Alytes_2015_32 - Amphibian Survival Alliance

Transcription

ISSN 0753-4973
VOLUME 32
|
DECEMBER 2015
VOLUME 32
DECEMBER 2015
Illustration. Fernando Correia (Chioglossa lusitanica)
Published
by the
International
Society for
the Study and
Conservation
of Amphibians
(ISSCA)
In partnership
with Amphibian
Survival Alliance
(ASA) and
IUCN/SSC
Amphibian
Specialist Group
(ASG)
2
OBITUARY
2015 | VOLUME 32 | PAGES 3-5
Leszek Berger
1925-2012
Krzysztof Kolenda1*, Mikołaj Kaczmarski2
1.
Department of Evolutionary Biology and Conservation of Vertebrates, Institute of Environmental Biology, Wroclaw University, ul. Sienkiewicza 21, 50-335 Wroclaw, Poland
2.
Institute of Zoology, Poznan University of Life Sciences, Wojska Polskiego 71 C, 60-625 Poznań, Poland
Fabulous green frog’s professor
With the death of Professor Leszek Berger at the age of 87 on 8 July 2012, the world of herpetology and
the Polish Academy of Science lost a major figure. He was a great teacher and beloved colleague.
Leszek Berger was born 10 February 1925 in Pabianice. He spent his childhood and youth in Lewkowiec
and Ostrów Wielkopolski (southern Wielkopolska region) near the Ołobok River. In 1947, he graduated from
the Male Secondary School in Ostrów Wielkopolski. Inspired by the richness of the local wildlife, he decided to
study biology. He was the first member of his family to attend university, studying biology at the Mathematics and
Natural Science Faculty of Poznań University in the years 1947-50. As a fourth-year student he began working
as an assistant in the Natural History Museum in Poznań. His master’s and doctoral dissertations concerned
molluscs. Required by his supervisor to study amphibians for his doctorate, he decided to focus on green frogs,
which heretofore had been considered a single taxon, Rana esculenta Linnaeus, 1758. At the outset of his studies
he proved that three morphological forms lived in Poland, namely, Rana esculenta esculenta, R. e. lessonae
Camerano, 1882 and R. ridibunda Pallas, 1771 ‒ the last-mentioned of which, in 1963, he raised to the rank of
species, a designation that has not been accepted by contemporary researchers. Fascinated by his results, in 1963
he built his first farm for green frogs in Poznań (Fig. 1) and in 1988 another in his garden in Jaskółki. Based on the
first crosses between green frogs, he discovered that the edible frog Rana esculenta (today Pelophylax esculentus)
was not a species but an interspecies hybrid between the pool frog R. lessonae (P. lessonae) and the marsh frog R.
ridibunda (P. ridibundus). Further studies showed that the edible frog had been created through backcrosses with
one of its parental species and maintained its population thanks to hybridogenesis, a unique reproductive system.
The gametes of hybrids contain only one of the genomes of the parental species; the second set of chromosomes
Received 08 December 2014
*Corresponding author
Accepted 08 December 2014
Published Online 10 March 2015
© ISSCA and authors 2015
krzysztof.kolenda@uni.wroc.pl
KRZYSZTOF KOLENDA & MIKOŁAJ KACZMARSKI
Figure 1. First green frog farm built by Berger in Poznań (Professor Berger’s archive).
is rejected from the germline prior to meiosis. Thus, in meiosis, the phenomenon of independent segregation of
chromosomes is excluded, and the unrecombined genome of one parental species is transmitted to the gametes.
Discovery of the process of hybridogenesis became the theme of his research dissertation. However,
his work was rejected by Poznań University and Jagiellonian University of Cracow because it was incompatible
with Mendel’s laws of genetics. Put simply, nobody believed him. After several years of effort, he received the
title of doctor habilitatus in 1969 from the University of Agriculture in Poznań. Since 1971, many scientists have
confirmed his thesis. For his discovery of the laws of a new type of heredity, he received the first-degree award
from the Polish Academy of Science in 1973. The stress connected with the problems of defending his dissertation
cost him a heart attack. On the basis of Berger’s results, zoologists around the world have begun to conduct
research on green frogs and as a result have discovered many new frog species. One of them, the former Rana
bergeri Günther, 1985 (now Pelophylax bergeri), was named in his honour.
Berger, who became an associate professor in 1981 and a full professor in 1990, retired upon turning sixtyfive. About ten years later, as he was planning to close his frog farm, or ‘ranarium’, an albino green frog was born
there, and this event prompted him to continue his research to the end of his life.
Over the course of 39 years (1963-2001) Professor Berger bred and reproduced all 16 taxa of western
Palearctic green frogs. He obtained over 800000 offspring from approximately 1500 crosses. This was unique ‒
probably the most prolific scientific breeding of amphibians in the world.
His scientific output includes over 120 publications, including two articles in the international journal
Alytes (1992 & 1993):
• Berger L., Rybacki M. (1993). Growth and maturity of brown frogs, Rana arvalis and Rana temporaria
in central Poland. Alytes, 11: 17-24.
• Berger L., Rybacki M. (1992). Sperm competition in European water frogs. Alytes, 10: 113-116.
In 1989 he organised a herpetological meeting in Turew (Wielkopolska). Thanks to this event,
herpetological meetings are still organised at irregular intervals in the form of a nationwide conference in Cracow.
4
ALYTES 2015 | 32
Today Professor Berger’s research is being continued in
the Department of Evolutionary Biology and Conservation
of Vertebrates at the University of Wrocław.
Berger was not only an outstanding scientist, but
also a proponent of nature conservation and ecological
education (Fig. 2). He conducted talks about amphibians
in his ‘ranarium’ for many pupils and biology teachers as
well as lectures in schools and universities. He also wrote
articles for local newspapers about the need for nature
conservation. In 2008, Professor Berger published a book
on the subject of European green frogs and their protection
(in Polish and English) in which he suggested active
protection of European amphibians based on so-called
rearing-tandems:
• Berger, L. (2008). European green frogs and
their protection. Ecological Library
Foundation in Poznań, Poland.
Professor Berger was greatly respected by many
generations of Polish and foreign herpetologists. Apart
from his great achievements, he was a very generous
person and set an example of respect for people and nature.
Leszek Berger was a great storyteller and had an excellent
collection of anecdotes drawn from his life experiences. He
Figure 2. Retired Prof. Berger with his exposition about green
is
greatly missed by all who knew him. His resting place
frogs at the Poznan International Fair (Professor Berger’s archive).
is in a cemetery near the Żurawiniec reserve in Poznań,
where he began his research on amphibians.
In October 2013, the Leszek Berger Park of
Nature Education was opened in Poznań; in June 2014, a pond in the shape of a frog was created in Raszków (near
the village of Jaskółki) in honour of Professor Berger.
Professor Berger’s most important articles:
• Berger, L. (1966). Biometrical studies on the population of green frogs form the environs of Poznan.
Annales Zoologici, 23(11): 303-324.
• Berger, L. (1967). Embryonal and larval development of F1 generation of green frogs different combinations. Acta Zoologica Cracoviensia, 12(7): 123-160.
• Berger, L. (1973). Systematics and hybridization in European green frogs of Rana esculenta complex. Journal Herpetology, 7(1): 1-10.
• Uzzell, T., Berger, L. (1975). Electrophoretic phenotypes of Rana ridibunda, Rana lessonae, and their
hybridogenetic associate, Rana esculenta. Proceedings of the Academy Natural Sciences of
Philadelphia, 127(2): 13-24.
• Berger, L. (1988). Principles of studies of European water frogs. Acta Zoologica Cracoviensia, 31(21):
563-580.
• Berger, L., Uzzell, T., Hotz, H. (1988). Sex determination and sex ratios in western Palearctic water
frogs: XX and XY female hybrids in the Pannonian Basin? Proceedings of the Academy Natural
Sciences of Philadelphia, 140(1): 220-239.
• Pabijan, M., Czarniewska, E., Berger, L. (2004). Amelanistic phenotypes in westen Palearctic water
frogs from Poland. Herpetozoa, 17: 127-134.
• Tryjanowski, P., Sparks, T., Rybacki, M., Berger, L. (2006). Is body size of the water frog Rana esculenta
complex responding to climate change? Naturwissenschaften, 93: 110-113.
5
6
RESEARCH ARTICLE
2015 | VOLUME 32 | PAGES 7-15
Effects of three diets on development of
Mantidactylus betsileanus larvae in captivity
Jeanne Soamiarimampionona1, Sidonie Samina Sam1, Rainer Dolch1, Katy Klymus2,
Falitiana Rabemananjara3, Eric Robsomanitrandrasana4, Justin Claude Rakotoarisoa1,
Devin Edmonds1*
1.
Association Mitsinjo, Andasibe, Madagascar
University of Toledo, Lake Erie Center, Oregon OH, U.S.A.
3.
Amphibian Specialist Group and the Société pour la Conservation des Amphibiens de Madagascar (SCAM), Ambohimanga, Bongatsara,
Madagascar
4.
La Direction Générale des Forêts, Nanisana, Antananarivo, Madagascar
2.
Conservation breeding programmes are increasingly needed for amphibians given the ongoing amphibian
extinction crisis, yet key aspects of husbandry such as larval diet remain understudied and in many
cases completely unknown. In Madagascar, enacting such programmes can also be challenging due to
the unavailability of diets designed specifically for tadpoles. We tested three diets locally available in
Madagascar — mustard greens, spirulina algae, and atyid shrimp — on the larvae of Mantidactylus
betsileanus and recorded their growth and development. Tadpoles fed mustard greens took longer
to develop and completed metamorphosis at a smaller size. No difference was found in the survival
of tadpoles between treatments. These results suggest that mustard greens are a poor food source
for rearing M. betsileanus and similar species. Instead, spirulina and atyid shrimp should be used,
although other alternatives such as commercially manufactured tadpole and fish foods might yield even
better results.
INTRODUCTION
Captive breeding programmes can be effective in combating amphibian extinctions and population
declines, especially in light of the threat presented by emerging infectious diseases, such as chytridiomycosis
(Gagliardo et al., 2008; Griffiths & Pavajeau, 2008; Tapley et al., in press).To help offset population declines,
offspring produced in captivity can be used to supplement declining wild populations, selected for increased disease
resistance or reproductive capacity, or reintroduced at created habitat in disease-free locations (Scheele et al.,
2014). In order to have the capacity to implement these types of programs, it is essential to have an understanding
of the natural history of the target species and ideally to have already on hand a protocol developed for best captive
care practices (Michaels et al., 2014).
The nutritional requirements of amphibians in captivity are one of the most important aspects of captive
management, yet they remain understudied compared to other taxa such as reptiles or fish (McWilliams, 2008;
Pessier, 2010). The diet of larvae in particular is important because it has significant effects on their survival,
growth, and development, the relationship of which has been studied in a number of anuran species (Steinwascher
& Travis, 1983; Leips & Travis, 1994; Carmona-Osalde, 1996; Álvarez & Nicieza, 2002). Special care needs to
be taken to ensure that the food offered to larvae produces healthy adult animals, in particular when long-term
sustained viability of a captive population is needed (McCallum & Trauth, 2002).
Understanding the diet of a species in nature can help guide captive rearing techniques (Pramuk &
Gagliardo, 2012), however, the natural history and feeding habits of the majority of amphibian species in their
Received 01 May 2015
Accepted 04 August 2015
Published Online 05 October 2015
devin@amphibiancare.com
JEANNE SOAMIARIMAMPIONONA et al.
larval stage remain largely unknown (Altig et al., 2007; Wells, 2007). Many questions remain regarding the
optimal diet for most species in captivity, providing an opportunity to test hypotheses about different foods and
their effects on development of offspring. What constitutes the optimal diet may vary depending on the goals of the
breeding programme, but in general it is advantageous for tadpoles to complete metamorphosis at a large size and
in a short time to increase their overall fitness (Martins et al., 2013). It’s also important to consider survivorship
post metamorphosis and avoid unintentional captive selection pressures and genetic bottlenecks, especially if the
purpose of the captive population is to supply stock for reintroductions (Christie et al., 2012). Determining the
best food takes both of these points into consideration, alongside the availability of different food types and costs
to the breeding programme.
We developed a facility in Andasibe, Madagascar in 2011 to enact captive breeding programmes as part of
the national amphibian conservation strategy known as the Sahonagasy Action Plan (Andreone & Randriamahazo,
2008; Edmonds et al., 2012). One of the goal’s of the project is to determine the captive care and husbandry
requirements for the numerous Malagasy amphibian species that have yet to be managed in captivity (see
García et al., 2008) so that if population declines or extinctions are detected survival assurance colonies can
rapidly be established. This is especially important in light of the recent news that the amphibian chytrid fungus
Batrachochytrium dendrobatidis is widespread on the island (Bletz et al., 2015). Because of its somewhat remote
location, the breeding facility relies on locally available materials as much as possible to avoid being dependent
on imported supplies since commercial tadpole diets are not available in Madagascar and aquarium fish foods can
only occasionally be found for sale, and even then are often already expired or are sold at exaggerated prices. As a
result, we reviewed products available locally in and around Andasibe that could be used to feed tadpoles produced
at the breeding facility.
Our focal species for the study, the Betsileo Madagascar Frog (Mantidactylus betsileanus), is known from
the areas around Andasibe and Ranomafana in eastern Madagascar. It is semi-aquatic and found in the vicinity of
rainforests, where it breeds in slow-moving streams, swampy areas, and associated shallow water bodies (Glaw &
Vences, 2007; Vences & Nussbaum, 2008). The larvae of M. betsileanus have been raised in captivity a few times
(Arnoult & Razarihelisoa, 1967; Blommers-Schlösser, 1979; Scheld et al., 2013), with tadpoles in the latter study
being fed a diet of fish flake and algae. It is not clear whether the two earliest reports were actually M. betsileanus
or one of the numerous similar cryptic species only recently recognized through DNA-barcoding (Vieites et al.,
2009; Perl et al., 2014), nevertheless there is still much to be learned about this species’ optimal requirements in
captivity.
In this article we describe the results of an experiment to determine the effects of three diets on the
survival and development of M. betsileanus larvae in captivity. Based on qualitative observations raising larvae on
mixed diets previously, we predicted that a diet of dried atyid shrimp would result in higher survivorship, faster
growth, and larger size at metamorphosis compared to diets of spirulina algae or mustard greens. We conclude by
discussing the importance of diet during the larval stage of development in anurans and the significance of captive
husbandry and zoo-based research considering the ongoing amphibian extinction crisis.
MATERIALS AND METHODS
Captive rearing methods and experimental design
Two egg masses from different breeding groups were used to source larvae for the study. Both egg masses
were located on a substrate of gravel on land near water, one concealed under a segment of PVC plastic pipe and
the other under half of a coconut shell. The first egg clutch was discovered on 2 January 2013 and contained 83
Table 1. Summary of breeding events that supplied tadpoles for the study.
8
Breeding group
Location of eggs in terrarium
Date eggs
found
Date larvae
emerged from
eggs
MABE-B
On gravel under half of a coconut
shell
02-Jan-2013
08-Jan-2013
83
8
5
MABE-A1
On gravel under PVC plastic pipe
segment
16-Jan-2013
16-Jan-2013
44
44
39
# Eggs # Tadpoles
# Tadpoles in
experiment
ALYTES 2015 | 32
Figure 1. Tadpoles housed
individually in 16 oz. plastic cups.
eggs, only 8 of which were fertile. The 8 larvae emerged from the egg mass in a separate container outside the
terrarium one week later. The second egg mass was found on 16 January 2013 and contained 44 well developed
larvae which were starting to hatch the day they were found (tab. 1). On 24 January 2013 we moved all surviving
tadpoles to individual 16 oz. (473 ml) plastic cups filled with 2-3 cm (~60-120 ml) of water (fig. 1). We assigned
separate identification numbers to each tadpole, with a B preceding the number to indicate the individuals from
the first egg clutch that was largely infertile. Tadpoles were later moved on 28 March 2013 to larger aquariums to
prevent escapes, which had by this point caused the loss of 7 individuals. The new aquariums measured 45 x 25 x
25 cm and were filled with about 12 cm (~13.5 l) of water.
Water for the study was sourced from the tap and was unfiltered, originating from a stream ~ 3km away in
Andasibe National Park. We performed complete water changes daily while tadpoles were in the 16 oz. (473 ml)
cups, and complete water changes weekly once moved to aquariums. Once per week each tadpole was examined
closely with a magnifying glass and their Gosner stage (Gosner, 1960) recorded. During this time we also recorded
the weekly minimum and maximum water temperature as well as pH, ammonia, nitrite, and nitrate of our water
source using colormetric aquarium water tests. Water temperature was allowed to fluctuate naturally with the
ambient room temperature, which varied seasonally to as low as 12.8°C in June and as high as 27.5°C in February.
