given following
Blommers-Schlösser (1979).
We calculated the body condition of each adult in-
dividual with the relative mass (Wr) condition index as
described in Sztatecsny and Schabetsberger (2005).
The obtained ratios were statistically analysed by
Mann-Whitney-U test implemented in R (v 3.2.4).
The distinctive ventral and dorsal natural markings
displayed by each metamorphosed frog were used for
identifying recaptured individuals.
Tadpole description
Morphological description and measurements of tad-
poles (to the nearest 0.1 mm) were obtained using a
stereomicroscope (Zeiss Discovery V12) following
landmarks, terminology and denitions of Altig and
McDiarmid (1999). Developmental stages were identi-
ed following Gosner (1960). Labial tooth row formu-
la (LTRF) follows Altig and McDiarmid (1999) but
labial teeth are named ‘keratodonts. For a list of mor-
phological characters measured and abbreviations
used, see tadpole description in Appendix 3.
15Contributions to Zoology, 86 (1) – 2017
Vocalisa tions
Calls probably representing advertisement calls were
recorded in February 2015 from two adult males (SVL
114 and 121 mm) kept in a terrarium (L × W × H = 71
× 50.5 × 40 cm; lled with water (7 cm depth), with
some reed and grass material) overnight together with
two females (SVL 103 and 128 mm). Calls were re-
corded above water with the built-in microphone of a
waterproof camera (Pentax; WG-II) and an acoustic
recorder (SM2+; Wildlife Acoustics Inc.) that also re-
corded air temperature. Water temperature was meas-
ured by a Thermochron iButton datalogger. Record-
ings were analysed at a sampling rate of 44.1 kHz and
16-bit resolution with Cool Edit Pro 2.0. We measured
interval length between calls and six acoustic features
for each call, including call duration, expiratory note
duration, inspiratory note duration, dominant frequen-
cy of the total call, and of presumed expiratory and
inspiratory notes (Glaw and Vences, 1991). Vocalisa-
tions were illustrated using the R package ‘seewave
(Sueur et al., 2008). Call recordings were submitted
to the FonoZoo sound archive (www.fonozoo.org);
ac
cession numbers 9850–9854.
Bacterial communities and Bd/Bsal screening
A screening for the two chytridiomycete pathogens,
Bd and B. salamandrivorans Martel, Blooi, Bossut
and Pasmans, 2013 (Bsal), was conducted in L. ni-
griventer, Pelophylax bedriagae (Camerano, 1882),
Hyla savignyi Audouin, 1827 and Salamandra in-
fraimmaculata Martens, 1885 from seven localities in
and around the Hula Valley, to identify the possible
presence or absence of these two amphibian pathogens
from this region in Israel. Skin microbial communities
were analysed for L. nigriventer and the syntopic Le-
vant water frogs (P. bedriagae).
DNA was extracted from swabs with the Power-
Soil® DNA Isolation Kit (Mo Bio Laboratories, Carls-
bad, Ca, USA), following the Earth Microbiome pro-
ject protocol (http://www.earthmicrobiome.org) except
that centrifuge conditions were adjusted to accommo-
date reduced rotor speed.
Bd/Bsal screening was performed by duplex quan-
titative PCR (in duplicate) according to the protocol of
Blooi et al. (2013).
To characterise skin bacterial communities, we
PCR-amplied the V4 region of the bacterial 16S
rRNA gene with dual-indexed primers (Kozich et al.,
2013). PCRs were completed in duplicate following
Sabino-Pinto et al. (2016). Negative controls were in-
cluded to check for contamination. PCR products were
combined for each sample and roughly quantied on
1% agarose gels. Approximately equal concentrations
of PCR amplicons from each sample were pooled, gel
puried using the MinElute gel extraction kit (Qiagen).
DNA concentration was determined using a Qubit 2.0
and equimolar amounts were sequenced on an Illumina
MiSeq platform at the Helmholtz Center for Infection
Research in Braunschweig, Germany, using paired-
end 2x250 v2 chemistry. Sequences were deposited in
the NCBI Short Read Database (SRA BioProject PRJ-
NA326938).
Sequences were processed wit h the Quantitat ive In-
sights Into Microbial Ecology (QIIME; v 1.9.1.) pipe-
line for Linux (Caporaso et al., 2010). Raw forward
and reverse reads of each sample were joined using
Fastq-join under default settings (Aronesty, 2011,
2013). After quality ltering with default settings to
remove low-quality sequences, we further ltered the
reads by length (250–253 bp; usegalaxy.org) and re-
moved chimeric sequences on a per sample basis using
de novo usearch61 chimera detection within QIIME
(http://drive5.com/usearch/usearch_docs.html; Edgar
et al., 2011). Sequences were clustered into bacterial
operational taxonomic units (OTUs) with a sequence
similarity threshold of 97% using an open reference
OTU-picking strategy (Rideout et al., 2014, http://qiime.
org/tutorials/open_reference_illumina_processing.
html). SILVA 119 (24 July 2014 release; https://www.
arb-silva.de) served as reference database and UCLUST
(Edgar, 2010) was used in the de novo clustering steps.
The most abundant sequences of each OTU were se-
lected as representative sequences and aligned using
PyNAST (Caporaso et al., 2010). OTUs with less than
0.005% of total reads were removed from the data set
following Bokulich et al. (2013). Taxonomy was as-
signed using the RDP classier (Wang et al., 2007)
with the SILVA 119 taxonomy and representative se-
quences as reference, and a phylogenetic tree built us-
ing FastTree (Price et al., 2010) adhering to QIIME’s
standard procedures. We also used QIIME to generate
a rarefaction curve to conrm that an asymptote was
reached for all samples (see S1 in the Supplement).
Lastly, we rareed the data to 1000 sequences to cor-
rect for sample depth heterogeneity and calculated
beta diversity using the weighted UniFrac distance
metric in QIIME.
We tested for (i) differences in dorsal and ventral
skin bacterial communities of L. nigriventer, (ii) sea-
sonal variation of the skin microbial community, and
16 Bina Perl et al. – Natural history and conservation of the Hula painted frog
(iii) species-specic differences between L. nigriventer
and P. bedriagae. Statistical analyses were done with
PRIMER v7 software (Plymouth Routines In Multi-
variate Ecological Research; Clarke and Gorley, 2015).
PERMANOVA analyses were performed using 999
permutations and associated plots were generated by
principal coordinates analysis (PCoA). Core bacterial
communities, dened as OTUs present in at least 75%
of the samples and overlap among core communities
was visualised with Venn diagrams drawn with VEN-
NY 2.0 (http://bioinfogp.cnb.csic.es/tools/venny/).
Skin peptides collection and anti-Bd growth inhibi-
tion assays
Skin secretions of two L. nigriventer individuals were
collected from two sterile polyethylene bags (Whirl-
Pak, Nasco) in which they were kept prior to measure-
ment, by washing the bags with ~50 ml of sterile water.
Samples were then frozen until further processing.
Af
ter defrosting, 2 ml of the skin secretion wash was
removed for direct testing against Bd. The remaining
volume from each sample was acidied to a nal vol-
ume of 1% HCL to inactivate potential endogenous
peptidases (Resnick et al., 1991; Steinborner et al.,
1997) and immediately loaded onto C-18 Sep-Pak car-
tridges (Waters Corporation, Milford, Massachusetts,
USA) which were then stored in vials with 2–5 ml of
0.1% HCL until further processing.
We eluted the peptides bound to the Sep-Paks with
70% acetonitrile, 29.9% water, and 0.1% triuoracetic
acid (v/v/v) and centrifuged them under vacuum to con-
centrate them to dryness. After Sep-Pak purication, we
determined the total concentration of the recovered skin
peptides by Micro BCA Assay (Pierce, Rockford, Illi-
nois, USA) following manufacturer’s instructions, ex-
cept that we used bradykinin (RPPGFSPFR; Sigma) to
establish a standard curve (Rollins-Smith et al., 20 02).
Skin peptides were analysed by matrix-assisted laser de-
sorption/ionisation mass spectrometry (MALDI MS) as
described in Pask et al. (2012). We analysed each sample
by averaging signals from 250 consecutive laser shots.
Mass spectrometry data was acquired in the mass/
charge (m/z) range 500 to 7,000, truncated at m/z 4,000
and baseline-corrected with Data Explorer v4.4 (Ap-
plied Biosystems). The peak values shown represent the
monoisotopic mass, [M+H]+. A few signals may show
secondary peaks 22 mass units greater than the primary
peak and probably represent a peptide plus sodium ad-
duct [M+Na]+. Spectra may also show peaks at m/z 568.1
and 650.0 because of matrix or background signal.
We conducted in vitro growth inhibition assays of
the enriched skin peptide mixtures against two Bd
isolates (JEL 197 and ‘Section Line’) (Longcore et
al., 1999; Piovia-Scott et al., 2015) as described pre-
viously (Rollins-Smith et al., 2006; Ramsey et al.,
2010; Holden et al., 2015). Briey, B. dendrobatidis
zoospores were grown on 1% tryptone agar for one
week at 23°C. Freshly isolated zoospores were added
(5 × 104/50 µl, 5 replicates) in tryptone broth to a 96-
well at-bottom microtiter plates with 50 µl of a seri-
ally diluted mixture of skin peptides dissolved in
HPLC-grade water. Positive control wells contained
zoospores and 50 µl HPLC water. Negative control
wells contained heat-killed zoospores (60°C for 10
minutes) and 50 µl of HPLC water. Plates were incu-
bated at 23°C for one week, and growth was meas-
ured as an increased optical density at 490 nm (OD490)
using an MRX Microplate Reader (Dynex Technolo-
gies, Inc., Chantilly, VA, USA). Percent growth was
calculated as follows: OD at Day 7 – OD at Day 0 for
test sample / OD at Day 7 – OD at Day 0 for positive
control.
We performed Bd growth inhibition assays with the
direct skin secretion solution following the methods
explained above, with 50 µl of the direct skin secretion
solution being added to the microtiter plate instead of
the peptide dilutions. The assays were completed with
the isolate Bd VMV 813.
Results
Range and habitat
We detected L. nigriventer at the known and newly
identied sites within the Hula Nature Reserve, and at
another location, about 1 km southeast of the reserve
borders near the small village Yesod HaMa’ala (Fig. 1
C–F). In total, we observed 64 adult females, 42 adult
males, 29 juveniles (Fig. 2) and 40 tadpoles. Of these,
six middle-sized to large individuals (SVL 33.8–76.8
mm) were discovered within the reserve, while 112
medium to large-sized individuals (SVL 43.0–128.4
mm), 19 small individuals (SVL 16.2–30.1 mm; Fig. 2
M) as well as tadpoles at Gosner stages 25–34 were
recorded at Yesod HaMaala. Based on our specimen
records we estimate L. nigriventer to occupy an area
of at least 6.5 km², but the number of active re
produc-
tive sites is uncertain.
The species was found to exploit different kinds of
terrestrial and aquatic habitats:
17Contributions to Zoology, 86 (1) – 2017
(1) In the Hula Nature Reserve all individuals were
discovered in terrestrial habitats. As a former part of the
Hula marshes and lake, the organic soil at this site is
peaty, damp and loose, and covered by a ca. 20–30 cm
layer of humid decomposed leaf litter. Most individuals
(including 9 of the 14 individuals found prior to our sur-
veys) were found beneath this layer within a dense
thicket of blackberry (Rubus sanguineus Fri valdszky,
1835), reeds (Phragmites australis (Cavanilles) Trinius
Ex Steudel, 1840) and occasionally g trees (Ficus car-
ica Linnaeus, 1753). The majority of the reserve’s water
bodies are lentic and about 20–30% of the permanently
ooded area is covered by dense stands of reeds and
Papyrus sedge (Cyperus papyrus Linnaeus, 1753). It is,
however, uncertain which of the water bodies of the
Hula Nature Reserve are used by L. nigriventer as no
individuals were found here in their aquatic habitat, and
no eggs or larvae were detected.
(2) At the site near Yesod HaMa’ala, all individuals
were found in the water or at the slopes of a ~600 m
long ditch. This ditch has a permanent source of water
from a small spring and dense vegetation both in and
next to the water. The water is very slowly owing and
depth is substantial (up to ca. 150 cm) but starting at
depths of ca. 10–100 cm. A deep layer of mud covers
the bottom of the ditch and aquatic vegetation covers
much of the water’s surface (comprising dense P. a u s -
tralis growths, water lettuce (Pistia stratiotes Lin-
naeus, 1753), and duckweed, Lemna minor Linnaeus,
1753). At the edges of the ditch, the mineral soil is
compressed, sandy and in the dry season less damp
than in the reserve. Individuals in terrestrial habitat
were detected either beneath dried or half-dried grass
tufts, or in natural cavities or small burrows at the
wa
ter edge dug by semi-terrestrial freshwater crabs
(Potamon potamios (Olivier, 1804)) or small mam-
mals. The Latonia individuals encountered at this site
displayed a strikingly high percentage of injury. Of the
112 medium to large-sized frogs (> 42 mm), 28% had
old or recent minor injuries mainly on the hind limbs
(Fig. 3), while such injuries were not detected in juve-
nile frogs or in individuals found in the Hula Nature
Reserve. However, the ditch is less frequently visited
by migrating birds or larger mammals and has fewer
sh species than other, major water bodies in the Hula
Nature Reserve (see S2 in the Supplement).
Activity patterns and movement
All tadpoles were caught in shallow parts of the Yesod
HaMa’ala ditch. Metamorphosed individuals were ob-
served either within humid leaf litter in the close vicinity
to a perennial water body, directly at the water’s edge or
in the water. The majority of the individuals discovered
during winter months – especially during the rst long-
term eld tr ip from November 2013 th rough Mar ch 2014
– were land-dwelling juveniles of snout-vent lengths of
~20–30 mm. Adults were mostly detected in the water
from February through September, which may corre-
spond to the breeding period (Fig. 4).
In general, the frogs appeared to be mainly noctur-
nal and could frequently be observed in the water after
nightfall. Individuals were found solitarily and were
not observed in aggregations.
Upon capture, terrestrial individuals rst froze in
their movement and then tried to escape forward by
either slowly jumping or walking. Walking individuals
retracted their eyes. Whenever the density of the soil
allowed, they forced their way underground head rst
with retracted eyes by strongly pushing back all limbs
in a walking manner. The increased secretion of vis-
cous skin mucus further facilitated the movement
through soil or vegetation. Individuals observed at
night in the water were usually submerged with excep-
tion of the rostral portion of the head (Fig. 2 N). Upon
disturbance, they immediately tried to retreat into the
water and escape head rst into dense thickets of
aquatic plants and roots. They were more easily scared
(e.g. by our electric torches) than P. bedriagae or H.
savignyi. While being handled, adults of both sexes ut-
tered release calls that were similar to the presumed
advertisement calls described below but less regular
and less intense.