Average water temperature over the entire experiment was recorded as 16.5°C (weekly low) and 26.5°C (weekly
high).
Table 2. Summary of diet treatments.
No. tadpoles that survived through
metamorphosis
No. tadpoles that died during
metamorphosis
Spirulina
7
2
Atyid shrimp
12
1
13
1
Treatment
Diet
A
B
C
Mustard greens
Three different diet treatments were used to test our hypothesis that a diet of shrimp would result in higher
survivorship, faster development and larger frogs (tab. 2). Treatment A (14 tadpoles) were fed powder spirulina
algae, treatment B (15 tadpoles) were fed ground dried atyid shrimp, and treatment C (15 tadpoles) were fed sundried ground mustard greens. During the course of the study some of the tadpoles escaped their enclosure and were
lost. These individuals were not included in any measurements, thus the total number of individuals per treatment
were A (9), B (13), C (14). All foods were sourced locally from markets near Andasibe, the shrimp commonly
known in Malagasy as patsamena and greens ananamanitra. Each tadpole was fed a small pinch of the diet (about
0.05-0.10 g) daily, as much as they could eat without polluting the water.
9
When tadpoles reached Gosner stages 41-42, we moved them from aquariums to 32 oz. (946 ml) ventilated
plastic containers with less than 1 cm (~20-40 ml) of water and several dried leaves as surfaces to help prevent
drowning. Upon completing metamorphosis, individuals were weighed (0.01 g) with an Ohaus TAJ402 digital
scale and their snout to vent length measured with a plastic calliper measured to the nearest millimetre.
Statistical analysis
We tested for differences in days to metamorphosis, length at metamorphosis and weight at metamorphosis
among the three feeding treatments (tab. 3). These traits were chosen as they likely relate to an individual’s fitness.
For instance, the sooner they complete metamorphosis, the sooner the frog can reproduce, while completing
metamorphosis at a larger size (greater mass and length) might indicate an increased chance to survive compared
to smaller individuals (Formanowicz & Brodie, 1982; Werner, 1986; Newman, 1998; Altwegg & Reyer 2007).
We first ran Shaprio-Wilk tests to assess normality of each dataset. As the three datasets (days to
metamorphosis, length at metamorphosis and weight at metamorphosis) were found to not follow a normal
distribution, non-parametric Kruskal-Wallis tests were used to detect differences among the three feeding
treatments. Finally, post-hoc pairwise Mann-Whitney U tests with Bonferroni correction were run to assess which
treatments were statistically significant (p < 0.05).
Table 3. Days to metamorphosis, length at metamorphosis and weight at metamorphosis.
10
ID
Treatment
Days to metamorphosis
SVL at metamorphosis (mm)
Weight at metamorphosis (g)
B1
4
Spirulina
119
12.0
0.16
Spirulina
112
11.0
0.17
5
Spirulina
98
12.0
0.10
6
Spirulina
105
12.0
0.22
8
Spirulina
112
12.0
0.21
9
Spirulina
105
12.5
0.14
10
Spirulina
105
12.0
0.20
15
Shrimp
84
11.0
0.09
16
Shrimp
98
12.0
0.22
19
Shrimp
91
11.0
0.16
B20
Shrimp
119
12.0
0.12
B21
Shrimp
147
12.0
0.15
22
Shrimp
98
12.0
0.14
23
Shrimp
98
13.0
0.22
24
Shrimp
119
18.0
0.18
25
Shrimp
98
12.0
0.22
26
Shrimp
91
12.0
0.14
27
Shrimp
98
12.0
0.16
28
Shrimp
98
12.0
0.20
29
Greens
189
9.0
0.08
30
Greens
147
11.0
0.09
32
Greens
133
9.0
0.09
33
Greens
209
10.0
0.19
34
Greens
157
12.0
0.12
35
Greens
164
12.0
0.10
36
Greens
171
11.0
0.12
37
Greens
168
9.0
0.09
38
Greens
161
10.0
0.10
39
Greens
140
10.0
0.10
41
Greens
280
12.0
0.12
42
Greens
154
11.0
0.19
43
Greens
154
11.0
0.15
ALYTES 2015 | 32
Figure 2. Box plots showing days to metamorphosis, length at metamorphosis and weight at metamorphosis for the different diet treatments.
RESULTS
Feeding treatment led to a significant difference in the amount of time it took for tadpoles to reach
metamorphosis (H(2) = 22.68, p < 0.05) (tab. 4). Specifically, post-hoc pairwise comparisons revealed that tadpoles
fed mustard greens had significantly longer larval stages (more days before metamorphosis) than those fed on the
other two diets (p < 0.05) (tab. 5), whereas there was no difference in time to metamorphosis between the tadpoles
fed spirulina or those fed shrimp (fig. 2). Larval diet also affected length and weight at metamorphosis, (H(2) =
13.50, p < 0.05) and (H(2) = 9.45, p < 0.05) respectively (tab. 4). Similarly to days to metamorphosis, pairwise
post-hoc analyses showed that the mustard green diet led to the significant differences in length and weight of
metamorphs (p < 0.05) (tab. 5), while no significant difference in either length or weight was found between
the groups fed shrimp and spirulina (fig. 2). Specifically, metamorphs fed mustard greens were shorter in length
and weighed less than those fed the other diets. Average and standard deviations among treatments are shown in
tab. 6. Four tadpoles died before metamorphosis, therefore we ran a Fisher exact test to analyse if survivorship
to metamorphosis differed among treatments. No statistically significant differences in survivorship among diet
treatments were found (p = 0.53) (tab. 2).
Table 4. Results of Kruskal-Wallis tests.
H
degrees of freedom
p value
22.6774
2
0.00012
Length at metamorphosis
13.5045
2
0.00117
Weight at metamorphosis
9.4500
2
0.00887
DISCUSSION
Diet treatments of spirulina and atyid shrimp both resulted in similar shorter times to and larger size
at metamorphosis, suggesting these locally available foods are better than mustard greens for raising larvae of
Mantidactylus betsileanus. This does not mean, however, that they are necessarily the best diets. It is possible
that other food options, such as manufactured tropical fish flakes and commercial tadpole diets, could result in
improved individual fitness or that using a variety of foods would result in better growth and development. For
instance, Altig et al. (2007) note that while generalized or omnivorous feeders may only consume low levels of
animal material as part of their diet, it is this highly nutritious component that contributes to high production and
rapid development, so while feeding only mustard greens slowed development and produced smaller frogs in our
study, combining greens with a portion of atytid shrimp or other animal material could produce very different
results.
11
It should also be noted that the traits that
we measured do not encompass the entire range
of characteristics that determine the fitness of an
individual tadpole. Weight and length at start of
metamorphosis or throughout the larval stage were
A
not recorded, and these traits could also be dependent
Greens
Shrimp
Shrimp
0.0001
on diet and have an effect on fitness of tadpoles and
Spirulina
0.0011
0.3216
metamorphs. Another consideration is that the growth
B
of tadpoles is highly dependent on temperature, and
Greens
Shrimp
while the varying temperature in our study was the
Shrimp
0.0044
same between treatments since they were run at the
Spirulina
0.0214
1.0000
same time and in the same location, it is possible that
C
under different environmental conditions the three
Greens
Shrimp
diet treatments could produce different results.
Shrimp
0.0260
Dietary protein has been shown to have a
Spirulina
0.0450
1.0000
significant effect on the development of amphibian
larvae. Though the optimal captive diet and its protein
content likely reflect the natural history of the species
being maintained, larval diets have been tested on a
number of anuran species and those with high protein contents have resulted in better growth and development
(Steinwascher & Travis, 1983; Carmona-Osalde et al., 1996; Martins et al., 2013). It is not so surprising then that
spirulina and atyid shrimp, both of which have protein contents upwards of 50% (Ciferri, 1983; Mugo-Bundi et
al., 2015), resulted in shorter times to and larger size at metamorphosis than mustard greens, which have a protein
content of less than 3% (USDA, 2014). Although mustard greens in our study did not affect survivorship of
Mantidactylus bestileanus and various other leafy greens have been used and recommended as a captive diet for
tadpoles in literature (Briggs & Davidson, 1942; Martin, 1991; Banks et al., 2008; Tyler, 2009), there are clearly
better options available for feeding tadpoles. It should be noted, however, that we did not measure the nutrient
content of our experimental diets, and so cannot directly infer that the higher protein diets led to better growth and
development in our study.
Mantidactylus betsileanus has been assessed by the IUCN Red List as Least Concern and seems to be
widespread but it is also clear that the species is composed of a complex of undescribed species, many of which
are thus far known from only one or two localities (Glaw & Vences, 2007; Vences & Nussbaum, 2008). While
the status in the wild and larval life histories of these candidate Mantidactylus species is unknown, the results
from our study suggest that if ex situ conservation breeding programmes are needed in the future, a starting point
for a captive larval diet could be based on spirulina and atyid shrimp. However, future studies that measure the
nutritional composition of different diets and how the nutrients in those diets are assimilated by tadpoles would
expand our understanding of optimal diets for captive breeding. With this in mind, we hope to see future captive
husbandry studies conducted on other Malagasy anuran species by the international ex situ community which
could help inform conservation breeding programmes for other poorly known and potentially threatened species.
Table 5. A. Post-hoc pairwise comparisons of days to
metamorphosis among 3 diets. B. Post-hoc pairwise comparisons
of length at metamorphosis among 3 diets. C. Post-hoc pairwise
comparisons of weight at metamorphosis among 3 diets.
Table 6. Average and standard deviation for each treatment.
Treatment
Spirulina
Atyid shrimp
Greens
Weight at metamorphosis
Length at metamorphosis
AVG
STDEV
AVG
STDEV
AVG
STDEV
108.00
6.83
0.17
0.04
11.93
0.45
103.25
17.18
0.17
0.04
12.42
1.83
171.31
38.25
0.12
0.04
10.54
1.13
RESUMÉ
Des programmes d’élevage en captivité des amphibiens sont de plus en plus nécessaires en vue du déclin
et des risques d’extinction, auquel cette classe animale est confrontée à court terme. De plus, les principaux
aspects de l’élevage, tels que l’alimentation des larves, continuent d’être négligés, parfois même totalement
inconnu. A Madagascar, il peut être difficile d’adopter ces programmes, en raison de l’indisponibilité de régimes
12
ALYTES 2015 | 32
spécifiques aux têtards. Nous avons testé trois régimes, disponibles localement à Madagascar : la moutarde brune,
l’algue spiruline, ainsi que des crevettes de la famille des Atyidae, sur les larves de Mantidactylus betsileanus, et
enregistré leur croissance et leur développement. Il en résulte, que les têtards nourris aux feuilles de moutarde ont
eu un développement plus long et leur métamorphose s’est effectuée à une taille plus petite. Cependant, aucune
différence du taux mortalité des têtards n’a été observée entre les différents programmes. Ces résultat suggèrent
que les moutarde brune sont une source de nourriture pauvre, tels que la spiruline et les crevettes de la famille
des Atyidae, sont de meilleurs choix que les légumes ou végétaux pour l’élevage de M. betsileanus et des espèces
similaires. Bien que d’autres alternatives comme les aliments industriels destinés spécialement aux têtards ou
poissons pourraient donner des résultats de meilleures qualités.
ACKNOWLEDGEMENTS
We wish to express our gratitude to Woodland Park Zoo, Biopat, Tree Walkers International, Understory
Enterprises, Cleveland Metroparks Zoo Africa Seed Grants program, and the 2013 Sustainable Amphibian
Conservation of the Americas Symposium for their financial support of husbandry research at Mitsinjo leading up
to and during the duration of this study. We also would like to recognize Jennifer Pramuk and Brian Gratwicke for
their help in developing and improving the methodology of the experiment. Renaud Boulenger kindly improved
our French abstract. This project was carried out through a contract of collaboration between the Malagasy
authority of the Direction Générale des Forêts, Association Mitsinjo, and the IUCN SSC Amphibian Specialist
Group of Madagascar.
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16
RESEARCH ARTICLE
2015 | VOLUME 32 | PAGES 17-22
On the generic status of “Nyctimystes rueppelli”
(Anura: Hylidae), a tree frog of Halmahera Island,
Indonesia
James I. Menzies1*, Awal Riyanto2
2.
1.
University of Adelaide, Adelaide SA 5005, Australia
Museum Zoologicum Bogoriense, Research Center For Biology - The Indonesian Institute of Sciences (LIPI), Widyasatwaloka Building,
Jalan Raya Jakarta Bogor Km. 46, Cibinong 16911, Indonesia
Nyctimystes is currently diagnosed by a combination of two characters, namely vertical pupil and
a palpebral venation. In a newly collected series of “Nyctimystes rueppelli” this combination of
characters is found wanting and the species is therefore removed from Nyctimystes and transferred
to Litoria. Removal of this species from Nyctimystes now allows that genus to have a third diagnostic
character, unpigmented ova, and confines it to New Guinea and satellite islands.
INTRODUCTION
The tree frog Hyla rueppelli, currently known as Nyctimystes rueppelli (Menzies, 2006; Tyler & Davies,
1978; Zweifel, 1958), was described by Boettger in 1895 from a series of 48 specimens collected on Halmahera
Island by Dr. W. Kükenthal and deposited in the Senckenberg Museum, Frankfurt. No holotype was designated and
his description gave measurements only for five specimens but many of the others were subsequently distributed
to various museums in Europe and America, including Basel (adult male, labelled paratype); Vienna, four males,
one subsequently transferred to Adelaide; New York (two syntypes) and London, four specimens, labelled “types”.
The description was repeated by Boettger in 1900 with some additional information on material held in Frankfurt,
noting eight adult males, seven females and four young, still with tails, all from Soah Konorah, and two males,
one female from Galela, and 28 adults from Kau. There are now only two adult males, two adult females and
five young in the Senckenberg Museum. There is no mention, in the description, of pupil shape nor presence of a
pattern on the lower eyelid, and Boettger’s illustration (1900, plate 16, figure 12) shows an expanded, more or less
circular, pupil. One specimen - a gravid female with pigmented ova - had previously been collected by Bernstein
in 1866 on either Gebe or Gag Island. Gebe is approximately 60 km from the eastern arm of Halmahera while Gag
is 120 km to the south-east. All three localities are part of the same island arc system. Bernstein had spent some
time collecting on Halmahera before visiting Gebe and Gag (Wichman, 1906). Brongersma (1948) reported on
two males collected on Morotai Island, to the north of Halmahera, by H.J. Lam, and these are now in the Carnegie
Museum in Philadelphia. We are not aware of any other specimens of Nyctimystes rueppelli in any museum that
have not come from Kükental’s, Bernstein’s or Lam’s collections, other than recent collections on Halmahera
made by Riyanto in 2010. Fig. 1 illustrates all locations from which the species has been recorded. No other
Nyctimystes species have been recorded on Halmahera or Morotai Islands.
As a result of lack of recent material, and poor condition of many of Kükenthal’s specimens, Nyctimystes
rueppelli has remained somewhat of a mystery. The two primary diagnostic features of Nyctimystes are the
presence of a pattern of lines and/or dots on the transparent upper part of the lower eyelid and a vertical pupil, but
all the Papuan Nyctimystes species now known, except N. rueppelli, produce relatively large unpigmented ova
from which torrent-adjusted tadpoles emerge, though very few tadpoles have actually been described. By contrast,
Received 07 May 2015
Accepted 21 July 2015
james.menzies@adelaide.edu.au
JAMES I. MENZIES & AWAL RIYANTO
Figure 1. Known localities (*) for Litoria rueppelli.
the ova of Nyctimystes rueppelli are pigmented brown on the animal pole. This does not necessitate removal of
the species from Nyctimystes, as currently defined by Zweifel (1958), but does invite further investigation. Apart
from the works of van Kampen (1923) and Gorham (1963), which do not add any new information, Hyla rueppelli
received no further attention until 1958 when Zweifel transferred it to Nyctimystes. Tyler (1968) did not include it
in his revision of the Papuan Hyla (now Litoria) because it had already been transferred to Nyctimystes.
The genus Nytimystes Stejneger
Stejneger (1916) erected Nyctimystes to accommodate two species of Papuan tree frogs which, up to that
time, had been included, on account of their vertical pupils, in the South American genus Nyctimantis. Zweifel
(1958) published a revision of the genus Nyctimystes and commented that the sole character of a vertical pupil
was “a tenuous one for defining a genus” and therefore added the “presence of a vein-like network, the palpebral
reticulum”, on the lower eyelid. Neither of these characters, individually, is present in any of the Papuan Litoria
species, the only other hylid genus occurring in the region, and so could be regarded as synapomorphies of the
genus Nyctimystes. Zweifel (1958) noted that Hyla rueppelli had a palpebral reticulum and so transferred it to
Nyctimystes but was unable to determine the shape of the pupil, which was fully expanded in specimens that he
saw. He assumed, because all the other Papuan hylid frogs with palpebral reticula also had vertical pupils, that
Hyla rueppelli also did so and therefore felt justified in transferring it to Nyctimystes. Tyler & Davies (1979)
attempted to redefine the genus and made a detailed examination of the skulls of 17 species, comparing them with
18
ALYTES 2015 | 32
Australo-Papuan Litoria. They defined Nyctimystes by a suite of 39 characters but nearly all these characters were
equivocal such as, “well developed or reduced quadrate-jugal”. In the montane Litoria, to which Nyctimystes
species were apparently related by their unpigmented large ova and torrent-type tadpoles, this element was always
reduced but unreduced in those Litoria with pigmented ova. Despite Tyler & Davies’ detailed analysis, the genus
is left with only two defining characters, contracted pupil shape and eyelid venation. Beyond suggesting that
Nyctimystes rueppelli may have had an independent origin from other Nyctimystes species, they did not question
its inclusion in the genus.