For the frogs for which displacement was recorded
by capture-recapture, the distances moved were < 20 m
(N = 10), > 20 m < 100 m (N = 4) and 100 m (N = 5)
(Table 1). Three radio-tracked frogs moved no more
than 2 m over periods of 5, 5 and 18 days, respectively.
Adult morphology
Latonia nigriventer is a rather inconspicuously col-
oured, robust frog reaching large sizes of 69.0–128.4
mm in females and 66.6–121.4 mm in males. In the
observed individuals, webbing was strongly developed
suggesting a substantial aquatic adaptation, and hind
limbs were comparatively short. The head was rather
at and the iris heart-shaped. A distinct transversal
dermal fold was present in the neck. The colour pattern
was similar in all individuals despite differences in dis-
tinctness and contrast (Fig. 2). An incomplete mid-dor-
sal band of the lighter colour was almost always visible
18 Bina Perl et al. – Natural history and conservation of the Hula painted frog
in the posterior part of the dorsum. The venter was
black to grey with a distinct pattern of white spots
which corresponded to raised tubercles in adults. For a
detailed morphological description of adults, see Ap-
pendix 1; for data and measurements of the holotype,
see Appendix 2.
From mid-February until mid-September, i.e. dur-
ing the presumed breeding season, we observed dis-
tinct dark nuptial pads and more or less distinct black,
keratinised excrescences on the thorax, ventral part of
arms and thighs, plantar surfaces as well as on the
outer edge of the webbing in males. In single cases,
excrescences were also present on dorsal surface of
feet. In females, such structures were distinct only on
plantar surfaces and to some degree on the webbing
edge, and in very rare cases, single excrescences were
seen on the thoracic region (Fig. 5). The excrescences
on the body skin had the form of isolated spicules
whereas at the webbing edges they formed dense ag-
gregations. The smallest male with clearly developed
nuptial pads had a snout-vent length of 66.6 mm, and
Tab le 1. Details on recaptured Latonia nigriventer individuals. Averages (min–max) are given for number of days after previous cap-
ture and covered distances. F = female; J = juvenile; M = male; na = GPS data partly missing, no calculations possible; – = not relevant.
Number of individuals in square brackets.
Sex days after capture 1 days after capture 2 ~distance capture 1 and 2 (m) ~distance capture2 and 3 (m)
J [2] 66 (27–105) [2] 108 [1] 0 [1] / na [2] na [1]
F [13] 84 (9–203) [13] 70 (44–108) [3] 59 (5–180) [10] / na [3] 20 (10–30) [2]
M [6] 107 (15-191) [6] 71 (10–170) [4] / na [2]
Fig. 3. Types of injuries observed in
medium-sized and large individuals of
Latonia nigriventer captured in Yesod
HaMa’ala. Blue arrows indicate the re-
spective injuries: A) male individual
(SVL 121.4 mm) lacking index nger; B)
individual of unknown sex (SVL 65.8
mm) lacking third toe; C) male individu-
al (SVL 101.3 mm) lacking left foot;
D)
female individual (SVL 110.9 mm)
with old scarring stretching all over
neck; E) female individual (SVL 88.1
mm) with fresh injury on left fourth toe;
F) female individual (SVL 95.0 mm)
with old scarring on left lateral side;
G)
male individual (SVL 105.0 mm)
with fresh puncture wound on neck;
H)
male individual (SVL 106.2 mm)
with old scarring on right foot; I) male
individual (SVL 98.0 mm) with injured
webbing. Images not to scale.
Fig. 4. Number of individuals of Latonia nigriventer captured
across months. All individuals below 66 mm were considered
juveniles, males and females were distinguished on the basis of
their foot webbing and the presence of nuptial pads in males.
19Contributions to Zoology, 86 (1) – 2017
we therefore used this value as cut-off to distinguish
adults from juveniles. Based on specimens > 66 mm,
we found no signicant difference in the size-weight
distribution of males and females (body condition cal-
culated with the relative m ass (Wr) condition index;
Sztatecsny and Schabetsberger, 2005) using a Mann-
Whitney-U test (p = 0.615; N (female/male) = 64/44;
Fig. 6), nor did we detect distinct sexual dimorphism
in morphometric measurements (Table 2). Slight dif-
ferences were however seen in foot webbing (see S3 in
the Supplement): females had slightly weaker web-
bing, with a distinct broadening of the toe III webbing
usually at the 3rd phalanx, and on toe IV at the 3rd–4th
phalanx; whereas in males it usually was seen at the
2nd phalanx at toe III and the 3rd phalanx at toe IV.
Forearms were strong in both sexes and could not be
reliably used to differentiate females from males (for
raw measurements see S4 in the Supplement).
Tadpole morphology
Tadpoles collected at two distinct sites in the ditch
near Yesod HaMaala on 15 May 2015 agree with the
description of Mendelssohn and Steinitz (1943) in
hav
ing double keratodont rows and a network of black
epidermal lines. They were small (total length 14 mm
at Gosner stage 25 and 24 mm at Gosner stage 34),
uniformly brown, with an unpigmented ventral side
and ventral spiracle. The LTRF was 2/3(1), not consid-
ering double rows (Fig. 7). For a detailed description
of the tadpoles see Appendix 3, for raw measurements
see S5 in the Supplement.
The tadpoles of L. nigriventer are distinguished
from those of the three other anuran species in this
area (Bufotes variabilis (Pallas, 1769), H. savignyi and
P. bedriagae) as well as those of Pelobates syriacus
Boettger, 1889, by their medial ventral spiracle, dis-
tinct epidermal reticulations and double keratodont
rows. Such biserial rows of keratodonts are a unique
state for the basal anuran genera Discoglossus and As-
caphus, and are, with exception of Hoplobatrachus,
not found in more advanced frogs (Grosjean et al.,
2004). Dorsally, L. nigriventer tadpoles mostly resem-
ble the ones of Bufotes in the position of their eyes and
general appearance, but they lack the distinctly golden
speckled venter as observed in the latter. The unpig-
mented venter of L. nigriventer tadpoles likewise
serves as an immediate distinguishing characteristic
from both Hyla and Pelophylax tadpoles that display a
silvery venter.
Of the 40 tadpoles (Gosner stages roughly between
25 and 35) found on 15 May, 9 were successfully raised
Fig. 5. Nuptial pads and keratinised
ex
crescenses displayed by Latonia nigri-
venter individuals during breeding sea-
son. A) Hand of female individual with
keratinised excrescences between n-
gers 1–2 and 2–3 (May); B) hand of male
individual with distinct nuptial pads and
keratinised excrescences on arm in ven-
tral view (May); C) nuptial pads of male
individual in dorsolateral view (May);
D) webbing of female individual with a
narrow rim of keratinised excrescences
present on outer webbing edge (mid-
Februar y); E) webbing of male individu-
al with distinct rim of keratinised ex-
crescences on outer webbing edge (mid-
Februar y); keratinised excrescences on
thorax as displayed by some female (F)
and male (G) individuals in May. Blue
arrows indicate the webbing parts on
which excrescences have been observed
by us. Images were cut out of photos of
live individuals with Photoshop CS2 soft-
ware (v 9.0, Adobe Systems, San Jose,
CA) and are not to scale.
20 Bina Perl et al. – Natural history and conservation of the Hula painted frog
to post-metamorphs at the School of Marine Sciences
(Ruppin Academic Center), Michmoret, where meta-
morphosis took place between 3–15 June 2015. Re-
cently metamorphosed frogs had a SVL of 6–9 mm.
At the end of June, all post-metamorphs were trans-
ferred to the Garden of Zoological Research at Tel
Aviv University for further rearing.
Reproduction
Besides the observation of tadpoles in mid-May, no di-
rect observation of breeding has been made. The dis-
section of a dead L. nigriventer female that had been
overrun by a tractor in mid-January revealed several
hundred greyish-black oocytes with a diameter be-
Fig. 6. Latonia nigriventer growth curves including data-driven LOESS (locally weighted scatterplot smoothing; Cleveland and Dev-
lin, 1988) lines and weight gain/loss of recaptured individuals. A) Total captures including recaptures, B) captured individuals exclud-
ing recaptures, C) recaptured individuals, D) weight gain (blue) and loss (red) of recaptured females, E) weight gain (blue) and loss
(red) of recaptured males as well as weight loss of the single recaptured juvenile (black). Initial capture weight of respective individual
given in parentheses.
21Contributions to Zoology, 86 (1) – 2017
tween 1.5–2 mm. From the available data and phylo-
genetic relatedness it can be hypothesised that at least
some aspects of the reproduction are similar to Dis-
coglossus, which are opportunistic breeders with a
short and intense inguinal amplexus during which
several batches of eggs are deposited and adhere to
stones, aquatic plants or the bottom of the water body.
According to the observations of (i) males with dis-
tinct nuptial pads and other keratinised excrescences
from February to September, (ii) tadpoles in mid-
May (our observation) and August (Mendelssohn and
Steinitz, 1943), and (iii) the observed weight losses in
recaptured females of L. nigriventer, we hypothesise
a relatively prolonged reproductive period with egg
depositions potentially taking place at least from
March to June, and possible from February to Sep-
tember. This extended reproductive season, if con-
rmed, would be longer than in the three sympatric
amphibian species, i. e., February/March–May in B.
variabilis, March/April–June in H. savignyi, and
May–August/September in P. bedriagae (Degani and
Mendelssohn, 1984).
♀♀ ♂♂
SVL 97.2 (74.6–128.4) N = 24 98.9 (68.9–121.4) N = 19
HW 23.9 (19.1–32.3) N = 24 24.6 (11.8–33.1) N = 19
BW 54.7 (36.2–75.0) N = 24 53.0 (35.4– 66.5) N = 19
ED 6.4 (5.1–8.2) N = 24 7.0 (4.8–9.3) N = 18
IO 13.2 (7.8–18.7) N = 23 13.1 (8.5–18.9) N = 19
FS 26.1 (19.6–34.5) N = 23 27.0 (13.1–35.3) N = 19
CS 16.7 (12.2–29.9) N = 24 16.1 (11.8–22.0) N = 19
HL 21.9 (14.4–48.0) N = 24 27.9 (12.4–48.3) N = 19
EF 23.9 (19.1–32.3) N = 24 35.6 (21.3–46.4) N = 19
TL 38.1 (29.0–46.7) N = 20 39.2 (28.1–48.6) N = 12
Tab le 2. Morphometric data for live adults
of Latonia nigriventer. Measurements (in
mm) include mean (minimum–maxi-
mum) of following morphometric traits:
body width (BW); tip of characteristic
colour patch on forehead to snout (CS);
eye diameter (ED); length of elbow to n-
ger tip (EF); neck fold to snout (FS); hand
length (HA L); head width at eyes (HW);
minimal interorbital distance (IO); snout-
vent length (SVL); tarsal length (TL).
Number of measured individuals is indi-
cated for each measurement.
Fig. 7. Latonia nigriventer tadpole and
(post-) metamorphic juveniles. A) Live
L. nigriventer tadpole at Gosner stage
34 in dorsal view, B) ventral view and C)
lateral view; D) oral disk of live tadpole
displaying characteristic biserial kerato-
dont rows and dense epidermal network
of black lines E) oral disk of preserved
tadpole (Gosner stage 34); F) individual
shortly after metamorphosis. The tiny
post-metamorph (SVL ~6–9 mm) al-
ready displays the typical colour patch
on its forehead; G) metamorphosing in-
dividual.
22 Bina Perl et al. – Natural history and conservation of the Hula painted frog
Vocalisa tions
Similar to Discoglossus (Weber, 1974; Glaw and Venc-
es, 1991; Vences and Glaw, 1996), the calls heard from
L. nigriventer were of very low intensity, low spectral
frequency, and consisting of a presumed expiratory and
a presumed inspiratory note. As the release calls of han-
dled L. nigriventer sound very similar to the recorded
calls, we assume the calls to be uttered at the water sur-
face like in Discoglossus species. While we cannot ex-
clude that other, more intense call types can be emitted
by this species, the absence of externally visible vocal
sacs in males makes it likely that their vocalisations
mainly serve short-distance communication. Although
we could only achieve provisional recordings under
suboptimal conditions in captivity, which might have
distorted some of the call features, we were able to re-
cord the calls of several male L. nigriventer individuals.
Air temperature during recordings varied between
13.5–18 °C and water temperature between 14–15 °C.
Call duration (ms) Interval Dominant frequency (Hz)
(N = 72) between (N = 72)
calls (ms)
(N = 65)
expirator y inspiratory total expiratory inspiratory total
note note call note note call
Total 671 291 962 787 795.2 531.6 775.5
SD 115 28 123 609 65.3 299.3 80
Tab le 3. Call features of the presumed ad-
vertisement calls recorded from a group
of several individuals of Latonia nigri-
venter.
Fig. 8. Spectrogram and oscillogram of
series of A) two and B) nine presumed
advertisement calls of Latonia nigriven-
ter. Note the low frequency (< 1.5 kHz).
The two notes (presumed expiratory and
inspirator y) within each call are distin-
guishable, yet not separated by a silent
interval. Sampling rate 44.1 kHz.
23Contributions to Zoology, 86 (1) – 2017
As several males were kept together in the same aquar-
ium, the following descriptive statistics refer to calls of
various males in unknown proportions.
Calls were mostly uttered in a series and were sepa-
rated from each other by short intervals of silence
varying from 246 –1606 ms (mean + SD: 787 + 609
ms; N = 65). Each call consisted of two notes which we
assume represent sounds produced by expiration (rst
note) followed by inspiration of air into the lungs (sec-
ond note). Both notes were spectrally structured and
pulsatile, but a clear distinction and count of pulses
was not possible. The two notes of one call were not
separated by a silent interval or distinct decrease in
amplitude. Therefore, in the spectrogram the two notes
are mostly recognisable by the somewhat lower fre-
quency and higher intensity of the second (inspiratory)
note (Fig. 8). Dominant frequency peak averaged over
the total call (mean + SD) was 775.5 + 80 Hz (N = 72);
frequency range was roughly between 0–1500 Hz.
Call duration (N = 72) ranged between 725–1212 ms,
with the expiratory note being longer (671 + 115 ms)
than the inspiratory note (291 + 28 ms) (Table 3).
Bd/Bsal screening, skin bacterial community, and
de
fensive skin peptides
The pathogen Bd was detected in 32% of the tested
amphibian individuals (n = 87) from northern Israel,
while none were positive for Bsal. We found Bd in two
amphibian species (L. nigriventer and P. bedriagae)
and in three of the seven examined locations within
the Hula Valley (Hula Nature Reserve, Kiryat Shmona
and Yesod HaMa’ala). Infection loads for Bd-positive
individuals ranged between 1–311 genomic equiva-
lents of zoospores per swab (Table 4).