Frost et al. (2006) returned all Nyctimystes species to Litoria on the basis of a molecular analysis that
included the Australian species, N. dayi and the Papuan N. pulcher. Wiens et al. (2010) also showed Nyctimystes to
be a paraphyletic genus with seven Papuan species forming a sister group to Litoria infrafrenata and N. dayi distant
from that group. However, Kraus (2013) saw that Nyctimytes dayi did not have a vertical pupil, was incorrectly
placed in that genus, and therefore the conclusions of Frost et al. and Wiens et al. were invalid. Nyctimystes
continues to be recognised as a valid genus (e.g. Kraus, 2013; Menzies, 2014a, b, c). Nyctimystes rueppelli was not
included in Wiens’ analysis and its molecular relationships remain unknown. Kraus (2013) also speculated on the
possible incorrect generic allocation of Nyctimystes rueppelli, noting, because pupil shape could not be determined
in any of the material then available, that “future study of the Halmaheran N. rueppelli may show that species to
be another mismatch”.
Recent collection of material on Halmahera by Riyanto, where the species is common, confirms these
suspicions and allows re-assessment of the generic status of Nyctimystes rueppelli.
This published work have been registered in ZooBank. The ZooBank LSIDs (Life Science Identifiers)
can be resolved and the associated information viewed through any standard web browser. The LSID for this
publication is: urn:lsid:zoobank.org:pub:F2B8791E-EDB9-4B94-8F46-D8223D827ACB
For this investigation, we confined ourselves to three characters, the shape of the pupil, the form of
the palpebral reticulum and the colour and size of the ova. Approximately 20 recently collected specimens of
Nyctimystes rueppelli were available, several of which had been photographed in life. Specimens from Kükenthal’s
original collection had already been examined in Europe and Appendix 1 lists all this material.
Relative eye size was estimated as the horizontal diameter of the eye/length of the body measured from the
tip of the snout to the end of the urostyle. Egg diameter was measured in a sample of approximately 20 ova taken
from each of the gravid females listed in Appendix 1.
RESULTS
Pupil shape
The eyes are large, mean horizontal eye diameter/body length = 0.15, the iris is dark brown. Fig. 2.A, B,
C shows enlargements of eyes of three of specimens of Nyctimystes rueppelli, photographed in life. The pupil is
rhomboid or diamond-shaped and in fig. 2.A and 2.B the horizontal dimension is clearly greater than the vertical.
In all the preserved specimens recently re-examined by Menzies, pupil shape was obscure. Fig. 2.D is the eye of
Litoria amboinensis, a species that bears some morphological resemblance to Nyctimystes rueppelli, photographed
in life. This pupil is also diamond or rhomboid in shape and is characteristic of that group of species that includes,
Figure 2. Eyes of A-C. Litoria rueppelli, D. Litoria amboinensis. Photo D. by Nick Baker.
19
Figure 3. Lower left and right eyelids of Litoria rueppelli, A, B. SAM R68247 and C, D. SAM R 68246; D. is drawn at a higher magnification.
in New Guinea, Litoria amboinensis, L. darlingtoni and L. rothi. Pupil shape in Litoria amboinensis confused
Brongersma (1953) who transferred the species, on that account, to Nyctimystes where it remained until Zweifel
(1958) returned it to Hyla (now Litoria).
Palpebral reticulum
Fig. 3.A and B show the left and right lower eyelids of one specimen, C and D are left and right eyelids of
another. The palpebral reticulum consists, in all cases, of thin, meandering, anastomosing, gold lines, supplemented by numerous, isolated pigment spots and covers the entire eyelid. Other specimens examined showed similar
patterns but with much variation in the relative number of lines or dots. The pattern, with its wavering lines and
dots, is unlike that of any Nyctimystes species known
(see Menzies 2006, figure 17 for ten different examples) and there is no “typical” eyelid pattern for the
genus. The eyes of a number of Litoria amboinensis
were carefully examined but no trace of a reticulum
could be seen in any.
Ova
Figure 4. Comparative egg sizes in some New Guinean hylid frogs.
La*, Litoria amboinensis; Lg*, Litoria genimaculata; Lt*, Litoria
thesaurensis; Lr*, Litoria rueppelli; Lm, Litoria micromembrana;
Ne, Nyctimystes eucavatus; Nf, Nyctimystes foricula. Horizontal
bars are the means and vertical bars are their standard deviations. *
denotes species with pigmented ova.
20
The pigmented ova of Nyctimystes rueppelli,
taken from two gravid females, have a mean diameter
of 1.52 mm (n = 17). For comparison, a sample
of ova was taken from gravid females of six other
Litoria and Nyctimystes species (fig. 4). The ova of
Nyctimystes rueppelli are smaller, on average, than
those of the three species that have unpigmented ova
and more in line with the pigmented ova of Litoria
amboinensis, L. genimaculata and L. thesaurensis.
The measurements are probably an underestimate,
due to shrinkage and distortion in preservative, but
remain useful for comparison.
The larvae of Nyctimystes rueppelli are still
unknown and the five shrivelled, young specimens in
Frankfurt show no larval characters except reducing
tails.
ALYTES 2015 | 32
DISCUSSION
Results described above show that Hyla rueppelli should never have been assigned to Nyctimystes, but the
limited material available, and with little likelihood of getting more from remote Halmahera Island, left Zweifel
(1958) with little choice but to place it there, as no known Papuan Litoria species displayed a palpebral venation.
Although it does have a palpebral reticulum, Hyla rueppelli does not have a vertical pupil, characters which,
in combination, are the two diagnostic features of the genus (Zweifel 1958). Hyla rueppelli fails to show the
synapomorphies of Nyctimystes and is therefore removed from that genus and transferred to Litoria as Litoria
rueppelli (Boettger). Other points of difference from Nyctimystes species are the pigmented ova and the occurrence,
on Halmahera Island.
With the removal of rueppelli, the genus Nyctimystes is now confined to New Guinea and satellite islands,
and all known species have unpigmented ova.
ACKNOWLEDGEMENTS
Menzies wishes to thank the Universities of Papua New Guinea and Adelaide for supporting his research
and the staff of the Museum Zoologicum Bogoriense for their help and hospitality during his visit to Indonesia.
Riyanto offers thanks to Prof. Gono Semiadi for inviting the Biodiversity Assessment Team of LIPI. Riyanto
thanks PT. Weda Bay Nickel on Halmahera for their support in carrying out fieldwork in their concession areas
during 2010. Also, Riyanto thanks PT Environmental Resources Management, Jakarta for inviting the LIPI team
to conduct the biodiversity assessment in Halmahera. Riyanto is grateful to the field staff of PT. Weda Bay Nickel
for their patient help during the field work. Nick Baker (EcologyAsia.com) for the photo of Litoria amboinensis.
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Wiens J.J., Kuczyinski, C., Xia, H., Moen, D. (2010). An expanded phylogeny of tree frogs (Hylidae) based on
nuclear and mitochondrial sequence data. Molecular Phylogenetics and Evolution, 55: 871-882.
Zweifel, R.G. (1958). Results of the Archbold Expeditions. No. 78. Frogs of the Papuan hylid genus Nyctimystes.
American Museum Novitates, 1896: 1-51.
APPENDIX 1
List of specimens examined
Litoria rueppelli
(AMNH, American Museum of Natural History, New York; BMNH, Natural History Museum, London;
CM, Carnegie Museum, Philadelphia; MZB, Museum Zoologicum Bogoriense, Cibinong, Indonesia; NHMB,
Naturhistorisches Museum, Basel, Switzerland; NMW, Naturhistorisches Museum, Vienna, Austria; RMNH,
Naturalis Museum, Leiden, Netherlands; SAM, South Australian Museum, Adelaide, Australia); SMF, Senckenberg
Museum, Frankfurt, Germany.
AMNH 23759-60 (data from Zweifel 1958). Kau, Halmahera
BMNH 95.10.26.19-22; North Halmahera
CM 25554-55 (data from Zweifel 1958); Morotai Island
MZB16517; 16519-16524; 16527-16528; 16531-16532-16534; 16536-16540; 16542-16543; 16546;
16548; I. Desa Iga, Kecamatan Wasile Utara, Kabupaten Halmahera Timur. 1.39N; E 128.26; 165 m asl; and II.
Tjetje, Halmahera, Weda Bay 1.39N; E 128.26; 161 m asl
NHMB 2383, North Halmahera
NMV 5899-5901, North Halmahera
RMNH 12368; Gebe (or Gag) Island
SAM R13800; R68246-47; central Halmahera.
SMF 2614-2617; North Halmahera.
Other species
Litoria amboinensis SAM R4890
Litoria genimaculata SAM R10747
Litoria micromembrana SAM R8924A, 13269
Litoria thesaurensis SAM R60645; 64785
Nyctimystes eucavatus SAM R5210B; 5210C; 5210Ae
Nyctimystes foricula SAM R5209A, 5209C
22
RESEARCH ARTICLE
2015 | VOLUME 32 | PAGES 23-29
The increase of an amphibian population:
11 years of Rana temporaria egg-mass monitoring
in 30 mountain ponds
Rocco Tiberti1,2*
2.
1.
Alpine Wildlife Research Centre, Gran Paradiso National Park, Degioz 11, 11010 Valsavarenche, Aosta, Italy
University of Pavia, DSTA-Dipartimento di Scienze della Terra e dell’Ambiente, Via Adolfo Ferrata 9, 27100 Pavia, Italy
Mount Guglielmo in the Italian Alps is an ancient transhumance area where man-made ponds for watering
cattle are an ancestral component of the landscape and represent the most important breeding sites
for common frogs (Rana temporaria) in the study area. Egg-masses in 30 mountain ponds from the
Mount Guglielmo were counted from 2005-2015 to monitor the population trends of R. temporaria,
after a putative population decline due to lethal bacterial infections which affected the tadpoles from
2002 to 2006. Egg-mass counts were also used to understand how the recovery of ponds from the
natural process of drying-out and the replacement of traditional ponds with pools waterproofed with
polymers influence the population dynamics of R. temporaria and the suitability of the ponds as
breeding sites. This R. temporaria population increased over the study period, suggesting that the
population was able to withstand long lasting larval mortality. Population increase was also sustained
by the recovery of some dried ponds, but the modern pools were less suitable than traditional ponds
for R. temporaria reproduction. These results suggest that the persistence of the traditional practices
related to transhumance (e.g. maintenance of man-made ponds) in the study area is probably of pivotal
importance for the conservation of the local herpetofauna.
INTRODUCTION
Amphibians diseases are a major cause of the global amphibian decline crisis (Daszak et al., 1999), and
are a primary factor determining the natural dynamics of wild populations (Anderson & May, 1986). To understand
the real extent of the global amphibian decline crisis, biologists need to distinguish between natural population
fluctuations and abnormal declines that may be directly or indirectly due to human actions (Pechmann et al., 1991;
Gardner, 2001). Long-term studies, encompassing population fluctuations or cycles, can help biologists learn more
about this problem (Meyer et al., 1998; Loman & Andersson, 2007). In any case, mortality events attributable to
infectious diseases should be carefully monitored to assess its impact on amphibian populations.
In the present 11-year study, egg-mass counts from 30 mountain ponds were used to assess the population
dynamics of Rana temporaria Linnaeus, 1758 on Mount Guglielmo massif (Italian Alps). The present study was
initiated to quantify the demographic effects of a series of widespread mass die-offs affecting R. temporaria
tadpoles at many ponds in the study area (Tiberti, 2011). Around 2002, die-offs of R. temporaria tadpoles were
reported for the first time, often resulting in mass mortality of the entire tadpole population within a pond and
affecting most ponds in the entire area (Tiberti, 2011). Other common amphibian species (Bufo bufo and Triturus
carnifex; Tiberti in press) were apparently unaffected by the epidemics. Die-offs have been ascribed to bacterial
infections caused by Aeromonas sp. (Tiberti, 2011), often associated to epidemics of red-leg disease (Rigney et al.,
1978). Aeromonas sp. is an ubiquitous bacterial genus living in free waters, on the skin and in the digestive tract of
Received 12 Mach 2015
Accepted 04 October 2015
rocco.tiberti@gmail.com
ROCCO TIBERTI
amphibians without causing infections, and is considered an opportunistic pathogen, infecting immunodepressed
hosts (Carey, 1993). Therefore, the spread of the epidemic could have been facilitated by other immunosuppressant
density dependent (e.g. overcrowding) or environmental factors (see Meyer et al., 1998), or by the presence of
other, not detected, pathogens (Tiberti, 2011). Despite not being able to determine the root causes, such die-offs
have not been reported in the study area (at least with the same severity and spatial extent) since 2007.
Mount Guglielmo is an ancient area of alpine transhumance (the seasonal transfer of livestock to high
altitude pastures). Due to its geology (dominated by calcareous rocks with many karst landforms), surface water
is largely absent above 1000 m a.s.l. and water for cattle was traditionally obtained by constructing ponds. These
anthropogenic habitats are an ancestral part of the cultural landscape (alpine transhumance dates back to Neolithic;
Festi et al., 2014) and represent virtually the only surface aquatic habitat in high altitude pasturelands in the
study area. In this context traditional land use practices (e.g. pond construction and maintenance) sustain valuable
ecosystems for amphibians. However modernization in the water supply for cattle has meant that some traditional
ponds (dug into the ground, with a clay bottom) were replaced with large artificial pools (waterproofed with
polymers sheets). Thus, a second objective of this study was to assess R. temporaria use of modern artificial ponds
compared to traditional transhumance ponds
Study area
Mount Guglielmo (1957 m a.s.l.) is an isolated massif between the Trompia and Camonica valleys
(Brescia Prealps, Italy) (fig. 1). In the study area there are 30 mountain man-made ponds for watering cattle, which
sustain Bufo bufo and Triturus carnifex populations and are the most important breeding sites for R. temporaria in
the study area. The mean altitude of the ponds was 1535 m a.s.l. (range: 1166-1863 m a.s.l. ), mean surface area
was 706 m2 (range: 140-3400 m2), and mean depth was 0.81 m (range: 0.2-2.0 m; values based on the maximum
seasonal depths measured in May-July 2006 ). Nearby ponds (situated less than 1 km apart) were clustered into
four groups (A-D; fig. 1), thus R. temporaria could probably disperse among ponds in the same group. Most of
the ponds had a clay bottom, but ponds A2, C2, C3, D3, D6, and D8 (fig. 1) had an artificial substrate made of
waterproof polymers.
Egg-mass counts
Obtaining accurate counts of R. temporaria egg-masses is possible because their clutch size is large (up to
4500 eggs; Nöllert & Nöllert, 1992), they are easily visible in shallow water, and laid simultaneously by numerous
frogs in a confined area (Bernini & Razzetti, 2006). For 11 years (2005-2015), one to three surveys per pond were
performed annually to count the egg-masses deposited in all mountain ponds in the study area (N = 30, fig. 1).
Surveys were conducted just after the putative breeding season by walking along the entire edge of each pond.
The date of the first survey was decided based on the weather conditions (e.g. persistence of the snow cover) and
on the pond features (e.g. altitude and exposure) during the usual breeding period in the study area, which was
highly predictable (between the 21 April and 10 May). Assuming that the time of reproduction should be the same
in the ponds closer together, repeat surveys were needed when the egg-masses had not yet been laid in a certain
area/group of ponds or when amplexing pairs were still observable in the ponds. The position and developmental
stage (freshly laid or not) of the egg-masses was recorded at each survey to not underestimate counts when repeat
surveys were conducted. Of 330 potential egg mass counts (30 sites for 11 years) over the study, only 15 were
not conducted, when i) the ponds were still ice-covered and a second survey was not performed, or ii) when the
survey was done after the eggs were already hatched, which rarely occurred. During the egg-mass surveys, the
number of adult Triturus carnifex along the shorelines was counted because their presence can potentially affect
the suitability of the ponds as a breeding site of R. temporaria (Tiberti, 2011). When surveys were repeated, the
maximum number of Triturus carnifex was used as an index of abundance.