Bacterial communities of L. nigriventer were com-
prised of Proteobacteria (56.7%) with a high represen-
tation of Gammaproteobacteria (33.4% of the overall
community), Bacteroidetes (25.3%) and Firmicutes
(6.7%) (Fig. 9 A). The 20 most abundant OTUs found
on the skin of L. nigriventer represented 42% of the
total reads (Fig. 9 B; S6 in the Supplement). The most
abundant OTU (7% of the total sequences) was as-
signed to an unspecied Chryseobacterium and pre-
sent in 100% of the samples, although in varying abun-
dance (< 1–24% of the reads). Comparisons based on
weighted UniFrac distances did not reveal signicant
differences between (i) microbial communities from
the ventral versus dorsal skin of L. nigriventer (PER-
MANOVA: N = 27; p = 0.117; Fig. 10 A), (ii) ventral
surfaces of females versus males (PERMANOVA: N =
15; p = 0.646; Fig. 10 B) or (iii) ventral surfaces of Bd-
positive versus Bd-negative individuals (PER MA NO-
VA: N = 22; p = 0.283; Fig. 10 C). However, we ob-
served a signicant shift in the ventral skin microbial
community over time.
While no signicant changes were observed be-
tween the ventral skin samples taken in mid-February
and mid-April (N = 13; p = 0.799) or between those
collected in mid-April and the end of June (N = 15; p
= 0.0.093), signicant differences were detected for all
other time-associated comparisons: mid-February –
late June (N = 15; p = 0.012); mid-February – mid-
September (N = 15; p = 0.001); late June – mid-Sep-
tember (N = 17; p = 0.001) (Fig. 10 D).
The results obtained for the skin-associated bacterial
communities of syntopic P. bedriagae were similar to
those of L. nigriventer: no signicant differences be-
tween ventral versus dorsal surfaces of the same indi-
viduals (N = 14; p = 0.898) nor between ventral sur
faces
Species Location No. of genomic Analysed/
equivalents of Positive
zoospores for Bd
per swab
Hyla savignyi Einan Reserve 0 8/0
Latonia nigriventer Yesod HaMa'ala 76 (2–311) 22/6
Pelophylax bedriagae Eastern part of valley 0 6/0
Handaj area 0 5/0
Kiryat Shmona 3 8/1
Hula Nature Reserve 45 (3–132) 7/4
Yesod HaMa'ala 9 (1–45) 22/15
Salamandra infraimmaculata Tel Dan 0 8/0
Total 86/26
Tab le 4. Number of individuals per spe-
cies tested for Batrachochytrium dendro-
batidis including mean (range) of genom-
ic equivalents of zoospores per swab for
positive tested specimen.
24 Bina Perl et al. – Natural history and conservation of the Hula painted frog
of Bd-positive versus Bd-negative individuals (N = 22;
p = 0.366).
A comparison of the ventral skin-associated com-
munities of L. nigriventer and P. bedriagae from the
same location and same time-point revealed differ-
ences between the two species (N = 17; p = 0.001; Fig.
10 E). The core bacterial communities contained 30
OTUs (88% of the core skin microbiota of L. nigrive-
nter and 57% of that of P. bedriagae) that were present
on the ventral skin of at least 75% of the individuals of
both species (Fig. 10 F).
The skin secretions collected from two different
individuals and examined for peptide composition
had signicant amounts of hydrophobic peptides re-
covered after C18 enrichment. We detected a number
of common peptide mass signals shared by both frog
individuals. The mass ranges are suggestive of pos-
sible antimicrobial peptides (Table 5; S7 in the Sup-
plement). In a growth inhibition assay, the mixture of
peptides inhibited the growth of two different Bd iso-
lates (JEL 197 and ‘Section Line’; Fig. 11). At the
highest concentration tested (500 mg/m l), Bd growth
inhibition ranged from 51% to 91.5% against the Sec-
tion Line isolate and 70–82% inhibition against the
original type isolate JEL 197. Both isolates are among
the global panzootic lineages (Schloegel et al., 2012;
Piovia-Scott et al., 2015). Furthermore, the direct
skin secretion solution was found to inhibit Bd by
35–36%.
Discussion
Although amphibians as a group are relatively well-
studied, data deciency still remains an issue that has
to be overcome before conservation efforts can be ef-
fectively developed. Prior to its supposed extinction,
the available information on the Hula painted frog was
merely descriptive and only based on three adult spec-
imens and two tadpoles. Without any details on its
habitat requirements, activity patterns and reproduc-
tive biology, this secretive species went unnoticed for
decades, despite numerous attempts to rediscover it in
its former, but highly modied habitat. Even though
habitat degradation remains a prime concern for most
species, recently emerged pathogens have led to an in-
creased awareness of disease-related threats. In this
multifaceted threat scenario, basic biological informa-
tion on the target species is still fundamental for devel-
oping risk assessments and conservation strategies, but
is not sufcient in itself anymore.
In our study, we have provided detailed morpho-
logical descriptions of both adults and tadpoles and
have compiled rst ndings on the behaviour, activity
Fig. 9. Relative abundances of major bacterial taxa obtained from Latonia nigriventer skin samples in Yesod HaMa’ala as identied by
the SILVA 119 database. The order of the taxa in columns corresponds to that in legends (ordered alphabetically). A) Abundances of
dominant bacterial phylotypes; B) abundances of the 20 most frequent bacterial OTUs. (For detailed OTU IDs see S5 in the Supplement).
25Contributions to Zoology, 86 (1) – 2017
Fig. 10. Principal coordinates analysis plots of weighted UniFrac distances of the microbial communities associated with Latonia
nigriventer and Pelophylax bedriagae. A) Comparison of dorsal and ventral surfaces in L. nigriventer; B) comparison of ventral skin
microbial communities found in female and male L. nigriventer; C) comparison of L. nigriventer individuals tested positive and nega-
tive for chytrid (ventral surfaces only); D) seasonal changes in the ventral skin microbial community associated with L. nigriventer;
E) comparison of the ventral skin microbial communities found in P. bedriagae and L. nigriventer; F) Venn diagram depicting the
overlap of core microbial communities as obtained from ventral skin swabs of L. nigriventer and P. bedriagae captured at the same
location and day. The minimum fraction of samples an OTU must be observed in was set to 75%.
26 Bina Perl et al. – Natural history and conservation of the Hula painted frog
patterns and vocalisation of L. nigriventer as well as
initial information on its habitat. Together with our
preliminary results on the presence of Bd on the Hula
painted frog, our ndings are important means to di-
rect future research and conservation management of
this critically endangered amphibian.
Our surveys in the Hula Valley and adjacent regions
of northern Israel conrmed that L. nigriventer is a
localised species that occurs within and in close vicin-
ity to the Hula Nature Reserve. This wetland site be-
came Israel’s rst natur e reserve in 196 4 (Israel Nature
and Parks Authority 2015) and is a closely monitored
haven for a rich vertebrate fauna (Table S1) that – with
exception of a fenced circular path – is not accessible
to the public. By contrast, the site at Yesod HaMaala is
subject to heavy anthropogenic inuences, e.g. exten-
sive rubbish dumping, petrol spills, near agricultural
pesticide applications as well as occasionally free-
roaming horses. The newly discovered large popula-
tion at this heavily disturbed site not only demonstrates
the adaptability of this rare frog to different environ-
mental conditions, but is furthermore likely to be cru-
Fig. 11. Bd growth inhibition assays of two female Latonia nigriventer individuals against two Bd strains. A) Female 1 (SVL 90 mm; weight
86 g) against Bd 197; B) female 2 (SVL 128 mm; weight 187 g) against Bd 197; C) female 1 against Bd Section Line strain; D) female 2
against Bd Section Line strain. Asterisks (*) indicate signicantly less than positive control for growth by Student’s t test (p < 0.05 ).
Sample SVL (mm) Mass (g) Total Peptides per Peptides/ml
Peptides (µg) gram body of mucus
weight (µg/g)
L. nigriventer 1 90 86 6247.6 72.6 10418.1
L. nigriventer 2 128 187 7824.6 41.8 8445.7
Tab le 5. Quantity of peptides detected in
the mucus of two Latonia nigriventer
females.
27Contributions to Zoology, 86 (1) – 2017
cial for its overall survival. In addition, the conrma-
tion of recent reproductive events at that site as well as
the fact that adult individuals can reach snout-vent
lengths of up to almost 130 mm while juveniles and
tadpoles are comparatively tiny, illuminate aspects of
the species’ life history that will guide future conser-
vation planning.
Latonia nigriventer is shy in its aquatic habitat, rare-
ly seen active during the day, and apparently not emit-
ting loud vocalisations. Given these elusive habits, ad-
ditional undetected populations might exist in the Hula
Valley. Nevertheless, it is most likely that L. nigriventer
will be restricted to particular kinds of habitats that al-
low the large adults to hide in the aquatic habitat (e.g.,
in deep layers of aquatic vegetation and mud) as well as
in the terrestrial environment. It is yet unclear whether
our observation of less adult individuals staying in the
water from September through January is associated
with the rather sparse vegetation along and within the
water during the colder months or simply a reection of
our sampling effort. Based on our knowledge on Disco-
glossus species we initially directed our sampling ef-
fort mainly on the terrestrial habitat. We therefore can-
not rule out the possibility of L. nigriventer being pre-
sent in its aquatic habitat year-round.
We acknowledge that our radio-tracking attempt
only yielded preliminary insights and implanted or di-
gested devices will be necessary to conrm the results.
However, our capture-recapture data also reect a re-
duced movement. This apparently low dispersal capa-
bility of the Hula painted frog and its extended pres-
ence in the breeding waters are a major difference in
comparison to species of its sister genus Discoglossus.
These smaller-sized frogs (SVL 36–80 mm) breed in a
large variety of shallow and often ephemeral water
bodies following episodes of rain and an increase of
temperatures (Vences and Grossenbacher, 2012). While
Discoglossus males might stay close to or in water
bodies during some weeks for reproduction, females
usually show aquatic habits only for reproduction. In
contrast, the large-sized Hula painted frog was so far
without exception found in the proximity of and most-
ly in permanent water bodies.
These seemingly more aquatic habits of L. nigriven-
ter convey risks as well as opportunities for conserva-
tion. On one hand, the year-round dependence on spe-
cic aquatic habitats might leave the individuals par-
ticularly vulnerable to local environmental changes,
especially during the hot and dry summer months. On
the other hand, if indeed small, deep, densely vegetat-
ed, muddy and permanent ditches represent an optimal
habitat for this species, it should be easy to engineer
exactly this kind of habitat in different parts of the Hula
Valley where drainages and canals are omnipresent.
Like the closely related Discoglossus species, we
found L. nigriventer to apparently breed opportunisti-
cally whenever conditions are advantageous. However,
the role of rainfall for triggering egg deposition in this
rare frog that mostly dwells in permanent water bodies
remains unclear. The comparatively few tadpoles and
juveniles found (Fig. 12) suggest that reproduction and
recruitment in Latonia is comparatively low, and pop-
ulations might survive less favourable periods with
low reproductive success due to the longevity of the
adults. Although the large size of this frog suggests a
rather long lifespan, this hypothesis requires further
testing. In contrast, the rather aquatic habits and repro-
duction in permanent water bodies might reduce the
dependence of Latonia on rainfall compared to spe-
cies of its sister genus Discoglossus.
Even though a large number of Hula painted frogs
were found in the newly discovered site near Yesod
HaMa’ala (131 out of 137 individuals), the conditions
at this site might not be optimal. We observed a vari-
ety of detrimental factors of anthropogenic origin that
might affect larval stages and productivity of adults.
One of those factors is the sheer quantity of plastic and
metal waste in the ditch, the degradation by-products
Fig. 12. Total numbers of detected Lato-
nia nigriventer individuals per size co-
hort.
28 Bina Perl et al. – Natural history and conservation of the Hula painted frog
of which have already been found to affect larval de-
velopment and reproduction in amphibians (Oehlmann
et al., 2009; Severtsova and Gutierrez, 2013). Another
potentially detrimental factor is the nearby pesticide
application. Studies that addressed the effect of pesti-
cides on amphibians have shown that exposures even
to low concentrations of the most commonly applied
pesticide atrazine result in the feminisation of male
frogs and cause the retardation of larval development
and growth (Hayes et al., 2002, 2006; Boone and
James, 2003; Carr et al., 2003). So far, however, the
impact of both rubbish deposition and pesticide appli-
cation at that new site on the development and repro-
duction of Latonia remains purely speculative.
Apart from anthropogenic inuences, the high per-
centage of injury found in adults and the overall low
numbers of small individuals might indicate a high
predatory pressure on the early life stages of L. ni-
griventer. Besides White-throated kingshers (Halcy-
on smyrnensis (Linneaeus, 1758)) that are known to
prey upon adult L. nigriventer (Biton et al., 2013), ju-
veniles might fall prey to a plethora of widespread and
abundant animals that are known or suspected to prey
on amphibians and present at the locality, such as
Western mosquitosh (Gambusia afnis (Baird & Gi-
rard, 1853); Goodsell and Kats, 1999; Komak and
Crossland, 2000; Segev et al., 2009), freshwater crus-
taceans (Gherardi and Micheli, 1989; Capolongo and
Cilia, 1987), dragony nymphs (Stav et al., 2007), Cas-
pian turtles (Mauremys caspica (Gmelin, 1774); Sidis
and Gasith, 1985), lycosid spiders and carabid beetles
(McCormick and Polis, 1982; Toledo, 2005), or larger
frog individuals comprising conspecics (Men-
delssohn & Steinitz, 1943) and P. bedriagae (ow n ob-
servations).
Our data show that the fungal pathogen, Bd, is pre-
sent on L. nigriventer (27%) as well as on P. bedriagae
in the Hula Valley. During our surveys in northern Is-
rael, we did not observe mass mortality events in any
amphibian species, nor did we notice skin lesions in
any individual. This nding is not unexpected given
Bd has not wreaked havoc in amphibian populations in
much of the Old World, with the exception of Austral-
ia. Also for Europe only a limited number of incidenc-
es with Bd-induced mortalities have been documented
so far (Bosch et al., 2001; Bosch and Martínez-Solano,
2006; Bosch and Rincón, 2008). All in all, many spe-
cies appear to coexist with Bd at low infection intensi-
ties (Spitzen-van der Sluijs et al., 2014). Albeit our
study has relatively low sample size and we conducted
Bd screening only during one season, we can hypoth-
esise that the low infection levels observed in our study
could be associated both with environment and host-
associated factors. In temperature experiments with
Bd isolates, Piotrowski et al. (2004) showed that Bd
optimally grows at temperatures between 10–25 °C,
and suggest that lower or higher temperatures may not
induce epidemics. The high water temperatures (> 25
°C) prevailing in the investigated ditches during the
summer months, could thus be one factor explaining
the low prevalence and intensity of Bd. Our observa-
tions that Latonia individuals do not seem to form
dense aggregations, but keep some distance to each
other might also have affected the ability of Bd to
spread from individual to individual.
The presence of antimicrobial peptides in L. ni-
griventer could also, in part, explain the observed low
Bd prevalence and intensity, although we acknowledge
that two screened individuals are not an adequate sam-
ple size for broad conclusions to be made. However,
our results provide a baseline for all future studies that
endeavour to analyse the innate immunity of L. ni-
griventer. Furthermore, the cutaneous bacterial com-
munity could play a role in mitigating chytrid infection
in these frogs; as a perspective for future studies it is
worth mentioning that seven of the most abundant bac-
terial OTUs found on the skin of L. nigriventer belong
to species or genera known to potentially exhibit anti-
fungal activity (Harris et al., 2006; Woodhams et al.,
2007; Lauer et al,. 2008; Becker et al., 2015). The in-
fection dynamics of Bd are likely a complex interac-
tion of external environmental parameters, host behav-
iour, and host immunity factors that may differ across
life stages. However, continued monitoring of Bd in-
fection rates as well as an identication of the Bd strain
present in northern Israel remain important. Further-
more, minimising the anthropogenic impact at the lo-
calities and preventing the introduction of new am-
phibian pathogens by carefully disinfecting utilised
equipment before and after usage at the respective
sites are imperative.
Our ndings have direct relevance for the ongoing
restoration project in the Hula Valley which offers
enormous opportunities for improving and extending
this rare species’ suitable habitat. In close collabora-
tion with local stakeholders and the Israel Nature and
Parks Authority we will design a prospective monitor-
ing system for this species and continue to raise local
awareness in order to improve conservation at known
and potential other locations. Environmental DNA
studies to detect further populations of L. nigriventer
are currently underway. Even though none of the Lato-
29Contributions to Zoology, 86 (1) – 2017
nia individuals discovered within the Hula Nature Re-
serve was found in an aquatic habitat, rst eDNA re-
sults indicate that individuals are at least crossing one
of the water bodies within the north-eastern part of the
reserve (Renan et al., 2016).
As our rst tadpole-rearing attempt proved fruit-
ful, a further step will be to set up a captive breeding
programme. Such a programme could tide this unique
frog over the most critical early tadpole and juvenile
stages enabling translocation of individuals to re-
stored and newly created habitats within its presumed
former range, which would help to increase the popu-
lation size and promote the survival of the Hula
painted frog.
Acknowledgements
This study was carried out with the help of funds granted by
B
IOPAT – Patenschaften für biologische Vielfalt e.V. and the
Amphibian Conservation Fund by Stiftung Artenschutz and
VDZ to MV, BP, SG and EG; by the Mohamed bin Zayed Spe-
cies Conservation Fund (MBZ) to MV and BP; and by the U.S.
Fish and Wildlife Services (USFWS) and the Israel Nature and
Parks Authority (INPA) to SR, SG and EG. Skin peptide studies
in the Rollins-Smith lab were supported by National Science
Foundation (USA) grant IOS-1121758. We are grateful to Yael
Ballon, Eliane Küpfer, Asaf Moran, Noa Truskanov (in alpha-
betic order), who have provided assistance during eldwork, as
well as Gilad Amir for reporting the rst specimen found in
Yesod HaMa’ala, and Shai, Muli and Yehuda Dabush for report-
ing the L. nigriventer roadkill. Furthermore, we thank the staff
of the Hula Nature Reser ve for providing access to areas not
open to the public and patiently helping wherever help was
needed; special thanks go to Yifat Artzi for logistic assistance
and provision of ofce space. We also thank Giora Gigis, cura-
tor at the Beit Ussishkin Nature Museum located in Kibbutz
Dan for assisting in night surveys and providing access to old
documents on L. nigriventer. Above this, we are grateful to
Meike Kondermann as well as Joana Sabino-Pinto for help with
labwork and Boaz Shacham who allowed the examination of
specimens in the HUJ collection under his care. Thanks are also
due to Sabin Bhuju, Robert Geffers and Michael Jarek, who per-
formed Illumina sequencing and sequence pre-processing. Last
but not least, we thank the reviewers and editorial staff for their
detailed comments and suggestions that improved our manu-
script. Permits and authorisations were granted by the Israel
Nature and Parks Authority. Tadpoles and metamorphs were
reared under Permit number 2015/40926.
References
Altig R, McDiarmid RW. 1999. Body plan: development and
morphology. Pp. 24-51 in: McDiarmid RW, Altig R, eds,
Tadpoles: The Biology of Anuran Larvae. Chicago Univer-
sity Press, Chicago.
Aronesty E. 2011. ea-utils: command-line tools for processing
biological sequencing data. Available at http://code.google.
com/p/ea-utils (accessed 20 Nov 2015).
Aronesty E. 2013. Comparison of sequencing utility programs.
The Open Bioinformatics Journal 7: 1- 8.
Baillie JEM, Grifths J, Turvey ST, Loh J, Collen B. 2010. Evo-
lution Lost: Status and Trends of the World’s Vertebrates.
Zoological Society of London, London.
Becker MH, Walke JB, Murrill L, Woodhams DC, Reinert LK,
Rollins-Smith LA, Burzynski EA, Umile TP, Minibole
KPC, Belden LK. 2015. Phylogenetic distribution of symbi-
otic bacteria from Panamanian amphibians that inhibit
growth of the lethal fungal pathogen Batrachochytrium
dendrobatidis. Molecular Ecology 24: 1628 -16 41.
Biton R, Geffen E, Vences M, Cohen O, Bailon S, Rabinovich
R, Malka Y, Oron T, Boistel R, Brumfeld V, Gafny S. 2013.
The rediscovered Hula painted frog is a living fossil. Nature
Communications 4: 1959.
Blommers-Schlösser R MA. 1979. Biosystematics of the Mala-
gasy frogs. I. Mantellinae (Ranidae). Beaufortia 352 : 1-7 7.
Blooi M, Pasmans F, Longcore JE, Spitzen-van der Sluijs A,
Vercammen F, Martel A. 2013. Duplex Real-Time PCR for
rapid simultaneous detection of Batrachochytrium dendro-
batidis and Batrachochytrium salamandrivorans in am-
phibian samples. Journal of Clinical Microbiology 51: 4173 -
417 7.
Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI,
Knight R, Mills DA, Caporaso JG. 2013. Quality-ltering
vastly improves diversity estimates from Illumina amplicon
sequenci ng. Nature Methods 10: 57-59.
Boone MD, James SM. 2003. Interactions of an insecticide, her-
bicide, and natural stressors in amphibian community meso-
cosms. Ecological Applications 13: 829- 841.
Bosch J, Martinez-Solano I, Garcia-Paris M. 2001. Evidence of
a chytrid fungus infection involved in the decline of the
common midwife toad (Alytes obstetricans) in protected
areas of central Spain. Biological Conservation 97: 331-337.
Bosch J, Martínez-Solano I. 2006. Chytrid fungus infection
related to unusual mortalities of Salamandra salamandra
and Bufo bufo in the Peñalara Natural Park, Spain. Oryx 40:
84 -89.
Bosch J, Rincón PA. 2008. Chytridiomycosis-mediated expan-
sion of Bufo bufo in a montane area of Central Spain: an
indirect effect of the disease. Diversity and Distributions
14: 637-643.
Boulenger GA. 1891. A synopsis of the tadpoles of the Europe-
an batrachians. Pp 593-678 in: Proceedings of the Zoologi-
cal Societ y of London, Blackwell Publishing Ltd, London.
Capolongo D, Cilia JL. 1987. Potamon uviatile lanfrancoi, a
new subspecies of a Mediterranean freshwater crab from the
Maltese Islands (Crustacea, Decapoda, Potamidae). Anna-
len des Naturhistorischen Museums in Wien. Serie B 91: p
215-224.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman
FD, Costello EK, Fierer N, Gonzalez Peña A, Goodrich JK,
Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE,
Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung
M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Wid-
man J, Yatsunenko T, Zaneveld J, Knight R. 2010. Qiime
allows analysis of high-throughput community sequencing
data. Nature Methods 7: 335-336.
30 Bina Perl et al. – Natural history and conservation of the Hula painted frog
Carr JA, Gentles A, Smith EE, Goleman WL, Urquidi LJ,
Thuett K, Kendall, RJ, Giesy JP, Gross TS, Solomon KR,
van der Kraak G. 2003. Response of larval Xenopus laevis
to atrazine: Assessment of growth, metamor phosis, and go-
nadal and laryngeal morphology. Environmental Toxicology
and Chemistry 22: 396-405.
Clarke KR, Gorley RN. 2015. PRIMER v7: User Manual/Tuto-
rial. PRIMER-E Ltd., Plymouth.
Cleveland WS, Devlin SJ. 1988. Locally weighted regression:
an approach to regression analysis by local tting. Journal
of the American Statistical A ssociation 83: 596 -610.
Cohen-Shacham E, Dayan T, Feitelson E, de Groot RS 2011.
Ecosystem service trade-offs in wetland management:
drainage and rehabilitation of the Hula, Israel. Hydrological
Sciences Journal 56: 158 2-1601.
Colombo BM, Scalvenzi T, Benlamara S, Pollet N. 2015. Micro-
biota and mucosal immunity in amphibians. Frontiers in
Immunology 6: 111.
Culp CE, Falkinham III JO, Belden LK. 20 07. Identication of
the natural bacterial microora on the skin of eastern newts,
bullfrog tadpoles and redback salamanders. Herpetologica
63: 66-71.
Degani G, Mendelssohn H. 1984. Amphibia. Pp 190-221 in:
Alon A, eds, Plants and animals of the land of Israel. Mis-
rad Habitachon Pub, Tel-Aviv.
Dimentman CH, Bromley HJ, Por FD. 1992. Lake Hula: recon-
struction of the fauna and hydrobiology of a lost lake. The
Israel Academy of Sciences and Humanitites, Jerusalem.
Edgar RC. 2010. Search and clustering orders of magnitude
faster than BLAST. Bioinformatics 26: 2460-2461.
Edgar, RC, Haas, BJ, Clemente JC, Quince C, Knight R. 2011.
UCHIME improves sensitivity and speed of chimera detec-
tion. Bioinformatics 27: 2194-2200.
Gherardi F, Micheli F. 1989. Relative growth and population
structure of the freshwater crab, Potamon potamios pal-
estinensis, in the Dead Sea area (Israel). Israel Journal of
Zoolog y 36: 133-145.
Glaw F, Vences M. 1991. Bioacoustic differentiation in Painted
frogs (Discoglossus). Amphibia-Reptilia 12: 385 -394.
Goodsell JA, Kats LB. 1999. Effect of introduced mosquitosh
on Pacic treefrogs and the role of alternative prey. Conser-
vation Biology 13: 921-924.
Goren M, Ortal R. 1999. Biogeography, diversity and conserva-
tion of the inland water sh communities in Israel. Biologi-
cal Conservation 89: 1-9.
Gosner KL. 1960. A simplied table for staging anuran em-
bryos and larvae with notes on identication. Herpetologica
16: 183-190.
Grosjean S, Vences M, Dubois A. 2004. Evolutionary signi-
cance of oral morphology in the car nivorous tadpoles of ti-
ger frogs, genus Hoplobatrachus (Ra nid ae). Biological
Journal of the Linnean Society 81: 171-181.
Harris, RN, James TY, Lauer A, Simon MS, Patel A. 2006. Am-
phibian pathogen Batrachochytrium dendrobatidis is inhib-
ited by the cutaneous bacteria of amphibian species. Eco-
Health 3: 53-56.
Hayes T, Haston K, Tsui M, Hoang A, Haeffele C, Vonk A.
2002. Herbicides: Feminization of male frogs in the wild.
Nature 419: 895-896.
Hayes TB, Case P, Chui S, Chung D, Haeffele C, Haston K, Lee
M, Mai VP, Marjuoa Y, Parker J, Tsui M. 2006. Pesticide
mixtures, endocrine disruption, and amphibian declines:
Are we underestimating the impact? Environmental Health
Perspectives 114: 4 0-50.
Holden WM, Hanlon SM, Woodhams DC, Chappell TM, Wells
HL, Glisson SM, McKenzie VJ, Knight R, Parris MJ, Rollins-
Smith LA. 2015. Skin bacteria provide early protection for
newly metamorphosed southern leopard frogs (Rana spheno-
cephala) against the frog-killing fungus, Batrachochytrium
dendrobatidis. Biological Conservation 187: 91-102 .
Hyatt AD, Boyle DG, Olsen V, Boyle DB, Berger L, Obendorf
D, Dalton A, Kriger K, Hero M, Hines H, Phillott R, Cam-
bell R, Marantelli G, Gleason F, Colling A. 2007. Diagnostic
assays and sampling protocols for the detection of Batra-
chochytrium dendrobatidis. Diseases of Aquatic Organ-
isms 73: 175-192.
Israel Nature and Parks Authority. 2009. Israel Nature and
Parks Authority. Available at http://www.parks.org.il/parks/
ParksAndReserves/hula/Pages/default.aspx (accessed 25
Sep 2015).
Johnson ML, Berger L, Philips L, Speare R. 2003. Fungicidal
effects of chemical disinfectants, UV light, desiccation and
heat on the amphibian chytrid Batrachochytrium dendroba-
tidis. Diseases of Aquatic Organisms 57: 255-26 0.
Kaplan D. 2012. Instability in newly-established wetlands? Tra-
jectories of oristic change in the re-ooded Hula peatland,
northern Israel. Mires and Peat 9 : 1-10.
Kaplan D, Oron T, Gutman M. 1998. Development of macro-
phytic vegetation in the Agmon wetland of Israel by sponta-
neous colonization and reintroduction. Wetlands Ecology
and Management 6: 143 -15 0.
Komak S, Crossland MR. 2000. An assessment of the intro-
duced mosquitosh (Gambusia afnis holbrooki) as a pred-
ator of eggs, hatchlings and tadpoles of native and non-na-
tive anurans. Wildlife Research 27: 185 -189.
Kozich JJ, Westcott SL, Baxter N T, Highlander SK, Schloss
PD. 2013. Development of a dual-index sequencing strategy
and curation pipeline for analyzing amplicon sequence data
on the MiSeq Illumina sequencing platform. Applied and
Environmental Microbiology 79: 5112-5120.
Lauer A, Simon MA, Banning JL, André EA, Duncan K, Har ris
RN. 2007. Common cuteaneous bacteria from the Eastern
Red-backed salamander can inhibit pathogenic fungi. Co-
peia 3: 63 0- 640.
Lauer A, Simon MA, Banning JL, Lam BA, Harris RN. 2008.
Diversity of cutaneous bacteria with antifungal activity iso-
lated from female four-toed salamanders. The ISME Jour-
nal 2: 14 5 -1 5 7.
Longcore JE, Pessier AP, Nichols DK. 1999. Batrachochytrium
dendrobatidis gen. et sp. nov., a chytrid pathogenic to am-
phibians. Mycologia 91: 219-227.
McCormick S, Polis GA. 1982. Arthropods that prey on verte-
brates. Biological Reviews 57: 29-58.
Mendelssohn H, Steinitz H. 1943. A new frog from Palestine.
Copeia 4: 231-233.