Statistical analysis
A linear mixed effects model (LME) was used to test if the observed trend in the time series of egg-mass
counts was significant, including the effects of some potentially important covariates, and accounting for the
repeated counts in the same ponds and groups of ponds. Transformed of annual egg-mass counts (log+1) were
added to the model as a dependent variable, the group of ponds as a random effect and each pond as a nested
24
ALYTES 2015 | 32
Figure 1. Mount Guglielmo summit (triangle) bordered by the 1000 m a.s.l. contour line. Dotted lines delimit a 500 m buffer area around the
numerated ponds belonging to the groups A-D.
random effect. The year of the survey (from year 1 to 11), the pond altitude (centered at 1500 m a.s.l.), the pond
area (m2), the number of adult newts along the shoreline, the substrate of the pond (artificial vs. natural), and the
mean temperature and the precipitation during i) the breeding season (from 21 April to 10 May), ii) the previous
hibernating season (November-April), and iii) the previous activity season (May-October), were added to the
model as covariates. In particular, the counts of Triturus carnifex were added to the model as their presence can
affect the suitability of the ponds as a breeding site for R. temporaria (Tiberti, 2011), while the climatic variables
were added as they could affect the survival and the energy allocated to reproduction of overwintering frogs, the
survival and fat reserves of the frogs at their feeding grounds, and the mildness of the climatic conditions during the
breeding period (Meyer et al., 1998). Meteorological covariates were measured at the weather station of Pisogne,
Brescia (45°49’02”N, 10°09’00”E, 842 m a.s.l.), 6.8 km from the peak of Mount Guglielmo. An “autoregressive
moving average” class (corARMA) was used to model the temporal autocorrelation structure of the residuals,
previously calculated with the function ACF (Autocorrelation Function) (Crawley, 2012). The LME was fitted by
Maximum Likelihood following Zuur et al. (2009) using the nlme package of the statistical environment R version
3.1.1 (R Development Core Team, 2010). The MuMIn package (Bartoń, 2011) was used following Grueber et
al. (2011) to select the best fitting models (ΔAICc < 4) among the models including all possible combinations of
the fixed covariates. We report the 95% confidence intervals of the averaged parameter estimates and the relative
importance of the covariates provided by the function model.avg of MuMIn. Linear regression was used to check
if the used climatic covariates showed a significant trend during the study period.
25
ROCCO TIBERTI
RESULTS
The number of egg-masses oviposited in the study area increased significantly from 2005 to 2015 (fig. 2,
tab. 1). The number of egg-masses oviposited in natural ponds (38.2 ± 58.5; mean ± sd) was significantly higher
than artificial ponds (3.5 ± 11.2; table. 1). In 2014, a decline of the egg-mass counts was observed (fig. 2), although
this could be partially influenced by missing data (4 missing counts from ponds B7, D5, D6, and D7 due to icecovered conditions during the last survey, which, due to the impossibility of performing a second survey, made it
impossible to account for the probable later ovipositions).
There was no evidence that climatic variables (mean temperature or precipitation) had a significant effect
on oviposition (tab. 1) and there was not a relationship between climatic variables and egg-mass counts. Olenly
precipitation during the hibernating season showed a significant increasing trend over the study period (tab. 2), but
LME results exclude a causal relationship with the increase in the number of egg-masses in the study area (tab. 1).
Figure 2. Time series of the egg-mass counting in the whole Mount Guglielmo area and divided by groups of ponds (A-D).
26
ALYTES 2015 | 32
Table 1. Fixed effect results from a generalized mixed effect model testing the effect of several covariates on the log+1 transformed of the abundance of R. temporaria egg-masses in pastureland ponds of Mount Guglielmo. All the observations (N = 285) have been collected over 11 years
(2005-2015) in 30 ponds. Legend: Beta = Averaged parameter estimate; Lower 95% CI and Upper 95% CI = lower and upper 95% confidence
intervals; RVI = Relative Variable Importance (from 0 to 1). Fixed terms whose 95% confidence intervals around parameter estimates do not
include 0 are considered significant and have been marked in bold. Temp.: mean temperature; Prec.: precipitation amount.
Fixed term
Beta
Lower 95% CI
Upper 95% CI
Intercept
0.838
-2.779
4.455
RVI
-
Year
0.148
0.078
0.218
1.00
Altitude
0.000
-0.002
0.002
0.10
Area
0.000
0.000
0.001
0.19
Newts
0.003
-0.003
0.017
0.38
Substrate (artificial vs. natural)
-1.592
-2.560
-0.624
1.00
Temp. breeding season
0.003
-0.201
0.232
0.19
Prec. breeding season
0.001
-0.001
0.004
0.35
Temp. previous Winter
-0.074
-0.230
0.015
0.69
Prec. previous Winter
0.000
-0.001
0.000
0.48
Temp. previous Summer
0.068
-0.059
0.375
0.43
Prec. previous Summer
0.000
-0.001
0.001
0.14
DISCUSSION
Because the breeding biology of R. temporaria (e.g. aggregate breeding and single clutch per female)
suggest that egg-mass counts provide an accurate estimate of the number of reproductive females (Crouch &
Paton, 2000), the observed increase in the number of egg-masses likely indicates an increase of the number of
breeding females, and probably of the effective population size over this 11-year study.
The climatic variables (mean temperatures and precipitations, tab. 1) were chosen as potentially important
factors affecting the survival of hibernating R. temporaria, their survival/resource allocation during the activity
period (Lardner & Loman, 2003) and the suitability of the breeding season. Climatic factors may affect effective
population sizes of several amphibian species (McCaffery & Maxell, 2010), but the present results are consistent
with those from other studies, where R. temporaria populations were relatively unaffected by a number of climatic
variables (Elmberg, 1990; Meyer et al., 1998; Tattersall & Ultsch, 2008). Probably the effects of climatic factors
are masked by the importance of density dependent regulation within the population. For example, an amphibian
population experiencing a rapid expansion can be relatively unaffected by climatic factors, of course in the absence
of catastrophic climatic events.
It is likely that the observed population increase was the results of the end of the epidemic (Tiberti, 2011).
These results suggest that some populations of R. temporaria have the capacity to withstand high larval mortality.
Unfortunately historical data on the abundance of R. temporaria before the observed larval mortality events are not
available and it is impossible to determine if the observed increases represent post-epidemic recovery. Therefore, it
is not possible to know how the observed population increases compares to population sizes before the epidemic.
Female R. temporaria take 2-5 years to reach sexual maturity (Guarino et al., 2008). Therefore, the effects
of the epidemic on the population size might not become evident for years. Such process may explain the pattern
of ongoing decline observed in pond group D in 2005-2008 (only 9 egg-masses were counted in ponds group D in
2008) after the end of epidemic in 2006. It is possible that in group D the monitoring period included part of the
putative decline.
The persistence of the studied population after high tadpole mortality could be explained in several ways.
First R. temporaria has a high reproductive potential, which allows populations to recover from a small number of
surviving individuals. Moreover this species is distributed widely (Kuzmin et al., 2009) and could re-colonize the
extirpated habitats. Also R. temporaria longevity (6-15 years; Guarino et al., 2008) could explain the persistence
of the studied population: adults can breed for multiple years and, if recruitment failure only occurs for a limited
number of years, it is possible that adult persistence could buffer populations against tadpole mortality. Indeed,
compared to the mortality of post-metamorphic stages, increased mortality in anuran larvae is less likely to affect
the overall population growth rate and the population viability (Biek et al., 2002). However, recurrent epidemic
events can sometimes cause persistent declines and local extinctions in R. temporaria populations (Teacher et al.,
2010), suggesting not to rely too much on their capacity to withstand epidemics.
There are other factors potentially contributing to the observed trend in egg-mass counts. For example,
27
ROCCO TIBERTI
Table 2. Linear regression of climatic variables against time (Year). Temp.: mean temperature; Prec.: precipitation amount. Significant P-values
(<0.05) have been marked in bold.
Model
Beta
t-test
P
Temp. previous Summer ~ Year
-0.102
-1.50
0.17
0.11
Prec. previous Summer ~ Year
28.48
1.72
0.12
0.16
Temp. previous Winter ~ Year
0.03
0.26
0.80
-0.10
Prec. previous Winter ~ Year
40.61
2.27
<0.05
0.29
Temp. breeding season ~ Year
-0.08
-0.76
0.47
-0.04
Prec. breeding season ~ Year
6.29
1.13
0.29
0.03
Adjusted R2
the sudden onset of R. temporaria egg-masses in group C is the result of the increased hydroperiod of a dry pond
(C4, fig. 1) in an area where R. temporaria was usually absent, probably due to the presence of a particularly large
population of Triturus carnifex, which could prevent the reproduction of R. temporaria (Tiberti, 2011). This new
pond was quickly colonized by R. temporaria, which established a small population.
Without a regular maintenance (e.g. removal of vegetation and sediments and re-waterproofing of the
bottom), most of the mountain ponds in the study area will probably disappear within a few years to decades, due
to their progressive burial or to the infiltration of water at the clay bottom. Pond recovery is a common practice
among the local farmers (nine pond restorations were detected in ten years) in the study area, where transhumance
still produce an actual water demand for cattle. The survival of alpine transhumance is probably favorable for
amphibians and their breeding habitats, preventing the progressive abandonment of the rural landscape, which is
often viewed with concern for the likely negative consequences for biodiversity due to land use change (e.g. Hartel
& von Wehrden, 2013). However, sometimes the modernization of agricultural practices has meant that traditional
ponds - much more suitable as breeding sites for R. temporaria (tab. 1) - were replaced by artificial large pools
with bottoms made of waterproofing polymers. These pools, due to their steep shore, may also become a lethal trap
both for amphibians and other animals, such as small mammals, marmots, and reptiles which can be often found
therein drowned (personal observation). This is somehow in contrast with the results from other study areas, where
the widespread construction of artificial ponds - less prone to drying out than natural ones - produced a population
increase in R. temporaria (Cooke, 1972). Probably artificial ponds can become more attracting for R. temporaria
in a context of general loss of natural ones, but in the area of the Mount Guglielmo frogs can find both the habitats
within a short distance, and their choice falls on natural ponds.
ACKNOWLEDGEMENTS
I thank Fiorenzo and Paola for their help and company during the field work.
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29
30
REVIEW ARTICLE
2015 | VOLUME 32 | PAGES 31-45
Ecología y comportamiento de las ranas venenosas
del género Oophaga en Costa Rica y Panamá
Heike Pröhl1*, Beatriz Willink2,3
1.
Instituto de Zoología, Universidad Veterinaria de Hannover, Bünteweg 17, 30559 Hannover, Alemania
2.
Departmento de Biología, Universidad de Lund, SE-223 62 Lund, Suecia
3.
Escuela de Biología, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, 2060 San
José, Costa Rica
The poison dart frogs Oophaga granulifera and Oophaga pumilio are distributed in Nicaragua (only
O. pumilio), Costa Rica and Panama. The ecology and behavior of both species has attracted scientific
attention for several reasons. These frogs exhibit toxicity in combination with bright coloration and
diversification into different color morphs. Moreover, they display highly aggressive and territorial
behavior, and have a complex mating and parental care system. In this article we summarize recent
published data from numerous researchers. We emphasize the link between the behavior (reproduction,
territoriality) of the frogs and their resource and habitat use. Additionally we demonstrate how within
species variation in the strategies used for predator avoidance (aposematism and crypsis) is associated
with the genetic population structure, and correlated with behavioral divergence. We conclude that
evolutionary forces like natural and sexual selection have contributed to diversification within the
species and that these processes might result in the formation of new species. These evolutionary
processes involved in speciation need more attention in conservation planning.
Las ranas venenosas Oophaga granulifera y Oophaga pumilio se encuentran en Nicaragua (solo O.
pumilio), Costa Rica y Panamá. La ecología y el comportamiento de las dos especies han sido de gran
interés científico por varias razones. Estas ranas poseen toxicidad en combinación con colores brillantes
y se han diversificado en diferentes morfotipos de coloración. Además presentan elevada agresividad,
territorialidad y un sistema de apareamiento y cuido parental altamente complejo. En este artículo
resumimos los datos publicados por numerosos investigadores en años recientes. Hacemos énfasis en
el vínculo entre el comportamiento (reproducción, territorialidad) de las ranas y el uso de sus recursos
y sus hábitats. Además demostramos cómo, dentro de las especies, la variación en las estrategias
para evitar depredadores (aposematismo y cripsis) está asociada a la estructura genética poblacional,
y correlacionada con la divergencia en el comportamiento. Concluimos que fuerzas evolutivas como
la selección natural y la selección sexual han contribuido a la diversificación dentro de las especies y
que estos procesos podrán resultar en la formación de nuevas especies. Dichos procesos evolutivos
involucrados en la especiación merecen más atención en los planes de conservación.
INTRODUCCIÓN
Las ranas venenosas de la familia Dendrobatidae forman una familia neotropical con numerosas
especies que se distribuyen en áreas boscosas tropicales desde Nicaragua en el norte hasta Bolivia y Brasil en
el Sur. En Costa Rica y Panamá la familia abarca ocho géneros y 18 especies (Savage, 2002; Grant et al., 2006;
Frost, 2015). Además hay una especies, Allobates talamancae, que pertenece a la familia Aromobatidae que en
Received 07 July 2015
Accepted 13 October 2015
heike.proehl@tiho-hannover.de
HEIKE PRÖHL & BEATRIZ WILLINK
conjunto con la familia Dendrobatidae forma la superfamilia Dendrobatoidea (Grant et al., 2006). Estas ranas son
reconocidas por su interesante y complejo comportamiento. Todas demuestran cierto tipo de cuidado parental y
territorialidad. Además, muchas poseen colores llamativos, son tóxicas y diurnas. Podemos clasificar a las especies
de dendrobátidos en dos grupos según sus defensas contra posibles depredadores: 1) el grupo de las especies poco
tóxicas y de colores crípticos (Summers & Clough, 2001), y 2) el grupo de las especies aposemáticas, que cuentan
con una coloración vistosa y toxinas potentes en glándulas de su piel (Santos et al., 2003, 2009; Saporito et al.,
2012). Las especies de los géneros Allobates, Colostethus y Silverstoneia pertenecen al grupo críptico, mientras
los Dendrobates, Phyllobates y Oophaga son conocidas como especies aposemáticas. Entre ellas, las más tóxicas
son las del género Phyllobates, también conocidas como ranas dardo, ya que los indígenas del Chocó colombiano
usaban las secreciones de su piel para envenenar dardos de caza (Myers et al., 1978). El nombre Oophaga se refiere
al hecho de que los renacuajos se alimentan únicamente de los huevos infértiles que su madres les suministran
(Bauer, 1988, 1994; Grant et al., 2006).
Muchas especies de dendrobátidos han atraído la atención científica de investigadores, quienes se han
interesado en la composición de los alcaloides en la piel de estas ranas (e.g. Daly et al., 1987; Daly et al., 1994), en
su comportamiento territorial y sexual (Rothmair, 1994; van Wijngaarden & von Goo, 1994; Bourne et al., 2001;
Medina et al., 2013), en sus estrategias de cuidado parental (Summers, 1992; Juncá et al., 1994), en sus diversos
sistemas de apareamiento (Brown et al., 2010; Ursprung et al., 2011) y su ecología, particularmente la relación
entre sus tipos de presa, estrategias de forrajeo y evasión de depredadores (Toft, 1995; Santos et al., 2003; Darst et
al., 2005). Sin duda la rana venenosa O. pumilio (en inglés “strawberry poison frog”), ha sido una de las especies
de ranas más estudiada en Panamá y Costa Rica debido a sus patrones de coloración extremadamente variables
(Summers et al., 2003), la toxicidad de su piel (Saporito et al., 2007a) y su comportamiento sumamente complejo
(Donnelly, 1989; Pröhl & Hödl, 1999; Bee, 2003). Sin embargo, la especie hermana O. granulifera ha ganado
terreno en los últimos años por su biología igualmente fascinante y compleja (Crump, 1972; Wang, 2011; Brusa
et al., 2013; Willink et al., 2014a,b). Dedicamos este trabajo al increíble modus vivendi de estas dos especies, que
en su tamaño varían alrededor de 20 mm y la mayoría de sus poblaciones muestran un color rojo fuerte conspicuo
(Lötters et al., 2007). Además incluimos información de tres especies panameñas menos conocidas para discutir
los patrones de especiación dentro del género Oophaga. Recientemente, se han descubierto novedosos aspectos
en su biología y filogenia. Para explicarlos, hacemos referencia a los procesos evolutivos y ecológicos que nos
permiten entender cómo se originan y mantienen las características tan peculiares de estos anfibios.