Oehlmann J, Schulte-Oehlmann U, Kloas W, Jagnytsch O, Lutz
I, Kusk KO, Wollenberger L, Santos EM, Paull GC, Van
Look KJW, Tyler CR. 2009. A critical analysis of the bio-
logical impacts of plasticizers on wildlife. Philosophical
Transactions of the Royal Society B 364: 2047-2062.
Pask JD, Woodhams DC, Rollins-Smith LA. 2012. The ebb and
ow of antimicrobial skin peptides defends northern leop-
31Contributions to Zoology, 86 (1) – 2017
ard frogs (Rana pipiens) against chytridiomycosis. Global
Change Biology 18: 1231-1238.
Payne RJ. 2012. A longer-term perspective on human exploita-
tion and management of peat wetlands: the Hula Valley, Is-
rael. Mires and Peat 9 : 1-9.
Piotrowski JS, Annis SL, Longcore JE. 2004. Physiology of Ba-
trachochytrium dendrobatidis, a chytrid pathogen of am-
phibians. Mycologia 96: 9-15.
Piovia-Scott J, Pope K, Worth SJ, Rosenblum EB, Poorten T,
Refsnider J, Rollins-Smith LA, Reiner t LK, Wells HL, Re-
jmanek D, Lawler S, Foley J. 2015. Correlates of virulence
in the frog-killing fungal pathogen: evidence from a Cali-
fornia amphibian decline. The ISME Journal 9: 1570 -
1578.
Price MN, Dehal PS, Arkin AP. 2010. FastTree 2 - Approxi-
mately maximum-likelihood trees for large alignments.
PLoS ONE: e949 0.
Ramsey JP, Reinert LK, Harper LK, Woodhams DC, Rollins-
Smith LA. 2010. Immune defenses against a fungus linked
to global amphibian declines in the South African clawed
frog, Xenopus laevis. Infection and Immunit y 78: 3981-
3992.
Rebollar EA, Hughey MC, Harr is RN, Domangue RJ, Medina
D, Ibáñez R, Belden LK. 2014. The lethal fungus Batra-
chochytrium dendrobatidis is present in lowland tropical
forests of far eastern Panamá. PLoS ONE: e95484.
Renan S, G af ny S, Geffen E. 2016. Using genetic monitoring to
detect the Hula painted frog populations in the Hula Valley
and testing the method efciency for the monitoring of am-
phibians in Israel. In press in: Proceedings of the 52th Meet-
ing of the Zoological Societ y of Israel, Israel Journal of
Ecology and Evolution 62, Taylor & Francis Online.
Resnick NM, Maloy WL, Guy HR, Zasloff M. 1991. A novel
endopeptidase from Xenopus that recognizes alpha-helical
secondar y structure. Cell 66: 541-554.
Rideout JR, He Y, Navas-Molina JA, Walters WA, Ursell LK,
Gibbons SM, Chase J, McDonald D, Gonzalez A, Robbins-
Pianka A, Clemente JC, Gilbert JA, Huse SM, Zhou H-W,
Knight R, Caporaso JG. 2014. Subsampled open-reference
clustering creates consistent, comprehensive OTU deni-
tions and scales to billions of sequences. Pe erJ 2 : e545.
Rollins-Smith LA, Reinert LK, Miera V, Conlon JM. 2002. An-
timicrobial peptide defenses of the Tarahumara frog, Rana
tarahumarae. Biochemical and Biophysical Research Com-
munications 297: 361-367.
Rollins-Smith LA, Woodhams DC, Reinert LK, Vredenburg
VT, Briggs CJ, Nielsen PF, Conlon JM. 2006. Antimicrobial
peptide defenses of the mountain yellow-legged frog (Rana
muscosa). Developmental and Comparative Immunology
30: 831-842.
Sabino-Pinto J, Bletz MC, Islam MM, Shimizu N, Bhuju S,
Geffers R, Jarek M, Kuraabayashi A, Vences M. 2016. Com-
position of the cutaneous bacterial community in Japanese
amphibians: effects of captivity, host species, and body re-
gion. Microbial Ecology 2016: 1-10.
Schloegel LM, Toledo LF, Longcore JE, Greenspan SE, Vieira
CA, Lee M, Zhao SA, Angen CW, Ferreira CM, Hipolito M,
Da Vies AJ, Cuomo CA, Daszak PD, James TY. 2012. Nov-
el, panzootic and hybrid genotypes of amphibian chytridio-
mycosis associates with the bullfrog trade. Molecular Ecol-
ogy 21: 5162-5177.
Segev O, Mangel M, Blaustein L. 2009. Deleterious effects by
mosquitosh (Gambusia afnis) on the endangered re sal-
amander (Salamandra infraimmaculata). Animal Conser-
vation 1 2(1) : 29-3 7.
Severtsova EA, Aguillon Gutierrez DR. 2013. Postembryonic
development of anurans in ponds littered with metal-con-
taining refuse (simulation experiments). Biology Bulletin
40: 738 -747.
Sidis I, Gasith A. 1985. Food habits of the Caspian terrapin
(Mauremys caspica rivulata) in unpolluted and polluted
habitats in Israel. Journal of Herpetology 19: 108 -115.
Spitzen-van der Sluijs A, Martel A, Hallmann CA, Bosman W,
Garner TWJ, van Rooij P, Jooris R, Haesebrouck F, Pasmans
F. 2014. Environmental determinants of recent endemism of
Batrachochytrium dendrobatidis infections in amphibian
assemblages in the absence of disease outbreaks. Conserva-
tion Biology 28: 1302-1311.
Stav G, Kotler BP, Blaustein L. 2007. Direct and indi rect effects
of dragony (Anax imperator) nymphs on green toad (Bufo
viridis) tadpoles. Hydrobiologia 579: 85-93.
Steinborner ST, Waugh RJ, Bowie JH, Wallace JC, Tyler MJ,
Ramsay SL. 1997. New caerin antibacterial peptides from
the skin glands of the Australian tree frog Litoria xan-
thomera. Journal of Peptide Science 3: 181-185.
Steinitz H. 1955. Occurrence of Discoglossus nigriventer in Is-
rael. Bulletin of the Research Council of Israel B 5: 192 -193.
Sueur J, Aubin T, Simonis C. 2008. Equipment Review: See-
wave, a free modular tool for sound analysis and synthesis.
Bioacoustics 18: 213-22 6.
Sztatecsny M, Schabetsberger R. 2005. Into thin air: vertical
migration, body condition, and quality of terrestrial habitats
of alpine common toads, Bufo bufo. Canadian Journal of
Zoolog y 83: 788-7 96.
Toledo L F. 2005. Predation of juvenile and adult anurans by
invertebrates: cur rent knowledge and perspectives. Herpeto-
logical Review 36: 395-399.
Vences M. 2012a. Discoglossus galganoi Capula, Nascetti,
Lanza, Bullini und Crespo, 1985 – Iberischer Scheibenzün-
gler. Pp. 187-211 in: Grossenbacher K, ed, Handbuch der
Reptilien und Amphibien Europas. Vol. 5/1: Froschlurche
(Anura) I – (Alytidae, Bombinatoridae, Pelodytidae, Pelo-
ba ti dae). AULA-Verlag, Wiebelsheim.
Vences M. 2012b. Discoglossus pictus Otth, 1837 – Gemalter
Scheibenzüngler. Pp. 225-248 in: Grossenbacher K, ed,
Handbuch der Reptilien und Amphibien Europas. Vol. 5/1:
Froschlurche (Anura) I – (Alytidae, Bombinatoridae, Pelo-
dytidae, Pelobatidae). AULA-Verlag, Wiebelsheim.
Vences M, Glaw F. 1996. Further investigations on Discoglos-
sus bioacoustics: Relationship between D. galganoi galga-
noi, D. G. jeanneae and D. pictus scovazzi. Amphibia-Rep-
tilia 17: 333-34 0.
Vences M, Grossenbacher K. 2012. 2.2.1.2. Unterfamilie Disco-
glossinae Günther, 1858; Gattung Discoglossus Ot th, 1837
– Scheibenzüngler. Pp. 177-185 in: Grossenbacher K, ed,
Handbuch der Reptilien und Amphibien Europas. Vol. 5/1:
Froschlurche (Anura) I – (Alytidae, Bombinatoridae, Pelo-
dytidae, Pelobatidae). AULA-Verlag, Wiebelsheim.
Wang Q, Garrity GM, Tiedje JM, Cole JR. 2007. Naive Bayes-
ian classier for rapid assignment of rRNA sequences into
new bacterial taxonomy. Applied and Environmental Mi-
crobiology 73: 5261-5267.
32 Bina Perl et al. – Natural history and conservation of the Hula painted frog
Weber E. 1974. Vergleichende Untersuchungen zur Bioakustik
von Discoglossus pictus Otth 1837 und D. sardus Ts ch udi
1837 (Discoglossidae, Anura). Zoologische Jahrbücher
(Physiologie) 18: 40-8 4.
Woodhams DC, Vredenburg VT, Simon MA, Billheimer D,
Shakhtour B, Shyr Y, Briggs CJ, Rollins-Smith LA, Harris
RN. 2007. Symbiotic bacteria contribute to innate immune
defenses of the threatened mountain yellow-legged frog,
Rana muscosa. Biological Conservation 138: 390-398.
Received: 18 March 2016
Revised and accepted: 8 July 2016
Published online: 13 February 2017
Editor: J.W. Arntzen
33Contributions to Zoology, 86 (1) – 2017
Online supplementary material
S1. Rarefaction curve of bacterial OTUs from cutaneous bacterial communities of Latonia nigriventer compiled
using Chao1 estimation. All quality sequence reads (250–253 bp) were clustered with UCLUST (97% similarity
cut-off) prior to calculation of alpha diversity.
S2. List of species (vertebrates and selected macroinvertebrates) observed at the two main study sites.
S3. Webbing differences between female (left) and male (right) individuals of L. nigriventer. SVL (female –male):
A) 80–82 mm; B) 106–105 mm; C) 107–109 mm; D) 112–114 mm. Images were cut from original photos of living
individuals using Photoshop CS2 software (v 9.0, Adobe Systems, San Jose, CA) and are not to scale.
S4. Raw measurements of captured individuals of Latonia nigriventer. AH =air humidity; aqu (e) = aquatic, partly
exposed; aqu (s) = aquatic, fully submerged; AT = air temperature; BW = body width; CA = capture circumstances;
CL = cloud coverage; CS = tip of characteristic colour patch on forehead to snout; ED = eye diameter; EF = length
of elbow to nger tip; F = female; FS = neck fold to snout; HAL = hand length; HW = head width at eyes; IND =
individual ID; IO = minimal interorbital distance; J = juvenile; LT = tarsal length; M = male; RC = recapture; Res
= Reserve; SVL = snout-vent length; terr (e) = terrestrial, exposed; terr (h) = terrestrial, in hide; WE = Weight; YHM
= Yesod HaMa’ala; – = missing values; 1 = hidden beneath leaf/reed litter; 2 = hidden in small burrow or cavity; 3 =
hidden beneath grass tufts; 4 = hidden under wooden log.
S5. Measurements of three preserved Latonia nigriventer tadpoles. The term keratodont row always refers to a bise-
rial row. A1 (rst upper keratodont row), A2 (second upper keratodont row), A1-2 den (density of the keratodonts in row
A1-2), A1-2 d ist (distance between rows A1-2, measured between outer biserial rows at centre of oral disk), A1-2 num (num-
ber of keratodonts in A1-2), BH (maximal body height), BL (body length), BW (maximal body width), DF (dorsal n
height at mid-tail), DG (size of the dorsal gap of marginal papillae), DMTH (distance of maximal tail height from
the tail-body junction), ED (eye diameter), EH (eyes height – measured from the lower curve of the belly to the
centre of the eye), HAB (height of the point where the axis of the tail myotomes contacts the body – measured from
the lower curve of the belly), IND (inter-narial distance – measured from the centre), IOD (inter-orbital distance –
measured from the centre), JL (maximal jaw sheath length), LR (number of the lower rows of keratodonts), LTRF
(labial tooth row formula), MP (marginal papillae), MTH (maximal tail height), ND (naris diameter), NH (naris
height – measured from the lower curve of the belly to the centre of the naris), NP (naris-pupil distance), ODW
(maximum oral disk width), P1 (rst lower keratodont row), P1gap (medial gap in P1), P2 (second lower keratodont row),
P3 (third lower keratodont row), P1-3 den (density of the keratodonts in row P1-3), P1-3 dist (distance between rows P1-3,
measured between outer biserial rows at centre of oral disk), P1-3 num (number of keratodonts in row P1-3), RN (rostro-
narial distance), SBH (distance between snout and the point of maximal body height), SBW (distance between snout
and the point of maximal body width), SE (snout-eye distance), SL (spiracle length – measured from the visible
edges), SS (snout-spiracle distance), TAL (tail length), TH (tail height at the beginning of the tail), THM (tail height
at mid-tail), Thorn-pap (thorn-shaped papillae), TL (total length), TMH (tail muscle height at the beginning of the
tail), TMHM (tail muscle height at mid-tail), TMW (tail muscle width at the beginning of the tail), UR (number of
the upper rows of keratodonts), VF (ventral n height at mid-tail) and VL (vent tube length).
S6. List of the 20 most frequent bacterial OTUs obtained from Latonia nigriventer skin samples in Yesod HaMa’ala.
Ac = Actinobacteria; Ae = Aeromonadaceae; Al = Alphaproteobacteria; Ba = Bacteroidetes; Be = Betaproteobacte-
ria; C = Caulobacteriaceae; E= Enterobacteriaceae; F = Flavobacteriaceae; G = Gammaproteobacteria; Mi = Mic-
rococcaceae; Mo = Moraxellaceae; P = Pseudomonadaceae; S = Sphingobacteriaceae.
S7. MALDI-TOF MS of skin peptides of two female Latonia nigriventer individuals. Common mass signals are
indicated by arrows.
34 Bina Perl et al. – Natural history and conservation of the Hula painted frog
Appendices
Appendix 1 - Morphological description of adult
specimens of Latonia nigriventer
Body width about two times head width. Head at-
tened and about as long as wide with a projecting snout
that appears rounded in dorsal as well as lateral view.
Nostrils positioned dorsally and closer to the tip of the
snout than to the eyes. Eyes protruding, with heart-
shaped pupil and golden iris. A distinct transversal
fold present in the neck (absent from species of Disco-
glossus, the sister genus of Latonia), varying in depth
depending on the posture. In a bent-forward posture
with stretched dorsal skin the fold merely discernible
as a slight groove. Tympanic membrane indistinct and
comparatively small. Forearms robust and muscular
with four ngers. Fingers free with rounded or pointed
tips (order of length: 1 < 4 < 2 < 3). Hind limbs muscu-
lar with ve toes (order of length: 1 < 2 < 5 < 3 < 4).
Metatarsalia largely separated by webbing. Webbing
formula difcult to assess as subarticular tubercles ab-
sent: 1(1), 2(1), 3(1), 4(1), 5(1), with webbing starting
more or less inconspicuous as dermal ridges. Snout-
vent length (SVL; mean value (range)): ♀♀ 94.8 mm
(69.0–128.4 mm; N = 64), ♂♂ 98.0 mm (66.6–121.4
mm; N = 44). Skin smooth with numerous small round
tubercles on dorsal and ventral surfaces as well as
limbs, but no distinct rows of tubercles. Tubercles on
limbs closer to each other and less rounded, resulting
in a rougher surface.
Colouration in life: Dorsally with ochre, brownish
or auburn background colour and dark brown or olive-
grey colour patches on head and dorsum; rarely with
Fig. 13. Latonia nigriventer specimens
preserved in the collection of the Hebrew
University of Jerusalem. A) Subadult
holotype (collection number HUJ 236)
in dorsal view; B) HUJ 236 in ventral
view; C) specimen collected in 1955
(collection number HUJ 544) in dorsal
view; D) HUJ 544 in ventral view. Both
individuals are assumed to be females.
35Contributions to Zoology, 86 (1) – 2017
rusty coloured ecks. Characteristic dark V-shaped
pattern on head with broad end positioned between
eyes and posteriorly narrowing up to level of arms pre-
sent in all L. nigriventer individuals. Likewise charac-
teristic lateral stripes at head mostly starting posterior
(sometimes anterior) of eyes and extending up to level
of arms. Dark patterns often to some degree rimmed
by a light beige line. Almost all individuals with an
incomplete middorsal band of the background colour
in the posterior part of the dorsum. No distinctly ocel-
lated pattern, nor prominent middorsal band extending
over the whole dorsum as present in the two colour
morphs displayed by both, Discoglossus galganoi and
D. pictus (Vences, 2012a, b), in any L. nigriventer indi-
vidual examined. Ventrally, greyish-black with numer-
ous characteristic white dots corresponding to raised
tubercles and distributed more or less evenly across all
ventral surfaces including arms, legs and plantar sur-
faces. In small juveniles (SVL 20–30 mm), white spots
not raised as in adults. Border between ventral and
dorsal pattern mostly gradually and rather inconspicu-
ous; in some individuals colour transition associated
with marbling in different hues of ochre, brown and
grey that might include arms and legs. Femur and tibia
often with more or less conspicuous dark crossbands.
Single individuals almost without dorsal pattern and
rather uniform. Ventral side sometimes rather light
grey with inconspicuous white tubercles. Individuals
found within deep leaf litter during winter months of-
ten displayed such a rather pale colouration.
Appendix 2 - Morphological data on the holotype of
Latonia nigriventer
The holotype specimen of L. nigriventer (described as
Discoglossus nigriventer Mendelssohn & Steinitz,
1943) is preserved in the collection of the Hebrew Uni-
versity of Jerusalem under the collection number HUJ
236 (Fig. 13 A–B). It is a subadult specimen, possibly a
female. We here provide some additional morphologi-
cal data complementing those of Mendelssohn and
Steinitz (1943). Upon examination in November 2011
(measurements by MV) it had a SVL of 39.9 mm.
Webbing formula was 1(2), 2i(2), 2e(2), 3i(2.5), 3e(2.5),
4i(2.5), 4e (2.75), 5(2.25). In addition, we also exam-
ined a second historical specimen collected in 1955
(no type status; Fig. 13 C–D). This specimen, HUJ
544, has faint remains of keratinised excrescences on
some ngers but no clear nuptial pads. It therefore is
likely a female. SVL is 76.6 mm, webbing formula is
1(0.5), 2i(1.5), 2e(1.5), 3i(2), 3e(2), 4i(2.75), 4e(2 .75),
5(2). Measurements of the two specimens (all in mm;
type specimen measurements followed by those of the
second specimen in parentheses) are as follows: head
width 13.3 (24.4), head length 14.5 (ca. 26.5), tympa-
num diameter 2.3 (4.6), ED 4.0 (6.7), eye-nostril dis-
tance 3.7 (6.1), nostril-snout tip distance 3.0 (5.0), nos-
tril-nostril distance 3.1 (4.7), HAL 9.4 (17.0), forelimb
length 17.5 (not measured), hind limb length 52.5 (not
measured), foot length including tarsus 25.5 (49.7),
foot length 15.7 (not measured), tibia length 16.5
(34.5 ).
Appendix 3 - Morphological description of tadpoles
of Latonia nigriventer
The following description refers to a single, preserved
tadpole in developmental stage 34, xed in 70% etha-
nol and preserved in 5% formalin (eld number ZCMV
12962, BL 8.9 mm, TL 23.8 mm). Abbreviations used:
A1 (rst upper biserial keratodont row), A2 (se con d up -
per biserial keratodont row), A1-2 den (density of the
keratodonts in biserial row A1-2), A1-2 dist (dist ance be-
tween biserial rows A1-2, between outer biserial rows at
centre of oral disk), A1-2 num (number of keratodonts in
A1-2), BH (maximum body height), BL (body length),
BW (maximal body width), DF (dorsal n height at
mid-tail), DG (size of the dorsal gap of marginal papil-
lae), DMTH (distance of maximal tail height from tail-
body junction), ED (horizontal eye diameter), EH (eyes
height – from the lower curve of the belly to the centre
of the eye), HAB (body height where axis of tail my-
otomes contacts body – from the lower curve of belly),
IND (inter-narial distance – from the centre), IOD
(inter-orbital distance – from the centre), JL (maxi-
mum jaw sheath length), LR (number of lower rows of
keratodonts), LTRF (labial tooth row formula), MP
(marginal papillae), MTH (maximal tail height), ND
(naris diameter), NH (naris height – from the lower
curve of the belly to the centre of the naris), NP (naris-
pupil distance), ODW (maximum oral disk width), P1
(rst lower biserial keratodont row), P1ga p (medial gap
in P1), P2 (second lower biserial keratodont row), P3
(third lower biserial keratodont row), P1-3 den (density of
keratodonts in biserial row P1-3), P1-3 di st (dista nce b e-
tween biserial rows P1-3, between outer biserial rows at
centre of oral disk), P1-3 num (number of biserial kerato-
donts in row P1-3), RN (rostro-narial distance), SBH
(distance between snout and point of maximal body
height), SBW (distance between snout and point of
36 Bina Perl et al. – Natural history and conservation of the Hula painted frog
maximal body width), SE (snout-eye distance), SL
(spiracle length – from the visible edges), SS (snout-
spiracle distance), TAL (tail length), TH (tail height at
beginning of tail), THM (tail height at mid-tail),
Thorn-pap (thorn-shaped papillae), TL (total length),
TMH (tail muscle height at beginning of tail), TMHM
(tail muscle height at mid-tail), TMW (tail muscle
width at beginning of tail), UR (number of upper rows
of keratodonts), VF (ventral n height at mid-tail) and
VL (vent tube length).
In dorsal view, body elliptical with small constric-
tions of the body wall at the plane of the spiracle,
maximal body width attained at anterior 1/3 of mid-
body length (SBW 28.4% of BL), and snout rounded.
In lateral view, body depressed (BW of 5.17 corre-
sponds to BH), maximal body height attained be-
tween 2/5 and 3/5 of body length (SBW 58% of BL),
and snout sloping. Eyes medium-sized (ED 8% of
BL), not visible from ventral view, positioned far dor-
sally (EH 64% of BH) and directed laterally, situated
between the 2/10 and 3/10 of the body length (SE 22%
of BL), medium distance between eyes (IOD 55% of
BW). Small rounded nares (ND 3% of BL), positioned
at medium height (NH 55% of BH) and oriented ante-
riorly, situated distinctly closer to snout than to eye
(RN 35% of NP) and lower than eye (NH 85% of EH),
rather close distance between nares (IND 45% of
IOD), dark spot posterior to nare present, other orna-
mentation absent. Spiracle crescentic, opening situat-
ed at centre of ventral side of the body (SL 4% of BL),
directed posteriorly with inner wall present as a slight
ridge, visible from ventral view, invisible from dorsal
and lateral view; opening situated between the 2/5 and
3/5 of the body length (SS 56% of BL), Long medial
vent tube (VL 19% of BL), attached directly to ventral
n. Tail moderat ely long (TA L 169% of BL), ma ximal
tail height lower than body height (MTH 89% of BH),
tail height at mid-tail lower than both body height and
maximal tail height (THM 83% of BH and THM 93%
of MTH), tail height at the beginning of tail slightly
lower than body height (TH 92% of BH). Caudal mus-
culature moderately developed (TMW 39% of BW,
TMH 50% of BH and 57% of MTH, TMHM 32% of
THM and 30% of MTH). Tail muscle does not reach
tail tip (distance of tail muscle to tail tip 6% of TAL).
Moderately high ns (DF 105% of TMHM, VF 110%
of TMHM), at mid tail ventral n higher than dorsal
n (VF 104% of DF). Dorsal n inserts at the tail
muscle posterior to the dorsal body-tail junction, rst
remains almost parallel to dorsal border of tail mus-
cle, then rises gradually until the posterior 1/3 of the
tail where it increases to attain its maximal height,
and then decreases gradually towards tail tip. Ventral
n or iginates at the vent ral term inus of the body, rises
minimally until middle of tail, and then remains al-
most parallel to the ventral border of the tail muscle
until 1/3 of the tail where it likewise decreases gradu-
ally towards the tail tip. Maximal tail height located at
anterior 1/3 of tail (DMTH 35% of TAL), lateral line
vein imperceptible, myosepta faintly discernible in
lateral view in the proximal 2/5 of the tail, point where
the axis of the tail myotomes contacts the body lo-
cated centred in body height (HAB 51% of BH). Tail
tip rounded. Oral disk relatively large (ODW 29% of
BL and 49% of BW), positioned and directed anter-
oventrally, not emarginated. Oral disk not visible from
dorsal view, upper labium is a continuation of snout.
Single row of marginal papillae interrupted by a nar-
row gap on the upper labium (DG 12% of ODW), gap
on the lower labium absent. Total number of marginal
papillae 64. Short and moderately sized conical papil-
lae, longest marginal papillae measured 0.1 mm.
LTRF 2/3(1) after Altig and McDiarmid (1999), with
two rows of keratondonts per ridge that are positioned
at a distance of 0.5 mm from each other. P3 row gener-
ally biserial, but occasionally additional keratodons in
between the two main rows. Very long A1 rows (88%
of ODW). Density of keratodonts varies from 64/mm
to 76/mm, A1 73/mm (total 206). Narrow gap in the
rst posterior interrupted P1 rows (P1g ap 6% of P1).
Rows alignment regular. Short discernible keratodont
(0.08 mm). Distal keratodont same length as those in
the middle. Space between marginal papillae and ker-
atodont rows only absent in P2. Partially keratinised
jaw sheath, with the half part close to the edge being
black and the remainder whitish coloured; nely
pointed serrations; moderately wide jaw sheath (JW
51% of ODW) without medial concavity on the upper
sheath. Lower jaw sheath V-shaped, partially kerati-
nised and partially hidden by the upper jaw sheath
(Table S3).
Colouration in life: Medium brown, translucent and
with a uniformly distinct reticulation, inner anatomy
well discernible. Dorsally, and with exception of the
eye region, body covered by homogenous dark brown
melanophoric pigments and golden speckles probably
corresponding to guanophores. Tail of a slightly light-
er hue than rest of body. Fins translucent, with dark
brown spots of variable sizes and irregularly distribu-
tion, spot density higher in dorsal n. Ventrally, oral
disk and gular region reticulated, branchial regions
reddish (Fig. 7).
37Contributions to Zoology, 86 (1) – 2017
Colouration in preservative: Uniformly dark brown,
body covered by homogenous dark melanophoric pig-
ments giving it a granular appearance, colour slightly
darker above brain and trunk region. Tail musculature
overlaid by dark brown speckles. Fins translucent and,
with exception of outer rim, covered with brown speck-
les. Venter including spiracle pale with irregularly dis-
tributed pale grey spots, spiracle hardly discernible,
intestinal coils visible. Besides the regular pigmenta-
tion, whole body distinctly reticulated as was previ-
ously known to be characteristic for the tadpoles of
only the genera Bombina, Discoglossus and Pelodytes
(Bou lenger, 1891). The n e pigment ary network is most
apparent on ns and paler ground colour, e.g. vent er.
Var iat ion: in total, three tadpoles were investigated,
two of which were in developmental stage 34 (ZCMV
12962 and ZCMV 12963) and one was at developmen-
tal stage 25 (ZCMV 12961). The observation of the
other voucher specimens from the same locality shows
the same general morphology and typical oral disk
conguration of the described specimen, independent
of their developmental stage.
S1. Rarefaction curve of bacterial OTUs from cutaneous bacterial communities of Latonia
nigriventer compiled using Chao1 estimation. All quality sequence reads (250–253 bp) were
clustered with UCLUST (97% similarity cut-off) prior to calculation of alpha diversity.
S2. List of species (vertebrates and selected macroinvertebrates) observed at the two main study sites. 1 Scientific names according to
Catalogue of Life (http://www.catalogueoflife.org/; accessed 09 June 2016). 2 Scientific names according to Amphibian Species of the
World 6.0 (http://research.amnh.org/vz/herpetology/amphibia/; accessed 09 June 2016). 3 Scientific names according to Avibase
(http://avibase.bsc-eoc.org/; accessed 09 June 2016).