INFORMACIÓN GENERAL, DISTRIBUCIÓN Y ESTRUCTURA
GENÉTICA DE POBLACIONES
Se estima que un ancestro del género Oophaga invadió Centroamerica desde América del Sur durante
el Mioceno hace alrededor de 20 millones de años (Santos et al., 2009). Estas ranas ancestrales se dividieron
en dos especies hace alrededor de 4 milliones de años (Amézquita et al., datos no publicados), para generar
O. granulifera en las tierras bajas de la vertiente Pacífica de Costa Rica, y el ancestro de O. pumilio y las otras
especies de Oophaga centroamericanas en la tierras bajas de la vertiente caribeña (fig. 1) (Noonan & Wray, 2006;
Galindo-Uribe et al., 2014). Actualmente, la distribución de O. granulifera se extiende desde el extremo oeste de la
vertiente Pacífica en Panamá hasta localidades entre Parrita y Quepos en Costa Rica (Savage, 2002; Köhler, 2011).
La especie habita sobretodo bosques naturales o pocos alterados cerca de quebradas. La distribución de O. pumilio
es más amplia: su rango se extiende desde Nicaragua, a través de la costa caribeña en Costa Rica hasta la región
de Bocas del Toro en el noroeste de Panamá (Savage, 2002; Köhler, 2011). También habita una mayor diversidad
de tipos de hábitat. La encontramos en bosque primario, y secundario, plantaciones de cacao, o banano, fincas
boscosas y hasta en áreas abiertas como herbazales (obs. pers.). Análisis genéticos con marcadores mitocondriales
y nucleares (microsatélites) ayudaron a descubrir la estructura genética en las dos especies. Las secuencias de
genes mitocondriales se utilizan para estimar el nivel y el tiempo de divergencia entre poblaciones mientras que
los microsatélites son secuencias de repeticiones en tándem que se utilizan para estimar la diversidad genética y el
flujo actual entre poblaciones. Un interesante hallazgo de estos análisis es que cada especie comprende dos grupos
genéticos. En O. pumilio el grupo genético del norte abarca las poblaciones de Nicaragua y el norte de Costa Rica,
y el grupo del sur comprende las poblaciones del sureste de Costa Rica y Panamá (Hagemann & Pröhl, 2007; Wang
& Shaffer, 2008). En O. granulifera el grupo del sur comprende las poblaciones del oeste de Panamá y suroeste de
Costa Rica, y el grupo de norte comprende poblaciones desde Palmar Norte hasta el límite norte de la distribución
(Wang, 2011; Brusa, 2013). Hay evidencia de que la formación de ríos caudalosos contribuyó a la separación de
los dos grupos hace alrededor de 1.5-2 millones de años: el Río Térraba en el caso de O. granulifera, y el Río
Reventazón en el caso de O. pumilio (Hagemann & Pröhl, 2007; Hauswaldt et al., 2011; Wang et al., 2011; Brusa
et al., 2013).
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Figura 1. Distribución de morfotipos y estructura genética en O. pumilio y O. granulifera: A. los grupos genéticos ancestrales se muestran en
verde oscuro y los recientes en verde claro; B. O. pumilio es más polimórfica que O. granulifera especialmente en el archipiélago de Bocas del
Toro en Panamá. Elaborada por Heike Pröhl, Beatriz Willink y Sönke van der Berg (2014).
ESTRATEGIAS ANTI-DEPREDADORES Y VARIACIÓN EN LA COLORACIÓN
En el grupo genético más reciente de ambas especies - el grupo Norte en O. granulifera y el grupo Sur
en O. pumilio - se ha originado, evolutivamente, una gran diversidad en la coloración de las ranas (fig. 1). Estos
procesos de divergencia entre poblaciones han dado lugar a un gradiente de coloraciones dorsales entre amarillo,
verde y rojo en O. granulifera (Willink et al., 2013), y a un mosaico de unos 16 distintos patrones de color en el
archipiélago y la tierra firme de Bocas del Toro en O. pumilio (Daly & Myers, 1967; Summers et al., 2003; Rudh et
al., 2007; Batista & Köhler, 2008; Wang & Shaffer, 2008). La importancia relativa de diferentes fuerzas evolutivas
en el proceso de diversificación es todavía un tema de debate entre investigadores. Sin embargo, existe un patrón
consistente entre estudios: en ambas especies la evolución del color ha ocurrido en conjunto con cambios en el
comportamiento y la toxicidad de las ranas (Pröhl et al., 2013).
La fascinante divergencia en color, comportamiento y toxicidad en O. pumilio y O. granulifera ha producido
poblaciones de ranas con estrategias anti-depredadores a lo largo de todo el continuo entre aposematismo y cripsis
(tab. 1). En ambas especies, la condición del grupo genético más antiguo y de algunas poblaciones del grupo
genético más reciente es el aposematismo (Pröhl et al., 2013). En estas poblaciones la coloración dorsal es rojo
brillante, lo que funciona como una señal de advertencia muy visible (sobretodo para aves) de la toxicidad de estas
ranas (Saporito et al., 2007; Paluh et al., 2014). También existen otras poblaciones de apariencia más críptica como
variedades de verde en ambas especies y azul en O. pumilio, estas ranas son más difíciles de detectar para las aves
(Maan & Cummings, 2012; Dreher et al., 2015). Las diferencias de coloración se correlacionan con diferencias de
comportamiento (Pröhl & Ostrowski, 2011; Willink et al., 2013). Nuestros estudios recientes mostraron que tanto
en O. pumilio como O. granulifera el morfotipo rojo es en general más activo que el verde: los machos rojos se
mueven más, comen más, cantan más y lo hacen desde posiciones más expuestas que los machos verdes (tab. 1;
Pröhl & Ostrowski, 2011; Rudh et al., 2011; Willink et al., 2013).
Adicionalmente, la divergencia en color y comportamiento ha sido acompañada por cambios en la toxicidad
de las ranas, pero de formas diferentes en cada especie. En O. pumilio, las ranas más visibles y activas son también
más tóxicas que las ranas más crípticas (Pröhl & Ostrowski, 2011; Maan & Cummings, 2012), mientras que en
O. granulifera las ranas verdes son las más tóxicas (Wang, 2011). La magnitud y variación en toxicidad entre
poblaciones de dendrobátidos se debe al consumo de insectos pequeños (e.g. ácaros, hormigas) que contienen
alcaloides venenosos que las ranas absorben para incorporarlos a las glándulas de su piel (Myers & Daly, 1983;
Daly et al., 1994; Saporito et al., 2004; Saporito et al., 2007; Saporito et al., 2012). Suponemos que las poblaciones
más tóxicas se alimentan de presas más tóxicas y que tienen una mayor capacidad de extraer y/o almacenar estas
toxinas en su piel. Anteriormente se ha encontrado que las especies de ranas más toxicas, de diferentes familias,
se especializan en el consumo de insectos venenosos (tab. 1; Caldwell, 1996; Savage, 2002; Darst et al., 2005).
En las dos especies existen además poblaciones que parecen ser intermedias entre los extremos de
aposematismo y cripsis. En O. granulifera estas poblaciones tienen toxicidad intermedia (Wang, 2011) y además
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Tabla 1. Resumen sobre las características de fenotipos aposemáticos, crípticos e intermedios en ranas. Ejemplos en las fotos: izquierda O.
granulifera y derecha O. pumilio.
Fenotipo aposemático
Fenotipo intermedio
Fenotipo críptico
Componentes del
fenotípo
Coloración/
Conspicuidad
Toxicidad
Comportamiento
Conspicua en su hábitat natural:
altos contrastes en color y brillo
contra los sustratos
Críptico en su hábitat natural: bajos
contrastes en color y brillo contra
los sustratos
(Altamente) tóxico*
Poco o no tóxico *
Comportamiento conspicuo
Depredadores activos; dieta
especializada (hormigas y ácaros)
Forrajeo
Comportamiento críptico
Intermedio
en todos los componentes del
fenotípo
Depredadores pasivos (“sit and wait
predators”); generalistas en su dieta
Actividad
Altas tasas de movimiento, activos
en áreas abiertas, perchas de cantos
expuestas
Bajas tasas de movimiento, más
tiempo escondidos, perchas de
canto más ocultas
Territorialidad
Altamente territorial/ territorios
grandes, defensa de presas tóxicas
Menos territorial/ territorios
pequeños, no defienden presas
tóxicas
* Normalemente en animales aposemáticos la toxicidad está correlacionada con una coloración brillante y conspicua. Sin embargo en O.
granulifera las ranas mas crípticas en coloración son las más toxicas (Wang, 2011).
comparten algunas características de color y comportamiento con cada extremo (Willink et al., 2013). En O.
pumilio, algunas de estas poblaciones utilizan posiciones para cantar donde están más escondidas que las ranas
aposemáticas, pero más expuestas que las ranas más crípticas (Rudh et al., 2011). Es decir, es posible que exista
un gradiente de estrategias en relación a los colores dorsales en ambas especies (tab. 1), aunque seguramente la
historia es aún más compleja en O. pumilio, donde la diversificación ha sido más extrema y las poblaciones de
ranas más aisladas. Estas ideas se respaldan en experimentos de depredación con modelos artificiales de ranas. En
estos estudios se encontró que en poblaciones de distintos morfotipos de O. granulifera, la coloración local provee
mayor protección contra aves depredadoraes que las coloraciones de otras poblaciones (Willink et al., 2014a).
Por otro lado, en O. pumilio el morfotipo local no es necesariamente el mejor adaptado contra depredadores y la
intensidad de depredación varía muchísimo más entre poblaciones (Hegna et al., 2012; Richards-Zawacki et al.,
2013; Dreher et al., 2015).
DISTRIBUCIÓN DE MACHOS Y HEMBRAS EN EL HÁBITAT
En las dos especies los machos son altamente territoriales (Crump, 1972; Bunnell, 1973; Pröhl, 2005a).
Ellos usan sus cantos de advertencia para indicar sus territorios a competidores y también para atraer hembras
(fig. 2.A). Cada macho ocupa uno o varios sitios de canto que pueden ser estructuras expuestas o escondidas
como raíces, bejucos, rocas, ramas u hojas entre 10 y 200 cm de altura. Al colocarse en sitios elevados los machos
facilitan la propagación de sus cantos para que más ranas puedan escucharlos (Forrest, 1994). Además, los machos
usan cantos agresivos para defender sus territorios contra intrusos, y cuando estos cantos son insuficientes para
resolver un conflicto recurren al combate físico como saltos uno encima del otro, volteretas, llaves y patadas (fig.
2.B). Estas luchas pueden ser cortas (unos segundos) o durar hasta horas, y es posible que un individuo pierda
su territorio después de varios días de repetidas peleas con otro (Bunell, 1973; Goodman, 1971; Bolaños, 1990).
Según la población, los territorios son desde pequeños (alrededor de 1 m2) hasta muy grandes (20-30 m2)
(Donnelly, 1989; Pröhl & Hödl, 1999; Pröhl & Ostrowski, 2011; B. Willink, obs. pers. para O. granulifera). La
calidad de los territorios depende de su capacidad de atraer hembras. Los machos intentan defender sitios con alta
densidad de hembras y sitios con cualidades que ayudan a maximizar la transmisión de sus cantos (Pröhl & Berke,
2001). En una población de O. pumilio del sureste de Costa Rica se observó que la calidad de los territorios estaba
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Figura 2. Defensa territorial de los machos en el género Oophaga. Los machos usan cantos de advertencia para defender sus territorios y atraer
hembras (A. O. pumilio de Hitoy Cerere). A veces los conflictos entre machos pueden pasar de cantos agresivos a combates físicos (B. O.
pumilio de Isla Colón). Fotos: Heike Pröhl y Corinna Dreher.
negativamente correlacionada con su tamaño, es decir que la densidad de las hembras que visitan o atraviesan los
territorios es mas alta en los territorios más pequeños (Meuche et al., 2012).
Sin embargo, las hembras no están vinculadas a los territorios de los machos (Pröhl & Berke, 2001).
Al contrario, ellas poseen rangos de acción muy grandes que se solapan con varios territorios de machos (fig.
3). La distribución de las hembras está influenciada principalmente por los recursos reproductivos - pequeñas
acumulaciones de agua donde depositan y crían a sus renacuajos (Donnelly, 1989; Pröhl & Berke, 2001). Estas
acumulaciones se encuentran sobre todo en las axilas de plantas como Dieffenbachia, bromelias, heliconias y
musáceas, y por lo tanto el área de uso más intensivo de las hembras está cerca de estas plantas. Aunque los machos
intentan establecer sus territorios cerca de estas plantas, normalmente no son capaces de monopolizar los recursos
reproductivos (Pröhl & Berke, 2001).
Estudios recientes mostraron que la distribución de los machos de O. pumilio está asociada a la distribución
de las presas en su hábitat (fig. 3) (Staudt et al,. 2010). Aparentemente, la abundancia de hormigas tóxicas como
Brachymyrmex y Paratrechina es más alta dentro de los territorios que en lugares no ocupados. En una población
se observó que también las hembras, dentro de sus extensos rangos de acción, defienden pequeños territorios, con
acumulaciones de hormigas tóxicas y no tóxicas, contra otras hembras. Al igual que los machos, ellas pueden
Figura 3. Distribución espacial de los sexos de Oophaga y sus recursos en el hábitat boscoso de las ranas. Los rangos de acción de las hembras
(azul) abarcan los territorios de los machos (rojo) donde se aparean con ellos, y los sitios donde crían a sus renacuajos (axilas de bromelias o
otras plantas). Los machos usan perchas de cantos (troncos, bejucos, raíces) para atraer a las hembras. La densidad de hormigas tóxicas de las
cuales se alimentan las ranas es más alta en los territorios que en otras áreas del hábitat.
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recurrir a combates físicos por estos territorios (Meuche et al., 2011; M. Scherm, datos propios no publicados).
Esto quiere decir que las presas que suministran alcaloides a las ranas como defensa contra sus depredadores son
un recurso crítico para los dendrobátidos (Staudt et al., 2010). Sin embargo, la distribución de ácaros tóxicos, que
son una fuente importante de alcaloides (Saporito et al., 2004), no ha sido estudiada en relación a los territorios
de las ranas. Estudios adicionales son necesarios para entender mejor el vínculo entre la distribución de insectos
tóxicos (ácaros, algunos escarabajos y hormigas; Saporito et al., 2012), la defensa y el consumo de insectos y la
toxicidad de las ranas. Estos aspectos son fascinantes ya que tanto la disponibilidad de diferentes toxinas (Saporito
et al., 2007c) como la toxicidad de las ranas (Maan & Cummings, 2012) varían geográficamente, lo que podría
generar un vínculo ecológico-etológico sumamente interesante con el comportamiento territorial de las ranas.
SISTEMA DE APAREAMIENTO Y CUIDADO PARENTAL
Tanto los machos como las hembras de las dos especies se aparean con varias parejas durante una estación
reproductiva (Limerick, 1980; Pröhl & Hödl, 1999). La estación reproductiva corresponde a la estación lluviosa,
durante la cual se observa más actividad reproductiva justo después de las lluvias. En la época seca casi no hay
reproducción y hay muy poca actividad de canto, ya que las ranas se esconden en sitios que mantienen alguna
humedad (Pröhl, 1997). Aunque los dos sexos tienen varias parejas, los machos que son exitosos suelen tener
más parejas que las hembras y la competencia dentro del mismo sexo por parejas es más fuerte entre los machos
debido a la proporción operacional de sexo (Pröhl & Hödl, 1999; Pröhl, 2002). La proporción operacional de sexo
es la proporción de machos comparada con la proporción de hembras que están receptivas, es decir, listas para
aparearse. Normalmente, hay más machos que están listos para cantar y atraer a hembras que hembras listas para
Figura 4. Como en muchas otras especies hay una asimetría entre los sexos en Oophaga en el tiempo y la energía que invierten en su cría. En
A) se observa que un macho puede producir suficientes espermatozoides para aparearse todos los días. Sin embargo, las hembras en promedio
producen una puesta de alrededor de 5 huevos cada 5 días. En B) se muestra esquemáticamente la asimetría en la inversión de recursos de
machos y hembras desde el apareamiento hasta la metamorfosis de los renacuajos. i) El apareamiento es en general más costoso para las
hembras. ii) Después de la ovoposición los machos se encargan de humedecer los huevos periódicamente, durante alrededor de dos semanas.
iii) Una vez que los embriones eclosionan, la hembra los transporta a pequeños cuerpos de agua donde iv) los alimenta con huevos infértiles por
algunas semanas hasta la metamorfosis y por ello no esta dispuesta a aparearse. El patrón temporal de reproducción y cuidado parental resulta
en una proporción operacional de sexos sesgado hacia los machos.
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ovular y poner huevos. Por lo tanto, puede haber competencia entre los machos para conseguir una hembra (Pröhl,
2002).