Species
Trivial name
Hula Nature Reserve
Yesod HaMa'ala
Fish1
Clarias gariepinus (Burchell, 1822)
African sharptooth catfish
+
-
Cyprinus carpio Linnaeus, 1758
Common carp
+
-
Gambusia affinis (Baird & Girard, 1853)
Western mosquitofish
-
+
Sarotherodon galilaeus (Linnaeus, 1758)
St. Peter's fish
+
-
Tilapia zillii (Gervais, 1848)
Redbelly tilapia
+
-
Amphibians2
Bufotes variabilis (Pallas, 1769)
Green toad
+
-
Hyla savignyi Audouin, 1827
Middle East tree frog
+
+
Latonia nigriventer (Mendelssohn & Steinitz, 1943)
Hula painted frog
+
+
Pelophylax bedriagae (Camerano, 1882)
Levant water frog
+
+
Reptiles1
Chamaeleo chamaeleon (Linnaeus, 1758)
Common chameleon
+
-
Daboia palaestinae (Werner, 1938)
Palestine viper
+
-
Hemorrhois nummifer (Reus, 1834)
Coin-marked snake
+
-
Stellagama stellio Linnaeus, 1758
Roughtail rock agama
+
-
Mauremys caspica (Gmelin, 1774)
Caspian turtle
+
+
Natrix tessellata (Laurenti, 1768)
Dice snake
+
+
Trionyx triunguis (Forskål, 1775)
African softshell turtle
+
-
Birds3
Alcedo atthis (Linnaeus, 1758)
Common kingfisher
+
+
Anas platyrhynchos Linnaeus, 1758
Mallard
+
-
Clanga clanga (Pallas, 1811)
Greater spotted eagle
+
-
Ardea alba Linnaeus, 1758
Great white egret
+
-
Ardea cinerea Linnaeus, 1758
Grey heron
+
-
Ardeola ralloides (Scopoli, 1769)
Squacco heron
+
-
Bubulcus ibis (Linnaeus, 1758)
Cattle egret
+
-
Ceryle rudis (Linnaeus, 1758)
Pied kingfisher
+
-
Ciconia ciconia (Linnaeus, 1758)
White stork
+
-
Circus aeruginosus (Linnaeus, 1758)
Marsh harrier
+
-
Corvus corone Linnaeus, 1758
Hooded crow
+
+
Corvus monedula Linnaeus, 1758
Eurasian jackdaw
+
-
Egretta garzetta (Linnaeus, 1766)
Little egret
+
-
Elanus caeruleus (Desfontaines, 1789)
Black-winged kite
+
-
Erithacus rubecula (Linnaeus, 1758)
European robin
+
+
Falco subbuteo Linnaeus, 1758
Eurasian hobby
+
-
Falco tinnunculus Linnaeus, 1758
Common kestrel
+
+
Fulica atra Linnaeus, 1758
Common coot
+
-
Gallinula chloropus (Linnaeus, 1758)
Common moorhen
+
+
Grus grus (Linnaeus, 1758)
Common crane
+
-
Halcyon smyrnensis (Linnaeus, 1758)
White-throated kingfisher
+
+
Himantopus himantopus (Linnaeus, 1758)
Black-winged stilt
+
-
Merops apiaster Linnaeus, 1758
European bee-eater
+
-
Microcarbo pygmaeus (Pallas, 1773)
Pygmy cormorant
+
-
Motacilla alba Linnaeus, 1758
White wagtail
+
-
Nycticorax nycticorax (Linnaeus, 1758)
Night heron
+
-
Passer domesticus (Linnaeus, 1758)
House sparrow
+
-
Pelecanus onocrotalus Linnaeus, 1758
Great white pelican
+
-
Phalacrocorax carbo (Linnaeus, 1758)
Great cormorant
+
-
Platalea leucorodia Linnaeus, 1758
Eurasian spoonbill
+
-
Plegadis falcinellus (Linnaeus, 1766)
Glossy ibis
+
-
Psittacula krameri (Scopoli, 1769)
Ring-necked parakeet
+
+
Pycnonotus xanthopygos (Hemprich & Ehrenberg, 1833)
White-spectacled bulbul
+
+
Tringa totanus (Linnaeus, 1758)
Common redhank
+
-
Saxicola torquatus (Linnaeus, 1766)
Common stonechat
+
-
Spilopelia senegalensis (Linnaeus, 1766)
Palm dove
-
+
Tachybaptus ruficollis (Pallas, 1764)
Little grebe
+
-
Upupa epops Linnaeus, 1758
Common hoopoe
+
+
Vanellus spinosus (Linnaeus, 1758)
Spur-winged plover
+
-
Mammals1
Canis aureus Linnaeus, 1758
Golden jackal
+
+
Felis chaus Schreber, 1777
Jungle cat
+
-
Herpestes ichneumon (Linnaeus, 1758)
Egypthian mongoose
+
+
Hystrix indica Kerr, 1792
Indian crested porcupine
+
-
Lutra lutra (Linnaeus, 1758)
European otter
+
-
Meles meles (Linnaeus, 1758)
Badger
+
-
Microtus socialis (Pallas, 1773)
Social vole
+
+
Myocastor coypus (Molina, 1782)
Coypu
+
+
Sus scrofa Linnaeus, 1758
Wild boar
+
-
Vulpes vulpes (Linnaeus, 1758)
Red fox
+
-
Invertebrates1
Anax imperator Leach, 1815
Emperor dragonfly
+
+
Crocothemis erythraea Brullé, 1832
Scarlet dragonfly
+
+
Potamon potamios (Olivier, 1804)
Feshwater crab
+
+
S3. Webbing differences between female (left) and male (right) individuals of L. nigriventer.
SVL (female male): A) 80–82 mm; B) 106–105 mm; C) 107–109 mm; D) 112–114 mm. Images
were cut from original photos of living individuals using Photoshop CS2 software (version 9.0,
Adobe Systems, San Jose, CA) and are not to scale.
S4. Raw measurements of captured individuals of Latonia nigriventer. AH =air humidity; aqu (e) = aquatic, partly exposed; aqu (s) =
aquatic, fully submerged; AT = air temperature; BW = body width; CA = capture circumstances; CL = cloud coverage; CS = tip of
characteristic colour patch on forehead to snout; ED = eye diameter; EF = length of elbow to finger tip; F = female; FS = neck fold to
snout; HAL = hand length; HW = head width at eyes; IND = individual ID; IO = minimal interorbital distance; J = juvenile; LT = tarsal
length; M = male; RC = recapture; Res = Reserve; SVL = snout-vent length; terr (e) = terrestrial, exposed; terr (h) = terrestrial, in hide;
WE = Weight; YHM = Yesod HaMa’ala; = missing values; 1 = hidden beneath leaf/reed litter; 2 = hidden in small burrow or cavity; 3 =
hidden beneath grass tufts; 4 = hidden under wooden log.
Date IND Sex RC SVL WE CS FS IO HW BW EF HAL LT ED CL AT AH CA Location
25.11.2013 L#14 J no 47.8 11.6 9.4 9.0 13.0 27.0 11.7 9.4 22.5 25.0 25 terr (h1) Res
05.12.2013 L#15 J no 33.8 3.5 4.7 10.5 4.5 9.6 16.4 7.0 6.0 15.0 3.5 80 terr (h2) Res
15.12.2013 L#16 J no 65.8 26.5 4.8 7.8 10.0 16.6 34.6 24.4 14.3 25.1 4.6 0 terr (h1) Res
22.12.2013 L#14 J yes 48.3 11.0 9.6 14.9 7.6 13.4 25.4 19.7 10.3 21.1 4.1 0 terr (h1) Res
29.12.2013 L#17 F no 80.7 55.0 15.3 24.4 12.1 22.3 43.3 33.9 18.0 37.2 5.4 95 terr (h1) YHM
07.01.2014 L#18 M no 80.0 58.0 15.7 23.7 13.0 21.1 36.6 33.7 19.0 34.5 6.6 10 terr (h3) YHM
12.01.2014 L#19 J no 22.3 1.5 5.6 7.9 4.5 7.5 10.7 9.4 5.0 10.0 2.8 100 terr (h4) YHM
12.01.2014 L#20 J no 22.5 1.5 5.4 7.9 4.6 7.7 11.8 9.3 5.1 10.2 2.9 100 terr (h3) YHM
13.01.2014 L#21 J no 23.5 1.5 5.7 8.0 4.5 7.4 9.9 9.5 5.6 10.1 2.9 90 terr (h3) YHM
16.01.2014 L#22 J no 25.0 1.5 5.7 8.0 4.5 7.8 12.8 9.5 5.4 10.3 3.0 30 20.5 63 terr (h3) YHM
21.01.2014 L#23 J no 23.6 1.4 5.3 7.7 4.5 7.8 12.5 9.5 5.5 10.6 2.9 30 19.9 55 terr (h3) YHM
26.01.2014 L#24 J no 22.2 1.3 5.5 8.6 4.2 7.6 10.6 8.4 5.5 10.3 3.4 100 20.9 43 aqu (s) YHM
26.01.2014 L#25 J no 23.0 1.3 5.6 7.8 4.3 7.4 10.3 9.3 5.4 10.4 3.0 100 20.9 43 terr (h3) YHM
26.01.2014 L#26 J no 16.2 0.5 4.3 6.2 3.3 5.3 8.4 6.4 3.2 7.0 2.1 40 21.0 38 terr (h3) YHM
29.01.2014 L#27 J no 21.3 1.3 5.1 7.8 3.9 7.0 11.0 8.9 4.5 9.5 2.5 30 18.0 78 terr (h3) YHM
03.02.2014 L#28 J no 21.1 1.3 5.1 7.5 3.9 7.0 10.3 9.0 4.3 10.0 2.8 85 15.9 52 terr (h3) YHM
03.02.2014 L#29 J no 22.0 1.3 5.5 7.8 4.6 7.1 10.1 9.5 5.1 10.1 3.0 85 15.9 52 terr (h3) YHM
03.02.2014 L#30 J no 21.7 1.3 5.2 7.4 3.9 7.0 11.1 8.4 5.0 9.9 2.8 85 15.9 52 terr (h3) YHM
05.02.2014 L#31 J no 21.2 1.0 4.8 7.0 4.4 6.6 9.7 8.4 4.9 9.6 2.6 0 12.4 48 terr (h3) YHM
08.02.2014 L#32 J no 26.6 1.8 6.3 8.9 5.0 8.4 11.7 11.8 6.5 12.6 3.2 0 19.2 30 terr (h3) YHM
25.02.2014 L#33 J no 21.7 1.0 5.0 7.4 4.0 6.9 8.2 8.0 4.9 9.8 2.8 80 21.3 51 terr (h3) YHM
25.02.2014 L#34 J no 20.5 1.0 5.0 7.2 4.0 6.6 8.2 8.1 4.9 9.1 2.8 80 21.3 51 terr (h3) YHM
01.03.2014 L#35 J no 23.8 1.5 5.4 7.7 4.7 7.4 11.7 9.0 5.5 10.5 2.8 50 24.2 35 terr (h3) YHM
07.12.2014 L#36 J no 26.4 1.8 5.7 8.5 4.7 8.3 12.4 10.2 6.0 11.9 2.9 0 22.0 53 terr (h1,3) YHM
10.12.2014 L#37 F no 101.3 93.5 15.4 26.9 14.9 24.9 57.3 35.5 18.3 38.5 6.0 14.2 83 terr (h1) YHM
20.12.2014 L#38 J no 30.1 3.0 7.1 10.0 5.9 9.7 16.9 12.5 6.8 20.3 3.4 40 14.5 78 terr (h1) YHM
26.12.2014 L#39 J no 51.9 15.0 10.0 14.8 7.8 13.8 27.1 19.3 11.2 24.5 4.7 0 17.9 55 terr (h1) YHM
29.12.2014 L#40 F no 74.6 49.5 12.4 19.6 11.0 19.9 47.2 26.9 14.4 29.0 5.5 0 15.5 85 terr (h1) Res
05.01.2015 L#41 M no 76.8 47.0 14.3 24.0 12.0 19.6 38.4 31.5 12.4 32.1 6.1 60 12.5 99 terr (h4) Res
08.01.2015 L#42 F no 98.8 94.4 17.1 28.3 15.3 25.5 48.6 37.6 20.0 38.5 6.8 99 12.0 64 terr (h2) YHM
18.01.2015 L#43 F no 100.8 100.1 16.5 22.4 14.8 24.4 55.3 29.3 19.2 37.8 6.6 1 15.3 86 terr (h2) YHM
18.01.2015 L#44 F no 75.2 44.9 12.2 9.3 19.1 36.2 22.7 16.6 5.1 terr (e) Res
27.01.2015 L#45 F no 95.4 85.3 16.2 26.6 14.4 23.1 55.7 35.8 19.0 39.4 6.4 100 20.3 48 terr (h2) YHM
11.02.2015 L#46 F no 90.3 74.8 14.1 25.4 12.3 23.4 54.0 34.3 20.5 35.6 6.1 100 13.1 52 terr (h1) YHM
13.02.2015 L#47 F no 103.3 114.0 18.3 28.8 15.8 26.5 55.3 42.3 21.5 44.6 7.0 100 8.8 99 aqu (e) YHM
13.02.2015 L#48 M no 114.0 140.0 19.0 31.2 17.1 28.3 56.5 46.4 22.8 42.9 7.2 100 8.8 99 aqu (e) YHM
13.02.2015 L#49 M no 121.4 190.0 22.0 35.3 17.6 31.2 64.2 45.8 25.6 48.6 8.2 100 8.8 99 aqu (e) YHM
13.02.2015 L#50 F no 95.7 88.0 17.0 27.4 15.0 24.6 50.3 38.5 20.6 40.2 6.3 100 8.8 99 aqu (e) YHM
14.02.2015 L#51 F no 88.0 79.5 15.0 25.8 13.7 23.1 51.2 35.6 18.9 34.6 6.1 10 6.9 94 aqu (e) YHM
14.02.2015 L#52 F no 90.4 86.0 16.3 25.7 13.2 21.7 51.7 34.7 18.3 37.8 5.8 10 6.9 94 aqu (e) YHM
14.02.2015 L#53 F no 128.4 187.0 22.7 34.5 18.7 32.3 71.1 45.1 23.4 46.7 8.1 10 6.9 94 aqu (e) YHM
14.02.2015 L#37 F yes 95.5 94.1 15.4 26.9 14.9 24.9 57.6 35.5 18.3 38.5 6.0 10 6.9 94 aqu (e) YHM
15.02.2015 L#54 M no 89.5 74.9 16.3 25.2 11.9 23.6 45.5 35.7 18.3 33.9 6.6 100 10.9 99 aqu (e) YHM
15.02.2015 L#55 F no 94.9 79.1 15.4 24.9 13.5 22.9 50.7 33.2 19.5 36.1 6.0 100 10.9 99 aqu (e) YHM
15.02.2015 L#56 F no 109.0 100.3 18.0 29.6 14.9 26.2 58.3 38.2 20.3 39.3 7.1 100 10.9 99 aqu (e) YHM
15.02.2015 L#57 M no 68.9 26.0 11.8 13.1 10.4 11.8 35.4 27.9 14.6 28.1 4.8 100 10.9 99 aqu (e) YHM
15.02.2015 L#58 M no 112.4 124.0 18.4 30.9 15.7 22.1 63.7 43.0 22.8 43.2 7.3 100 10.9 99 aqu (e) YHM
15.02.2015 L#59 J no 64.3 26.3 12.5 18.0 9.9 16.9 33.4 26.3 14.0 27.7 5.0 100 10.9 99 aqu (e) YHM
15.02.2015 L#60 J no 65.8 25.5 12.0 17.4 9.7 12.2 30.0 26.2 15.8 26.9 4.6 100 10.9 99 aqu (e) YHM