Esta asimetría se basa en el hecho de que las hembras tienen que invertir más energía en la producción
de los huevos que los machos en la producción del esperma y que las hembras invierten más tiempo en el cuidado
parental que los machos (fig. 4). Probablemente, el esperma que un macho gastó en un apareamiento se puede
reemplazar en tan solo un día, mientras que reemplazar una puesta que varía entre 3-6 huevos siempre tarda más,
entre unos días hasta una semana. Nuestros estudios indican que como mínimo la producción de un huevo dura 1
día, o sea la producción de una puesta de 5 huevos dura por lo menos 5 días (Pröhl & Hödl, 1999).
Cuando una hembra se acerca a un macho, el cortejo, que incluye muchas señales acústicas y táctiles,
se desarrolla dentro del mismo territorio. Durante el cortejo, el macho lleva a la hembra a sitios de oviposición,
también dentro del territorio, que muchas veces son hojas secas en la hojarasca (fig. 5.A) (Pröhl & Hödl, 1999).
Aquí los machos dejan su esperma en la superficie de la hoja y las hembras ponen los huevos encima (Limerick,
1980; obs. pers.). Después de la oviposición los machos visitan las puestas en sus territorios una vez al día y las
humedecen. Esta actividad no dura más de media hora. Las hembras recogen a los renacuajos en su espalda cuando
éstos eclosionan y los transportan a pequeños depósitos de agua donde los alimentan con huevos infértiles (fig.
5.B, C). El transporte de un renacuajo puede durar varias horas, pero la alimentación y la crianza de un renacuajo
dura varias semanas, ya que los renacuajos son estrictamente oófagos, es decir, que se alimentan exclusivamente
de huevos (Weygoldt, 1980; Brust 1993; Pröhl & Hödl, 1999; obs. pers.). Las hembras reencuentran a su renacuajo
según la ubicación del depósito de agua pero no reconocen a sus crías directamente (Stynoski, 2009). Durante estas
semanas la producción de huevos infértiles para la crianza de renacuajos impide la producción de huevos para
apareamientos (Pröhl & Hödl, 1999). Los renacuajos necesitan señales táctiles y visuales por parte de la madre
cuando les visita. Cuando esto sucede ellos también empiezan a moverse y vibrar lo que provoca la liberación de
huevos en la hembra (Stynoski, 2012). Recientemente se descubrió que, al igual que los adultos, los renacuajos
contienen alcaloides (Stynoski et al., 2014a) y por eso son rechazados por depredadores como larvas de escarabajos
(familia Elateridae), arañas (Cupiennius coccineus) y serpientes (Leptodeira septentrionalis, Rhadinea decorata)
(Stynoski et al., 2014b). Las hembras suministran los huevos nutritivos con las mismas toxinas que tienen en
sus glándulas granulares de la piel. Este es el primer caso conocido en que una madre o un padre activamente
suministra a su cría con defensas químicas (Stynoski et al., 2014a). A pesar del alto cuidado parental en estas ranas
la supervivencia de los huevos hasta el estado de renacuajo y después hasta la metamorfosis es baja por razones
como depredación (gusanos, caracoles), moho y desecación (Maple, 2002; Pröhl 2005b).
Figura 5. Cortejo y cuidado parental en O. pumilio y O. granulifera: Los machos cortejan a la hembras que entran a su territiorio (A. Oophaga
pumilio). De ser exitosos, la oviposición ocurre dentro del territorio del macho. Las hembras regresan por los renacuajos recién eclosionados y
los transportan hasta pequeños cuerpos de agua (B. O. granulifera), donde los alimentan por semanas con huevos infértiles (C. O. granulifera).
Fotos: Heike Pröhl (A) y Beatriz Willink (B y C).
SELECCIÓN SEXUAL: IMPORTANCIA DE CANTOS Y COLORES
La selección sexual es quizás el tema más investigado en O. pumilio. Gracias al exceso de machos
receptivos en poblaciones donde el número total de machos iguala al número total de hembras, las hembras tienen
la opción de seleccionar a los machos más atractivos, mientras los machos aceptan a cualquier hembra, y hasta
varias hembras en un día, para aparearse (Pröhl, 2002). Los primeros estudios correlacionaron la atracción y el
éxito reproductivo de los machos con sus características de comportamiento. Se encontró que los machos más
exitosos lograban a veces atraer varias hembras durante un mismo día y que su éxito era mucho más alto que el
de otros machos que no conseguían ni una hembra durante observaciones en el campo de varios meses (Pröhl
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& Hödl, 1999). El rasgo que más influye en el éxito de los machos parece ser su actividad de canto, cuánto más
canta un macho más hembras atrae (Pröhl & Hödl, 1999; Meuche et al., 2013). Lo que aún no está claro es si esto
se debe a que las hembras seleccionan machos que cantan mucho, o si los machos que defienden territorios en
áreas con mayor abundancia de hembras cantan más porque la presencia de hembras los estimula a cantar. En O.
granulifera, los machos incrementan su actividad de canto drásticamente cuando una hembra se aproxima a su
territorio e incluso los machos verdes, que normalmente cantan poco, dedican más del 70% de su tiempo a cantar
en estas condiciones (Willink et al., 2014b).
También existe una estrategia alternativa a la territorialidad que ayuda a los machos a conseguir hembras.
Parece que esta estrategia es poco común. Se han observado machos satélites que invaden los territorios de machos
que están cantando con varias hembras presentes. Estos machos satélite tratan de atraer a una de las hembras con
cantos muy suaves y guiarla afuera del territorio para aparearse (Meuche & Pröhl, 2011).
Además, se ha documentado variación en el comportamiento de las hembras. En una población con una
proporción igual de ambos sexos (1:1), las hembras visitaron territorios de varios machos y se observó que una
parte de las hembras rechazó al macho en la última parte del cortejo. Esto quiere decir que las hembras hicieron
una selección (Pröhl & Hödl, 1999). En una población con una proporción de sexos sesgada hacia las hembras
(3:1), las hembras no seleccionaron ni rechazaron machos, sino que se apareaban con el macho que estuviera
cantando más cerca. Experimentos de reproducción artificial de cantos a través de parlantes (play-back) en esta
población verificaron que las hembras prefieren a los machos cercanos sobre machos con cantos de baja frecuencia
dominante (frecuencia que contiene mas energía que otras frecuencias) y altas tasas de canto, características que
son atractivas para hembras en otras especies de ranas (Meuche et al., 2013).
La selección de pareja no sólo es importante para encontrar un macho de ciertas características ventajosas
dentro de una población, sino también para el reconocimiento de la propia especie o de la propia población. Los
dos mecanismos no se pueden distinguir fácilmente ya que una pareja de otra especie representa una pareja de mala
calidad. Sin embargo, hay que tomar en cuenta que hay variación entre poblaciones, grupos genéticos y especies
en los caracteres sexuales como cantos y patrones de color. Aunque los cantos de las especies de Oophaga tienen
una estructura similar (fig. 6), que se podría describir como “zumbido”, ellos varían en la duración de notas, la
tasa de notas y en la frecuencia dominante. Además dentro de cada especie hay variación en los cantos entre las
poblaciones y los grupos genéticos (Pröhl et al., 2007; Brusa et al., 2013). La variación en los morfotipos de
diferentes coloraciones se da sobre todo en la región Bocas del Toro en Panamá en el grupo genético Sur de O.
pumilio y el grupo genético Norte en O. granulifera (Pröhl et al., 2013).
La distribución de O. granulifera no se solapa en su distribución con la de otras especies de Oophaga,
pero la de O. pumilio ocurre en parapatría, es decir que su distribución se solapa parcialmente con al menos otras
dos especies: Oophaga arborea, en la vertiente Caribe del oeste de Panamá, y Oophaga vincentei, en el sureste de
la región de Bocas del Toro (Myers et al,. 1984; Ostrowski & Maan, 2015a). En estas zonas de parapatría, al igual
Figura 6. Presentación gráfica de los cantos de advertencia de A) O. granulifera y B) O. pumilio. Las figuras muestran un oscilograma
arriba y un espectrograma (= sonograma) abajo. El oscilograma muestra el amplitud del sonido contra el tiempo. El espectrograma indica la
distribución de energía a través de las frecuencias contra el tiempo. Se nota que en las dos especies la frecuencia dominante (área oscura rojoazul) se encuentra alrededor de 4 kilohercios. Las notas del canto son mas largas y se reproducen a una tasa más lenta en O. granulifera que
en O. pumilio.
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que en áreas donde los grupos genéticos se solapan (fig. 1: áreas sombreados) la selección de pareja basada en
señales sexuales podría evitar la hibridación y así asegurar la reproducción con una pareja compatible.
Algunos estudios trataron de investigar las preferencias de las hembras por cantos y colores locales versus
cantos y colores de otras poblaciones. Los resultados son un poco ambiguos, ya que se descubrió que en algunas
poblaciones de Panamá las hembras prefieren machos de la coloración local, pero en otras poblaciones no hay tales
preferencias (Summers et al., 1999; Maan & Cummings, 2008; Richards-Zawacki & Cummings, 2011; RichardsZawacki et al., 2012). Otros estudios indicaron que las hembras prefieren colores más brillantes sobre colores
opacos (Maan & Cummings, 2009; Dreher et al., datos no publicados), y que los machos más brillantes son más
agresivos (Crothers & Cummings, 2015). Al contrario otra investigación descubrió que los cantos locales son más
importantes que los colores para atraer a las hembras desde largas distancias, probablemente porque los cantos son
perceptibles desde distancias de varios metros mientras que el reconocimiento de los colores alcanza solamente
distancias cortas (Dreher & Pröhl, 2014). Los cantos permiten a las hembra reconocer a su propia especie o grupo
genético claramente, mientras los padrones de color parecen haber evolucionado por selección natural (Saporito
et al., 2007; Pröhl & Ostrowski, 2011; Maan & Cummings, 2012; Dreher et al., 2015) y/ o una combinación entre
selección natural, selección sexual y reducción en el flujo genético entre poblaciones (Cummings & Crothers,
2013; Gehara et al., 2013).
TRES ESPECIES DE OOPHAGA POCO CONOCIDAS EN PANAMÁ
Existen por lo menos otras tres especies de Oophaga que han sido descubiertas en Panamá: Oophaga
speciosa (Jungfer, 1985), O. arborea (Myers et al., 1984) y O. vincentei (Jungfer et al., 1996; Lötters et al. 2007).
Estas especies han sido poco estudiadas ya que su rangos de distribución son pequeños y sus hábitats menos
accesibles. Oophaga speciosa tiene un rango de distribución muy limitado en el Valle Chiriqui superior en el
Oeste de Panamá, donde habita bosques húmedos premontanos y bosques nublados. Las ranas miden entre 2630 mm y su coloración varia entre rojo vino claro hasta oscuro con manchas oscuras (Jungfer, 1985). Oophaga
arborea habita bosques húmedos de tierras bajas en el Caribe de Panamá hasta bosques premontanos lluviosos
y nublados en la provincia Ngäbe-Buglé (Myers et al., 1984). La coloración varia entre negro y café-bronce
salpicado con puntos amarillos, y el tamaño entre 20 y 22 mm (Myers et al. 1984). La tercera especie, O. vincentei
habita bosques caribeños de tierras bajas y premontanos húmedos en los distritos de El Copé, el Valle de Antón y
Santa Fé en Panamá central (Jungfer, 1996). A lo largo del Río Concepción hay una población simpátrica con O.
pumilio (Ostrowski & Maan, 2015a). Las ranas son pequeñas (19-20 mm) y las poblaciones de esta especie son
bastantes politípicas en cuanto a la coloración dorsal: hay poblaciones rojas, verdes, de color amarillo, turquesa
o verde menta. La mayoría de las ranas poseen barras o manchas oscuras. Además existe una población de ranas
muy pequeñas (~16 mm) rojas con piernas azules en la Isla Escudo de Veraguas y la Península Valiente. Estas
ranas difieren etológica y genéticamente de las otras especies de Ooophaga; pero todavía no se ha realizado una
descripción taxonómica para esta población (Steinmann & van der Lingen, 2014).
Aunque todos estos Oophaga de Panamá son pocos estudiados, su relación filogenética con respecto
a los dos grupos genéticos de O. pumilio son sumamente interesantes y proveen información valiosa sobre la
especiación dentro del género Oophaga. Mientras O. granulifera es la especie menos emparentada con las otras
especies en Costa Rica y Panamá (Clough & Summers, 2000) los otros dos linajes, el de O. pumilio y el de las
Oophaga spp. de Panamá, son géneticamente bastantes cercanos indicando una radiación reciente (Hagemann
& Pröhl, 2007). Curiosamente, O. pumilio no es monofilética en relación a las otras especies. Oophaga arborea
forma un clado con las poblaciones del Sur de O. pumilio, mientras O. speciosa y las poblaciones de Escudo de
Veraguas/ Peninsula Valiente se encuentran en un clado con el linaje del norte de O. pumilio (Hagemann & Pröhl,
2007); O. vincentei está situada genéticamente entre los dos linajes de O. pumilio, pero es más cercana a las O.
pumilio del norte (Hauswaldt et al., 2011). Debido a esta mezcla de diferentes clados se podría argumentar que
O. pumilio del norte y sur representan diferentes especies, sin embargo hay un flujo de genes pronunciado en el
centro de Costa Rica entre los dos grupos, verificando que la especiación no ha terminado (Hauswaldt et al., 2011).
Por otro lado, las especies de Panamá se distinguen ecológicamente de O. pumilio por sus cantos de
advertencia y/o su tamaño. Oophaga speciosa es más grande que O. pumilio, habita bosques a mayor altitud y
produce cantos con una frecuencia dominante más baja y notas un poco más largas (Jungfer, 1985). Oophaga
arborea y O. vincentei son arborícolas y habitantes de epífitas fitotélmicas. Aunque hay observaciones de parapatría
con O. pumilio en las tierra bajas, las dos especies están espacialmente separadas de O. pumilio en su microhábitat
(Myers et al., 1984; Jungfer, 1988; Jungfer, 1996). Además O. arborea produce cantos de una frecuencia dominante
más baja, y notas más largas, mientras O. vincentei produce cantos con una frecuencia dominante más alta y notas
de una duración mucho más larga que O. pumilio (Myers et al., 1984; Jungfer et al., 1996). Las dos especies son
más grandes que las O. pumilio de Panamá (Myers et al., 1984; Jungfer, 1996) pero más pequeñas que O. speciosa.
39
Como los cantos de advertencia juegan un papel importante en la selección sexual en los anuros (Gerhardt &
Huber, 2002) y particularmente en O. pumilio donde las hembras prefieren cantos de los machos de su propio grupo
genético (Dreher & Pröhl, 2014), se puede concluir que las especies de Oophaga en Panamá evolucionaron a causa
de presiones selectivas que resultaron en diferenciación ecológica y acústica, en ausencia de gran diferenciación
genética. Futuros estudios deberán aclarar si hay flujo de genes entre las poblaciones parapátricas. En cuanto al
comportamiento, los machos de O. arborea parecen ser poco territoriales (Myers et al., 1984) en comparación con
los machos de otras especies de Oophaga. Sin embargo el comportamiento sexual y el cuidado parental parecen
ser muy similar entre todos los Oophaga (Limirick, 1980; Weygoldt, 1980; Jungfer, 1985; Jungfer, 1988; Jungfer,
1996; van Wijngaarden & Bolaños, 1992; Brust, 1993). Igualmente la población de Escudo de Veraguas parece
demostrar un comportamiento territorial y reproductivo similar al descrito para O. pumilio, pero los cantos se
distinguen por tener notas mucho mas cortas (Ostrowski & Mahn, 2015b).
CONSERVACIÓN: ¿CUÁL ES EL FUTURO DE LAS RANAS OOPHAGA
EN COSTA RICA Y PANAMÁ?
Las dos especies de Oophaga en Costa Rica se encuentran en distintas categorías de amenaza según la
Unión Internacional para la Conservación de la Naturaleza (UICN): O. pumilio es una especie de preocupación
menor (Solis et al., 2010), mientras que O. granulifera se cataloga como vulnerable (Solis et al., 2008). Una de
las razones para esta diferencia es que O. pumilio se encuentra a menudo en hábitats alterados, como plantaciones
abandonadas de cacao y banano, jardines y matorrales. Por otro lado, O. granulifera se asocia más con bosques
y sobretodo a los lados de quebradas en el piedemonte. Además, la distribución de O. pumilio es más amplia y su
densidad poblacional a menudo es mayor.
Sin embargo, ambas especies comparten importantes amenazas. Según la UICN, la tendencia demográfica
en ambas especies es decreciente. Una de las razones es la continua destrucción del hábitat para asentamientos
humanos y agricultura convencional, aunque O. pumilio prevalece en ciertas plantaciones orgánicas (Solis et al.,
2008, 2010). Además la captura ilegal de estas ranas para el comercio de mascotas, sobretodo en Estados Unidos y
Europa, es un problema persistente y de dimensiones desconocidas (Steinmann & van der Lingen, 2014). Encima
de esto, estudios recientes sugieren que el cambio climático puede afectar las poblaciones de éstas y otras ranas,
incluso en zonas protegidas, debido a los cambios en la cantidad de hojarasca en el bosque (Whitfield et al., 2007).
En Costa Rica, el Sistema Nacional de Áreas de Conservación (SINAC) protege las poblaciones mejor
conocidas en ambas especies. Por ejemplo, hay poblaciones de O. pumilio en el Parque Nacional Tortuguero, en la
Reserva Biológica Hitoy-Cerere, en varios Refugios de Vida Silvestre estatales, mixtos y privados como GandocaManzanillo, Barra del Colorado y La Tirimbina y Zonas Protectoras como La Selva y la Cuenca del Río Banano.
Por otro lado, O. granulifera se encuentra protegida en el Parque Nacional Corcovado, el Parque Nacional Piedras
Blancas y Refugios de Vida Silvestre aledaños. Muy lamentablemente, las poblaciones del grupo genético Norte,
donde ha ocurrido la divergencia evolutiva en la coloración, están prácticamente desprotegidas, ya que este área
del país apenas cuenta con algunos Refugios de Vida Silvestre privados o mixtos, como Hacienda Barú y Portalón
y que son de los más pequeños en Costa Rica.
Igualmente en Panamá hay refugios y reservas que protegen una parte de su diversidad biológica.
Oophaga pumilio está protegida en el Parque Nacional Marino Isla Bastimentos dentro del archipiélago de Bocas
del Toro (Summers et al., 1997) que protege algunos morfotipos. Sin embargo la mayoría de los morfotipos no
están bajo protección en las islas, y más bien están en peligro por el creciente turismo y colecta comercial (Solis
et al., 2010a). Las especies O. speciosa y O. arborea están categorizadas como “En peligro” dado a sus pequeño
rangos y poblaciones con tendencia decreciente (Solis et al., 2010b,c). Una parte del rango de distribución de estas
especies se encuentra en dos áreas protegidas, el Bosque protector Palo Seco y el Parque Internacional La Amistad.
Lötters et al. (2007) suponen que las dos especies están en el riesgo de infectarse con el hongo Batrachochytrium
dendrobatidis, que ha arrasado con múltiples especies en la región en las últimas décadas (Lips, 1999). Oophaga
vincentei es clasificada como “Datos insuficientes” por la UICN. Otras amenazas incluyen la pérdida de hábitat
por tala, el desarrollo de asentamientos humanos, explotación pecuaria y colecciones para el comercio de mascotas
(Solis et al., 2004).
Se podría pensar que no hace falta proteger a una especie en todo su rango de distribución, sino solo
algunas poblaciones lo suficientemente grandes y/o conectadas como para permitir la persistencia de la especie.
Es posible que actualmente cumplamos con este objetivo básico para Oophaga. Sin embargo, los resultados de
más de 20 años de estudios científicos sugieren que si no protegemos a estas especies a lo largo de su extensión
perderíamos una gran diversidad de trayectorias evolutivas que se reflejan en una variedad de colores, cantos,
comportamientos, estrategias reproductivas y posiblemente otros rasgos que aún no se han estudiado. Pensamos
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que esta diversidad es sumamente valiosa, no sólo por su belleza inherente, sino también porque nos permite
entender cómo procesos de miles de millones de años moldean cada detalle en la naturaleza. La pregunta es: ¿Están
Costa Rica y Panamá listas para apreciar y defender tanto la diversidad de especies como los procesos evolutivos
que la generan?
AGRADECIMIENTOS
Agredecemos a todos aquellos estudiantes que en los últimos años ayudaron con la colecta de datos en los
estudios de la ecología de las especies O. granulifera y O. pumilio. Agradecemos también a los funcionarios del
MINAE (Costa Rica) y ANAM (Panamá) por haber colaborado con la obtención de los permisos de investigación,
y Sönke van der Berg por haber ayudado en la preparación de las figuras 1 y 6. Damos las gracias a los revisores,
Gonçalo M. Rosa y Félix Requena que amablemente ayudaron de mejorar el texto.
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46
RESEARCH ARTICLE
2015 | VOLUME 32 | PAGES 47-54
Diversity of amphibians in Wandoor, South Andaman,
Andaman and Nicobar Islands, India
S. R. Chandramouli1*, Tasneem Khan2, Roshni Yathiraj2, Nayantara Deshpande2,
Shreya Yadav2, Cara Tejpal2, Sanne de Groot2, Isabelle Lammes2
2.
1.
Centre for Ecological Sciences, Indian Institute of Science, Bangalore
Andaman and Nicobar Islands Environmental Team (ANET), Junglighat P.O., Port Blair, Andaman and Nicobar Islands.
In this study, for the first time, we present quantitative information about the species richness, habitat
associations and abundances of insular frog species in Wandoor, South Andaman Island, Bay of Bengal,
India. During our surveys spanning across pre and post-monsoon seasons, we recorded six species of
anurans including two endemics. Another endemic sub-species was observed opportunistically. The
Anuran community in the study site was found to be uneven (J’ = 0.34), dominated by the Dicroglossid
Limonectes cf. hascheanus. Of the different forest types surveyed, littoral forests were found to harbor
all the species recorded, followed by evergreen forests which were inhabited by five species; paddy
fields, occupied by four species and the mangroves supported just one specialist species, Fejervarya
cf. cancrivora. A considerable amount of similarity (82%) was observed between paddy fields and
secondary forests in their Anuran species composition, followed by primary evergreen forests (76%).
Possible reasons for the observed patterns in habitat associations of the frog species are discussed.
INTRODUCTION
Small island ecosystems are, in general, known to be “species-impoverished” due to the lack of space and
resources when compared to continental landmasses (Mac Arthur & Wilson, 1967). The Andaman Islands, forming
the major part of the Andaman and Nicobar archipelago, however, are known to harbor a rich assemblage of flora
and fauna, on par with tropical islands in other biodiversity hotspots. A considerable proportion of the fauna are
endemic, and herpetofauna are no exception, currently comprising about 90 species, of which nearly 60% are
endemic (Das, 1999). The number is still likely to rise owing to the incomplete extent of faunal exploration.
Information on the anuran fauna of this region has remained sparse in the past, but for a few sporadic reports
and description of two new species (Pillai, 1977; Mehta & Rao, 1987; Sarkar, 1990; Das, 1998; Chandramouli
et al., 2011). The Nicobar Islands, on the other hand, have been better explored in the recent past, leading to a
new regional record of Hylarana chalconata (see Das, 1996a) and the discovery of two new anurans namely
Limnonectes shompenorum, Polypedates insularis (see Das, 1995; 1996b). In this note, we present information
on species richness and habitat associations of amphibians along the Southwestern coast of the South Andaman
Island. Also, this article presents the first ever detailed quantitative study on amphibian fauna of the Andaman
Islands.
Study area
The Andaman archipelago is composed of five large islands, namely, North Andaman, Middle Andaman,
Accepted 04 November 2015
Published Online 16 November 2015
findthesnakeman@gmail.com
S. R. CHANDRAMOULI et al.
Baratang, South Andaman and Little Andaman. Apart from these, there are approximately 300 small islands that
surround them. Of the larger islands, the first four (i.e., the North, Middle, South Andaman and Baratang) are
relatively large and more or less contiguous, separated from each other only by narrow channels, while Little
Andaman Island lies about 80 km to the south, across the Duncan Passage. The South Andaman Island covers a
geographical land area of 134820 Ha of which 111376 Ha are forested and fall under the legal status of Reserved
Forest, Protected Forest, National Park and the Jarawa Tribal Reserve (Ganeshamurthy et al., 2002; Anonymous,
2007).
The study area is located within the village of Wandoor (c.a. 11.59°N, 92.61°E, 40 m a.s.l., ~ 12 km2) in
the southwestern corner of the South Andaman Island (fig. 1.A and B), and constitutes the eastern periphery of the
Mahatma Gandhi Marine National Park (MGMNP) and the Lohabarrack Crocodile Sanctuary. Though adjacent to
the protected areas mentioned above, the study area supports several fishing villages and farming is one of the most
important activities with respect to land-use. After the December 2004 Tsunami, most of the agricultural areas have
become inundated by sea water, leading to increased soil and water salinity (Raja et al., 2009). Forested areas have
been cleared in the past for cultivation of coconut, arecanut, banana, vegetables and predominantly paddy. The
range of forest types in Wandoor can broadly be described as tropical lowland evergreen, semi deciduous, littoral
and mangrove forests (for detailed descriptions, see Tikader & Das, 1985; Davidar et al., 2001; Anonymous,
2007). Of the above, we classified the tropical lowland evergreen type further into primary and secondary based
on the extent of human activities and disturbances. Our surveys were carried out in all the major habitat types
here namely, primary evergreen forests (PEF), secondary/disturbed forests (SF), littoral forests (LF), mangrove
forests (MF) and paddy fields (PF). Two of the above habitat types, namely, secondary forests and paddy fields are
human-impacted and modified landscapes while the others are natural. Among the above habitat types, primary
and secondary evergreen forests are relatively more extensive and are bordered by littoral habitat along the coast.
Patches of paddy fields are located near the edges between forests and human habitation. Our sampling locations,
surrounding the Andaman and Nicobar Islands Environmental Team (ANET) base-camp, do not fall under any of
the protected areas.
Figure 1. A) Map of the Andaman Islands, showing the location of Wandoor in South Andaman and B) the study area within Wandoor showing
the habitat types surveyed.
Species richness
Visual encounter survey method (Crump & Scott, 1994) was employed to document the anuran fauna in
this region. Typically, the surveys were conducted for a duration of one hour at dusk, wherein specific types of
habitats and microhabitats we carefully inspected by two observers. The sampling effort in terms of the number of
observers was kept constant (i.e. two observers) during all of the sampling sessions. However, the pair of observers
(RY-SY, ND-CT, SdG-IL) varied for each sampling session and they were trained for detecting amphibians
effectively in the field, prior to the initiation of this study. As conducting fieldwork in the Andaman Islands during
the monsoons is not feasible due to logistic reasons, surveys were carried out over a discontinuous period of three
months, i.e., April-May during pre-monsoon and October 2011 in the post-monsoon seasons respectively. No
voucher specimens were collected owing to the lack of collection permits, but the frogs were photographed to
confirm our observations and species identification. Photo-documentation was carried out in the natural habitat,
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upon capture. A total of 34 surveys were carried out, each of which were searches for anurans made over a period
of one hour in a specific type of habitat. Seven such surveys were conducted in each habitat type, with the exception
of mangroves, for which we could make only six surveys. The survey sites were randomly selected in and around
the base. Of these, 21 were during the pre-monsoon season and 13 were during post monsoon. For each survey,
the type of habitat surveyed, number of species, number of individuals per species observed and the microhabitats
occupied by the frogs were recorded. The observed species richness across those 34 surveys was subjected to
randomization and rarefaction using the software package Estimate S (Colwell, 2013) and Chao1 estimator was
used to obtain an estimate of species richness in the study area and to check the adequacy of our sampling.
Species abundance and habitat associations
Abundance of each species recorded during the surveys was quantified by their encounter rates, represented
as the number of individuals of a species observed over a period of one hour. Relative abundance of each species
was represented as the proportion of the number of individuals of that species observed across all the samples.
Hierarchical cluster analysis was used to discern clusters of species with similar habitat usage. Likewise, the
similarity/distinction between the different types of habitats in terms of their anuran species composition was
examined using hierarchical cluster analysis. For both these, the Bray-Curtis similarity index was used as the
measure to construct the dendrogram with paired-group algorithm using the software package PAST (Hammer et
al., 2001).
RESULTS
Species richness
A total of 681 individuals of six species of anurans belonging to four genera and three families were
recorded during the systematic surveys. These included two endemic species and one endemic sub-species,
accounting for 40% of endemism (tab. 1). Abundances of the six species observed during the surveys indicate
an uneven (J’ = 0.34) structure of the anuran community. The observed species richness (S = 6) for the survey
sample size of 34 was found to coincide exactly with the estimated value, indicating the adequacy of samples and
completeness of our inventory (fig. 2).
Table 1. Checklist of anuran species observed at Wandoor, South Andaman during this study. * indicates endemic species. ^ indicates that the
status applies to the conferring species.
Family
IUCN status
Bufonidae
Least concern
Kaloula baleata ghoshi Cherchi, 1854*
Microhylidae
Least concern
Microhyla chakrapanii Pillai, 1977*
Microhylidae
Data deficient
Fejervarya andamanensis (Stoliczka, 1870)*
Dicroglossidae
Least concern
Fejervaya cf. cancrivora
Dicroglossidae
Least concern^
Fejervarya cf. limnocharis
Dicroglossidae
Least concern^
Limnonectes cf. hascheanus
Dicroglossidae
Least concern^
Species
Duttaphrynus melanostictus (Schneider, 1799)
Species abundance and habitat associations
On average, 20.1 anurans were observed in each sample of one hour duration (fig. 3). There was a great
disparity in the number of frogs sighted during pre-monsoon (mean: 4 frogs/hour) and post-monsoon (mean: 29
frogs/hour) seasons. Limnonectes cf. hascheanus (fig. 4.F) was found to be the most common species with an
49
Figure 2. Samples based rarefaction curves of frog species in Wandoor.
encounter rate of 14.35 individuals/hour, followed by 3.52 for Fejervarya cf. limnocharis, 1.17 for Microhyla
chakrapanii (fig. 4.B), 0.47 for Fejervarya andamanensis (fig. 4.E) and 0.23 in the case of Fejervarya cf.
cancrivora (fig. 4.D) and Duttaphrynus melanostictus (fig. 4.A). Another endemic subspecies, Kaloula baleata
ghoshi (fig. 4.C), was observed opportunistically and was not recorded during the surveys. This was observed to
be an elusive species and was seen emerging out of tree-holes at night time within the base premises, thus making
it a hard species to detect during sampling.
Paddy fields were found to be inhabited by four species, namely, Fejervarya andamanensis, F. cf.
limnocharis, Limnonectes cf. hascheanus and Microhyla chakrapanii. Primary evergreen and secondary forests
were inhabited by most species except Fejervarya cf. cancrivora, the only frog species found in the mangrove
habitat, which is known to be relatively tolerant to salinity in the environment. Littoral forests were the only type of
habitat found to harbor all the six species of frogs recorded during this survey in this region (fig. 5). Species-wise,
Fejervarya andamanensis was observed mostly in primary evergreen forests, followed by paddy fields, secondary
forests and to a very low extent, in littoral forests. The common asian toad (Duttaphrynus melanostictus), being an
anthropophilic species, was recorded mostly in the disturbed secondary forests, followed by littoral forests, and to
a very low extent, in primary evergreen forests. The cricket frog (Fejervarya cf. limnocharis) was predominant in
the paddy fields, but was also observed in low numbers in secondary forests, primary evergreen and littoral forests.
Figure 3. Relative abundance of frog species in Wandoor, South Andaman.
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Figure 4. Some amphibian species found in Wandoor, South Andaman, Andaman and Nicobar islands, India: A. Duttaphrynus melanostictus;
B. Microhyla chakrapanii; C. Kaloula baleata ghoshi; D. Fejervarya cf. cancrivora; E. Fejervarya andamanensis, the first ever illustration of
this species in life; F. Limnonectes cf. hascheanus.
The narrow-mouthed frog (Microhyla chakrapanii) was most predominant in secondary forests, but was also
observed in littoral forests, primary evergreen forests and paddy fields to some extent. Limnonectes cf. hascheanus
was equally prevalent in paddy fields, evergreen and secondary forests, and to a lesser extent in littoral forests.
Fejervarya cf. limnocharis and Microhyla chakrapanii showed a similar pattern in habitat use, while Duttaphrynus
melanostictus and Fejervarya andamanensis showed a similar habitat use pattern. Fejervarya cf. cancrivora
showed a remarkable shift in habitat associations by being more abundant in mangrove forests, a habitat which
was used by no other anuran (fig. 6).
Complementarily, the different habitats also showed a specific pattern in their species composition.
Paddy fields and secondary forests showed a similar pattern in their species composition followed by primary
evergreen forests. Littoral forests were used to some extent by all the species, while the mangrove habitat was used
51
Figure 5. Habitat utilization by frogs in Wandoor.
exclusively by Fejervarya cf. cancrivora (fig. 7).
Terrestrial and semi-aquatic microhabitats such as ground, leaf litter and ephemeral puddles were used
by most of the species while arboreal niches were rather empty. Arboreal microhabitats such as tree holes were
occasionally used by Kaloula baleata ghoshi as a refuge, though much of its foraging and feeding activities
occurred on the ground. There was no significant difference in the pattern of habitat use between the endemic and
non-endemic species, as both utilized natural and human-impacted habitats to the same extent.