23.02.2015 L#53 F yes 128.4 186.0 22.7 34.5 18.7 32.3 73.5 45.1 23.4 46.7 8.1 15.4 51 aqu (e) YHM
25.02.2015 L#61 F no 88.1 69.1 16.6 24.8 7.8 22.7 51.0 28.5 17.7 35.1 6.0 5 11.7 78 aqu (e) YHM
25.02.2015 L#62 M no 101.3 107.0 17.9 29.5 16.3 26.5 53.0 43.4 21.4 38.6 6.7 5 11.7 78 aqu (e) YHM
25.02.2015 L#63 F no 84.7 60.2 13.6 22.9 12 20.5 49.9 31.2 15.8 32.0 5.4 5 11.7 78 aqu (e) YHM
25.02.2015 L#64 M no 110.8 149.0 19.6 31.7 16.8 23.3 66.5 43.5 23.9 43.3 7.3 5 11.7 78 aqu (e) YHM
08.03.2015 L#65 M no 119.0 145.5 14.8 35.0 8.5 33.1 63.0 33.6 48.3 0 aqu (e) YHM
08.03.2015 L#66 M no 97.0 82.7 15.6 28.2 9.7 25.1 63.0 28.2 44.0 9.3 0 aqu (e) YHM
09.03.2015 L#67 M no 97.0 78.5 13.3 23.0 10.4 24.4 57.0 28.7 34.4 6.6 10 aqu (e) YHM
09.03.2015 L#46 F yes 88.0 66.0 14.1 25.4 12.3 23.4 51.0 26.7 34.2 6.1 10 aqu (e) YHM
09.03.2015 L#68 F no 86.1 56.7 13.5 26.1 10.8 23.0 47.0 28.4 37.0 5.7 10 aqu (e) YHM
09.03.2015 L#69 M no 85.3 56.0 13.1 24.0 9.0 22.2 43.0 21.3 39.0 6.8 10 aqu (e) YHM
09.03.2015 L#70 M no 102.0 89.5 15.2 26.6 13.3 25.5 53.0 34.1 41.0 7.5 10 aqu (e) YHM
09.03.2015 L#71 M no 109.0 91.1 13.6 22.8 10.6 26.4 53.0 30.6 41.0 5.9 10 aqu (e) YHM
09.03.2015 L#72 F no 112.0 98.2 16.1 30.1 10.8 27.2 57.0 29.7 48.0 8.2 10 aqu (e) YHM
09.03.2015 L#73 F no 98.0 79.7 15.5 25.6 10.8 25.2 56.0 26.1 39.0 6.4 10 aqu (e) YHM
09.03.2015 L#74 M no 89.0 70.1 15.2 25.2 10.3 24.7 51.0 29.2 39.6 6.4 10 aqu (e) YHM
20.04.2015 L#75 M no 106.2 99.0 17.9 30.4 15.7 27.7 52.8 41.7 22.0 43.8 8.1 aqu (e) YHM
20.04.2015 L#76 F no 108.2 119.0 17.0 26.7 14.0 24.3 65.8 36.6 19.3 38.6 6.6 19.0 47 aqu (e) YHM
20.04.2015 L#77 M no 105.8 132.0 17.7 29.0 18.9 29.4 58.9 42.8 21.9 44.3 7.5 19.0 47 aqu (e) YHM
20.04.2015 L#54 M yes 90.8 83.4 16.3 25.2 11.9 23.6 41.6 35.7 18.3 33.9 6.6 19.0 47 aqu (e) YHM
20.04.2015 L#78 F no 118.9 159.5 20.3 30.5 22.0 75.0 40.3 22.5 42.5 7.4 19.0 47 aqu (e) YHM
20.04.2015 L#79 M no 93.3 80.1 15.3 24.9 12.5 21.6 52.3 34.8 18.8 37.0 6.5 19.0 47 aqu (e) YHM
20.04.2015 L#80 F no 98.5 88.3 15.5 24.4 13.0 22.4 57.5 34.6 17.2 36.2 6.7 19.0 47 aqu (e) YHM
21.04.2015 L#81 F no 110.9 135.0 29.9 20.0 15.2 26.8 66.3 39.2 20.5 43.0 7.4 17.8 65 aqu (e) YHM
21.04.2015 L#53 F yes 128.4 223.0 22.7 34.5 18.3 32.3 82.8 45.1 23.4 46.7 8.1 17.8 64 aqu (e) YHM
15.05.2015 L#82 M no 98.0 84.5 0 aqu (s) YHM
15.05.2015 L#83 M no 105.0 87.0 0 aqu (e) YHM
15.05.2015 L#84 J no 43.0 89.0 0 aqu (e) YHM
15.05.2015 L#85 M no 116.0 138.5 0 aqu (e) YHM
15.05.2015 L#81 F yes 113.0 112.5 0 aqu (e) YHM
31.05.2015 L#52 F yes 90.0 73.0 aqu (e) YHM
31.05.2015 L#86 F no 100.0 93.0 aqu (e) YHM
31.05.2015 L#87 F no 85.0 57.0 aqu (e) YHM
31.05.2015 L#88 F no 69.0 32.0 aqu (e) YHM
31.05.2015 L#89 J no 59.0 25.0 aqu (e) YHM
31.05.2015 L#60 M yes 73.0 46.0 aqu (e) YHM
31.05.2015 L#90 F no 78.0 47.0 aqu (e) YHM
31.05.2015 L#91 F no 90.0 71.0 aqu (e) YHM
31.05.2015 L#92 M no 82.0 64.0 aqu (e) YHM
31.05.2015 L#93 F no 84.0 62.0 aqu (e) YHM
31.05.2015 L#94 F no 90.0 70.0 aqu (e) YHM
31.05.2015 L#95 J no 54.0 14.0 aqu (e) YHM
31.05.2015 L#96 M no 83.0 53.0 aqu (e) YHM
31.05.2015 L#43 F yes 100.0 88.0 terr (e) YHM
31.05.2015 L#97 M no 115.0 115.0 aqu (e) YHM
31.05.2015 L#98 M no 96.0 83.0 aqu (e) YHM
31.05.2015 L#99 M no 97.0 90.0 aqu (e) YHM
31.05.2015 L#100 M no 92.0 74.0 aqu (e) YHM
31.05.2015 L#101 M no 99.0 85.0 aqu (e) YHM
31.05.2015 L#102 M no 115.0 118.0 aqu (e) YHM
31.05.2015 L#85 M yes 113.0 131.0 aqu (e) YHM
31.05.2015 L#103 M no 93.0 77.0 aqu (e) YHM
31.05.2015 L#73 F yes 98.0 95.0 aqu (e) YHM
31.05.2015 L#104 M no 90.0 85.0 aqu (e) YHM
31.05.2015 L#105 F no 112.0 151.0 aqu (e) YHM
31.05.2015 L#106 M no 110.0 122.0 aqu (e) YHM
29.06.2015 L#107 F no 103.5 88.8 0 2127 76 aqu (e) YHM
29.06.2015 L#108 F no 86.3 49.2 0 2127 76 aqu (e) YHM
29.06.2015 L#81 F yes 109.4 120.2 0 2127 76 aqu (e) YHM
29.06.2015 L#109 M no 105.2 106.4 0 2127 76 aqu (e) YHM
29.06.2015 L#110 F no 99.8 78.3 0 2127 76 aqu (e) YHM
29.06.2015 L#111 M no 100.3 88.7 0 2127 76 aqu (e) YHM
29.06.2015 L#112 F no 97.0 79.2 0 2127 76 aqu (e) YHM
29.06.2015 L#50 F yes 103.4 85.8 0 2127 76 aqu (e) YHM
29.06.2015 L#113 F no 106.9 116.2 0 2127 76 aqu (e) YHM
29.06.2015 L#114 F no 109.4 110.3 0 2127 76 aqu (e) YHM
29.06.2015 L#71 M yes 100.5 96.6 0 2127 76 aqu (e) YHM
29.06.2015 L#115 F no 98.5 91.4 0 2127 76 aqu (e) YHM
29.06.2015 L#75 M yes 106.0 84.9 0 2127 76 aqu (e) YHM
29.06.2015 L#116 F no 102.3 90.5 0 2127 76 aqu (e) YHM
29.06.2015 L#117 F no 79.5 51.2 0 2127 76 aqu (e) YHM
29.06.2015 L#118 F no 112.1 112.5 0 2127 76 aqu (e) YHM
29.06.2015 L#119 F no 105.3 94.6 0 2127 76 aqu (e) YHM
29.06.2015 L#120 F no 105.8 104.1 0 2127 76 aqu (e) YHM
29.06.2015 L#121 M no 107.4 98.1 0 2127 76 aqu (e) YHM
29.06.2015 L#76 F yes 110.3 103.5 0 2127 76 aqu (e) YHM
29.06.2015 L#122 F no 77.1 36.8 0 2127 76 aqu (e) YHM
29.06.2015 L#123 F no 90.7 76.6 0 2127 76 aqu (e) YHM
29.06.2015 L#124 M no 100.7 87.5 0 2127 76 aqu (e) YHM
29.06.2015 L#125 F no 103.7 85.5 0 2127 76 aqu (e) YHM
29.06.2015 L#126 F no 88.9 55.5 0 2127 76 aqu (e) YHM
29.06.2015 L#127 F no 76.0 36.2 0 2127 76 aqu (e) YHM
29.06.2015 L#128 M no 66.6 28.0 0 2127 76 aqu (e) YHM
29.06.2015 L#129 F no 102.3 84.2 0 2127 76 aqu (e) YHM
29.06.2015 L#130 F no 93.5 64.0 0 2127 76 aqu (e) YHM
29.06.2015 L#131 F no 93.0 66.0 0 2127 76 aqu (e) YHM
29.06.2015 L#132 M no 81.7 45.2 0 2127 76 aqu (e) YHM
29.06.2015 L#133 M no 84.2 60.0 0 2127 76 aqu (e) YHM
29.06.2015 L#134 F no 78.4 46.6 0 2127 76 aqu (e) YHM
29.06.2015 L#135 F no 91.9 75.6 0 2127 76 aqu (e) YHM
29.06.2015 L#136 F no 96.4 75.9 0 2127 76 aqu (e) YHM
29.06.2015 L#137 M no 102.0 105.5 0 2127 76 aqu (e) YHM
29.06.2015 L#138 F no 101.6 91.5 0 2127 76 aqu (e) YHM
16.09.2015 L#139 F no 98.5 83.0 0 2123 63 aqu (e) YHM
16.09.2015 L#74 M yes 101.3 104.2 0 2123 63 aqu (e) YHM
16.09.2015 L#127 F yes 78.4 51.3 0 2123 63 aqu (e) YHM
16.09.2015 L#140 M no 98.2 95.5 0 2123 63 aqu (e) YHM
16.09.2015 L#141 F no 113.8 100.5 0 2123 63 aqu (e) YHM
16.09.2015 L#110 F yes 99.5 75.5 0 2123 63 aqu (e) YHM
16.09.2015 L#142 F no 100.5 79.7 0 2123 63 aqu (e) YHM
16.09.2015 L#43 F yes 110.3 88.8 0 2123 63 terr (e) YHM
16.09.2015 L#143 F no 95.0 74.3 0 2123 63 aqu (e) YHM
16.09.2015 L#144 F no 79.3 64.2 0 2123 63 terr (e) YHM
16.09.2015 L#145 M no 110.6 93.0 0 2123 63 aqu (e) YHM
16.09.2015 L#146 J no 55.6 14.6 0 2123 63 aqu (e) YHM
16.09.2015 L#147 F no 73.6 35.8 0 2123 63 aqu (e) YHM
16.09.2015 L#70 M yes 103.0 93.3 0 2123 63 aqu (e) YHM
16.09.2015 L#63 F yes 91.8 65.5 0 2123 63 aqu (e) YHM
16.09.2015 L#148 M no 83.3 56.6 0 2123 63 aqu (e) YHM
16.09.2015 L#149 F no 74.6 32.7 0 2123 63 aqu (e) YHM
16.09.2015 L#60 M yes 76.3 68.5 0 2123 63 aqu (e) YHM
16.09.2015 L#134 F yes 84.7 57.5 0 2123 63 aqu (e) YHM
16.09.2015 L#150 F no 92.2 69.2 0 2123 63 aqu (e) YHM
S5. Measurements of three preserved Latonia nigriventer tadpoles. The term keratodont row
always refers to a biserial row. A1 (first upper keratodont row), A2 (second upper keratodont
row), A1-2 den (density of the keratodonts in row A1-2), A1-2 dist (distance between rows A1-2,
measured between outer biserial rows at centre of oral disk), A1-2 num (number of keratodonts in
A1-2), BH (maximal body height), BL (body length), BW (maximal body width), DF (dorsal fin
height at mid-tail), DG (size of the dorsal gap of marginal papillae), DMTH (distance of maximal
tail height from the tail-body junction), ED (eye diameter), EH (eyes height – measured from the
lower curve of the belly to the centre of the eye), HAB (height of the point where the axis of the
tail myotomes contacts the body – measured from the lower curve of the belly), IND (inter-narial
distance measured from the centre), IOD (inter-orbital distance measured from the centre), JL
(maximal jaw sheath length), LR (number of the lower rows of keratodonts), LTRF (labial tooth
row formula), MP (marginal papillae), MTH (maximal tail height), ND (naris diameter), NH
(naris height – measured from the lower curve of the belly to the centre of the naris), NP (naris-
pupil distance), ODW (maximum oral disk width), P1 (first lower keratodont row), P1gap (medial
gap in P1), P2 (second lower keratodont row), P3 (third lower keratodont row), P1-3 den (density of
the keratodonts in row P1-3), P1-3 dist (distance between rows P1-3, measured between outer biserial
rows at centre of oral disk), P1-3 num (number of keratodonts in row P1-3), RN (rostro-narial
distance), SBH (distance between snout and the point of maximal body height), SBW (distance
between snout and the point of maximal body width), SE (snout-eye distance), SL (spiracle
length – measured from the visible edges), SS (snout-spiracle distance), TAL (tail length), TH
(tail height at the beginning of the tail), THM (tail height at mid-tail), Thorn-pap (thorn-shaped
papillae), TL (total length), TMH (tail muscle height at the beginning of the tail), TMHM (tail
muscle height at mid-tail), TMW (tail muscle width at the beginning of the tail), UR (number of
the upper rows of keratodonts), VF (ventral fin height at mid-tail) and VL (vent tube length).
Tadpole ID Stage A1 A2 A1-2 den A1-2 dist A1-2 num BH BL BW
ZCMV12961 25
2.5 4.5 3.3
ZCMV12962 34 2.2 2.2 73 0.3 412 5.2 8.9 5.2
ZCMV12963 34
4.6 8.9 5.2
Tadpole ID DF DG DMTH ED EH HAB IND IOD JL
ZCMV12961 0.8
2.1 0.4 1.7 1.3
1.6
ZCMV12962 1.4 0.3 5.2 0.7 3.3 2.6 1.3 2.9 1.3
ZCMV12963 1.6
4.2 1.1 2.8 2.3 1.4 2.8
Tadpole ID MP MTH ND NH NP ODW P1 right P1 left P1 gap
ZCMV12961
2.6 0.1 1.4 0.8
ZCMV12962 0.1 4.6 0.3 2.8 1.4 2.6 1.0 1.0 0.1
ZCMV12963
4.6 0.3 2.6 1.6
Tadpole ID P2 P3 P1-3 den P1-3 dist P1-3 num RN SBH SBW SE
ZCMV12961
0.4 3.5 2.0 1.2
ZCMV12962 2.2 1.7 81 0.4 477 0.5 5.1 3.1 1.9
ZCMV12963