DISCUSSION
Till date, eight species of frogs are known from the Andaman Islands (Mehta & Rao, 1987; Sarkar,
1990; Pillai, 1997; Das, 1999). Our surveys were able to record most of the species known from this region
with the exception of Kaloula baleata ghoshi, which has been sighted here opportunistically (pers. obs.). Charles
Darwin’s frog (Ingerana charlesdarwini), a critically endangered species, was discovered from the forests of Mt.
Figure 6. Dendrogram showing the similarity between frog species based on their habitat occupancy. The relative abundance in each habitat
is represented within peranthesis.
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Figure 7 Similarity between different types of habitats based on their anuran species composition.
Harriet National Park, which lies on the eastern periphery of South Andaman (Das, 1998) and was not observed
in Wandoor during our study. Microhyla chakrapanii has been recorded for the first time from the island of South
Andaman since its description. This species has been known only from its type locality, Mayabunder, located
in the Middle Andaman Island and vicinity. It is noteworthy to mention that the observed pattern in the relative
abundance of species indicates that widespread, non-endemic taxa tend to dominate the community compared to
range-restricted, narrowly endemic species in this region. However, the taxonomic identities of many of these
species warrant further intensive investigation (pers. obs.). The lower abundance of the common toad observed
here probably indicates its sporadic distribution, making it more common in and around human habitation and
disturbed forests but rare in natural, pristine evergreen forests. The common toad is known to be an anthropophilic
species with a high degree of tolerance to human habitations (Daniel, 2002; Daniels, 2005). The differences in
seasonal abundances of frogs observed during our study could be attributed to the high frequency of courtship and
breeding activities which commence with the onset of early monsoons (making their presence more conspicuous),
and gradually subside as the rains recede, thereby making them more dormant. Relative abundances of the endemic
species, Microhyla chakrapanii and Fejervarya andamanensis in pristine and altered habitats observed during
this study, probably point at their incipient adaptive response to cope with the recent, dynamic habitat alterations.
However, further surveys across larger geographic extents would be essential to test for the consistency of this
pattern.
With regard to the conservation status of these amphibians, none of them are in any of the IUCN threatened
categories. However, it should be borne in mind that their status of being Least Concern holds good only at the local
regional scale. When considered from a global perspective, the restricted distribution of these endemic species
renders them vulnerable to extinction, due to threats such as deforestation and climate change. Even baseline
data on ecology and natural history of many of these poorly known species has largely been lacking and their
population trends after natural disturbances like the Tsunami need to be monitored for their long term survival.
ACKNOWLEDGEMENTS
We thank the ANET staff Saw John and Anita Prasad; especially Saw Tehsorow and Saw Agu for having
assisted with fieldwork. We are thankful to Kartik Shanker for his suggestions and comments on the manuscript.
LITERATURE CITED
Anonymous (2007). Forest statistics. Department of Environment and Forests, Andaman and Nicobar Islands,
Port Blair.
53
Chandramouli, S.R., Harikrishnan, S., Vasudevan, K. (2011). Little known endemic frogs of the Andaman islands.
Froglog, 98: 16-17.
Colwell, R.K. (2013). EstimateS: Statistical estimation of species richness and shared species from samples.
Version 9. User’s Guide and application available from http://purl.oclc.org/estimates (accessed November
2014).
Crump, M.L., Scott, N.J. (1994). Visual encounter survey. In: Heyer, W.R., Donnelly, M.A., Mcdiarmid, R.W.,
Hayek, L.C., Foster, M.S. (eds.). Measuring and monitoring biological diversity: Standard methods for
amphibians. Smithsonian Institution press, Washington, DC: 84-96.
Daniel, J.C. (2002). The book of Indian reptiles and amphibians. Bombay Natural History Society, Mumbai.
Daniels, R.J.R. (2005). Amphibians of Peninsular India. Indian Academy of Sciences University Press, Hyderabad.
Das, I. (1995). A new tree frog (genus Polypedates) from Great Nicobar, India (Anura: Rhacophoridae). Hamadryad,
20: 13-20.
Das, I. (1996a). Geographic distribution: Rana chalconata (copper cheeked frog). Herpetological Review, 27:
30-30.
Das, I. (1996b). Limnonectes shompenorum, a new frog from the Rana macrodon (Anura: Ranidae) complex from
Great Nicobar, India. Journal of South Asian Natural History, 2: 60-67.
Das, I. (1998). A remarkable new species of ranid (Anura: Ranidae), with phytotelmonous larvae, from Mount
Harriet, Andaman Island. Hamadryad, 23: 41-49.
Das, I. (1999). Biogeography of the amphibians and reptiles of the Andaman and Nicobar islands, India. In: Ota,
H. (ed.). Tropical island herpetofauna: Origin, current diversity and current status. Elsevier, Amsterdam:
43-77.
Davidar, P., Yoganand, K., Ganesh, T. (2001). Distribution of forest birds in the Andaman Islands: importance of
key habitats. Journal of Biogeography, 28: 663-671.
Ganeshamurthy, A.N., Dinesh, R., Nair, A.K., Ahlawat, S.P.S. (2002). Land resources of Andaman and Nicobar
Islands. Central Agricultural Research Institute, Port Blair.
Hammer, Ø., Harper, D. A.T., Ryan, P.D. (2001). PAST: Paleontological statistics software package for education
and data analysis. Paleontologia Electronica, 4: 1-9.
MacArthur, R.H., Wilson, E.O. (1967). The theory of island biogeography. Princeton University Press, New Jersey.
Mehta, H.S., Rao, G.C. (1987). Microhylid frogs of Andaman and Nicobar Islands. Journal of the Andaman
Science Association, 3: 98-104.
Pillai, R.S. (1977). On two frogs of the family Microhylidae from Andamans including a new species. Proceedings
of the Indian Academy of Sciences, 86: 135-138.
Raja, R., Chaudhuri, S.G., Ravisankar, N., Swarnam, T.P., Jayakumar, V., Srivastava, R.C. (2009). Salinity status
of tsunami-affected soil and water resources of South Andaman, India. Current Science, 96: 152-156.
Sarkar, A.K. (1990). Taxonomic and ecological studies on the amphibians of Andaman and Nicobar Islands, India.
Records of the Zoological Survey of India, 86: 103-117.
Tikader, B. K., Das, A.K. (1985). Glimpses of animal life of Andaman and Nicobar islands. Zoological Survey of
India, Calcutta.
54
SCIENTIFIC NOTE
2015 | VOLUME 32 | PAGES 55-58
The first record of the streamside frog Craugastor
rupinius (Anura: Craugastoridae) in Honduras,
confirmed by 16S DNA barcoding
Josiah H. Townsend1,*, Thomas J. Firneno, Jr.1, Dorian L. Escoto2,
Einstein A. Flores-Girón2, Melissa Medina-Flores1, Olvin Wilfredo Oyuela3
2.
1.
Department of Biology, Indiana University of Pennsylvania, Indiana, Pennsylvania 15705-1081, USA
Escuela de Biología, Universidad Nacional Autónoma de Honduras, Tegucigalpa, Departamento de Francisco Morazán, Honduras
3.
Herbario TEFH, Universidad Nacional Autónoma de Honduras, Tegucigalpa, Departamento de Francisco Morazán, Honduras
We report the first record of the streamside frog Craugastor rupinius from the Central American
Republic of Honduras. This species was previously known from adjacent areas of El Salvador and
Guatemala. Due to the highly conserved morphology of anurans in this group, we used sequence data
from the mtDNA gene 16S to confirm that the Honduran sample is conspecific with a verified sample
of C. rupinius from El Salvador.
Reportamos el primer registro de la rana de ribera Craugastor rupinius en la República Centroamericana
de Honduras. Esta especie ha sido reportada previamente en zonas adyacentes en El Salvador y
Guatemala. Debido a que la morfología de los anuros en este grupo es altamente conservada, utilizamos
datos de la secuencia del gen 16S del genoma mitocondrial para confirmar que la muestra de Honduras
es conspecífica con una muestra verificada de C. rupinius de El Salvador.
Craugastor rupinius (Campbell & Savage, 2000) is a terrestrial, riparian frog found along the Pacific
versant in southern Mexico, southern Guatemala, and most of El Salvador from 400-1760 m elevation (Campbell
& Savage, 2000; Köhler, 2011). This species is considered a member of the C. rugulosus series and the C.
punctolarius group (Hedges et al. 2008), a clade of 34 described species characterized by their conserved
morphology and elevated conservation status (Campbell & Savage, 2000; Percino-Daniel et al., 2014). Craugastor
rupinius has not been previously reported from Honduras, however McCranie & Castaneda (2007) listed it as a
species of probable occurrence in Honduras, due to the presence of C. rupinius on the El Salvadoran portion of
Cerro Montecristo, a mountain that also extends into the Department of Ocotepeque in extreme western Honduras.
From 24 June to 10 July 2011, four of the coauthors (MMF, EF, DE, and OO) sampled herpetofaunal
diversity on the Honduran side of Cerro Montecristo, within the boundaries of Montecristo-Trifinio National
Park, Depto. Ocotepeque, as part of a multidisciplinary project to promote biodiversity conservation and rural
development in the communities surrounding the park. On 29 June 2011, they collected a single specimen of
streamside Craugastor (fig. 1; Carnegie Museum of Natural History [CM] 158361) from a small stream above Los
Naranjitos (14°27’39.60”N, 89°18’21.60”W, 1695 m a.s.l.). We investigated the taxonomic status of this specimen
using sequence data from the mitochondrial gene 16S.
A tissue sample from CM 158361, along with tissue samples representing three other Craugastor species
collected from 2005-2013 in Honduras (tab. 1), were amplified for 16S using primers 16Sar-L and 16Sbr-H
(Palumbi et al., 1991) and sequenced using a BigDye Terminator v3.1 Cycle Sequencing kit (ABI) on an ABI
Received 08 October 2015
josiah.townsend@iup.edu
JOSIAH H. TOWNSEND et al.
Table 1. Samples used in phylogenetic analyses; CR, Costa Rica; ES, El Salvador; GT, Guatemala; HN, Honduras; PA, Panama.
Locality
GenBank
voucher
GenBank accession
number
HN: Ocotepeque, Cerro Montecristo
CM 158361
KR260377
ES: Usulutan, Cerro del Tigre
KU 289861
EU186669
HN: Atlántida, Pico Bonito
USNM 578582
KR260374
HN: Cortés, El Paraiso Valley
UF 149340
KR260375
HN: Cortés, Cerro Azul Méambar
UF (IRL023)
KR260376
CR: Heredia, Chompipe, Volcan Barba
MVZ 149762
EU186681
C. megacephalus
PA: El Copé
KRL0686
FJ784335
C. obesus
PA: Chiriqui
AMNH 124540
EU186737
C. punctariolus
PA: El Copé
KRL0954
FJ784448
C. rugulosus
CR: Guanacaste, Volcan Cacao
MVZ 207279
EU186680
C. sandersoni
GT: Izabal, Sierra de Santa Cruz
UTA A-49803
EF493712
C. fitzingeri
PA: El Copé
KRL 0720
FJ784344
C. rhodopis
MX: Oaxaca
JAC 22721
DQ283317
Taxon
Craugastor rupinius
C. rupinius
C. aurilegulus
C. charadra
C. laevissimus
C. angelicus
3730xl sequencer. Our samples were combined with sequences from GenBank representing other members of the
C. rugulosus series and three outgroup taxa: C. fitzingeri, C. megacephalus, and C. rhodopis (tab. 1). Sequences
were aligned using ClustalW (Thompson et al., 1994) within the program package MEGA 6.06 (Tamura et al.,
2013) using default parameters. Best fit models of nucleotide substitution were estimated using jModeltest 2.0
(Darriba et al., 2012). Uncorrected (p-distance) pairwise sequence divergence was calculated for all samples and
for the gene to provide an estimate of intra- and interspecific variation. Sequence divergence estimation was
performed in MEGA 6.06 (Tamura et al., 2013). Maximum likelihood (ML) analysis was carried out in RaxML
v8.0 (Stamatakis, 2014), with 1000 bootstrap pseudoreplicates under the GTR+GAMMA substitution model.
Bayesian Inference (BI) was performed using MrBayes3.2.2 (Hueslsenbeck & Ronquist, 2001), and consisted of
two parallel runs of four Markov chains (three heated, one cold) run for 20x106 generations and sampled every
10,000 generations, with a random starting tree and the first 2x106 generations discarded as burnin. External
measurements were taken on the preserved specimen using digital calipers to the nearest 0.01 mm.
Distance-based analysis for 16S yielded interspecific divergence distances that ranged from 2.1-19.4%,
with CM 158361 being 0.4% divergent from a reference sample of Craugastor rupinius (KU 289861) available
on Genbank. Both the ML and BI methods recovered a topology that indicates CM 158361 is conspecific with
the available C. rupinius sample from El Salvador (fig. 2). Measurements for CM 158361 are as follows (in
mm): snout-vent length = 57.11; head length = 20.76;
head width = 22.93; eye width = 4.98; interorbital
distance = 5.72; eye length = 6.88; tympanum width
= 3.58; crus length = 30.42. Based on morphological
comparisons and genetic analyses, we confirm the
taxonomic identity of CM 158361 as C. rupinius,
providing the first verified record of this taxon in
Honduras.
DNA barcoding has become an increasingly
utilized component of taxonomic studies over the
past decade (Hebert et al., 2003; Biju et al., 2014).
The use of short DNA sequences for preliminary
taxonomic assignment has proven useful with
taxa such as amphibians when morphological
identification can be confounded by conserved
phenotypic diversification and morphological
homoplasy (Emerson, 1986; Wake, 1991; Vences et
Figure 1. Craugastor rupinius (CM 158361), from Cerro Monteal., 2005). The mitochondrial 16S large subunit RNA
cristo, Depto. Ocotepeque, Honduras.
56
ALYTES 2015 | 32
Figure 2. Phylogram showing the phylogenetic relationships of the Craugastor rupinius sample from Honduras based on a single fragment of
16S rRNA; Bayesian posterior probabilities and maximum likelihood bootstrap scores are shown when > 0.6 and 60, respectively.
(16S) has been shown to provide sufficient resolution and robustness in most amphibian species (Vences et al.,
2005), and has been much widely applied in phylogenetic analyses of amphibians, thus providing a more robust
comparative reference dataset than the traditional metazoan barcoding locus COI. As the costs associated with
generating sequence data continue to decrease, we envision papers such as this one becoming more commonplace
and thus realizing the utility of DNA barcoding as a tool for assisting in taxonomic identification of unknown or
challenging samples.
ACKNOWLEDGEMENTS
Research in Honduras was carried out under permits issued by the Instituto Nacional de Conservación y
Desarrollo Forestal, Áreas Protegidas y Vida Silvestre [ICF] (Resolución DE-MP-086-2010 and Dictamen DVS
ICF-045-2010); we thank S. Laínez, I. Acosta, and R. Downing for assistance in obtaining these permits and for
granting permission to export this specimen (Constancia 009-2012-DVS-ICF). We thank José Padial and Steve
Rogers (CM) for accessioning the specimen.
LITERATURE CITED
Biju, S.D., Garg, S., Gururaja, K.V., Shouche, Y., Wilujkar, S.A. (2014). DNA barcoding reveals unprecedented
diversity in Dancing Frogs of India (Micrixalidae, Micrixalus): a taxonomic revision with description of
14 new species. Ceylon Journal of Science, 43: 37-123.
Campbell, J.A., Savage, J.M. (2000). Taxonomic reconsideration of Middle American frogs of Eleutherodactylus
rugulosus group (Anura: Leptodactylidae): a reconnaissance of subtle nuances among frogs. Herpetological
Monographs, 14: 186-292.
Darriba, D., Taboada, G.L., Doallo, R., Posada, D. (2012). jModelTest 2: more models, new heuristics and parallel
computing. Nature Methods, 9: 772-772.
Emerson, S.B. (1986). Heterochrony and frogs: the relationship of a life history trait to morphological form. The
American Naturalist, 127: 167-183.
Herbert, P.D., Cywinska, A., Ball S.L., deWaard, J.R. (2003). Biological identifications through DNA barcodes.
Proceedings of the Royal Society B: Biological Sciences, 270: 313-321.
Hedges, S.B., Duellman, W.E., Heinicke, M.P. (2008). New World direct-developing frogs (Anura: Terrarana):
Molecular phylogeny, classification, biogeography, and conservation. Zootaxa, 1737: 1-182.
Huelsenbeck, J.P., Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17:
57
JOSIAH H. TOWNSEND et al.
754-755.
Köhler, G. (2011). Amphibians of Central America. Offenbach, Germany, Herpeton.
McCranie, J.R., Castañeda, F.E. (2007). Guia de Campo de los Anibios de Honduras. Bibliomania, Salt Lake City,
Utah.
Palumbi, S.R., Martin, A., Romano, S., McMillan, W.O., Stice, L., Grabowski, G. (1991). The simple fool’s guide
to PCR, version 2. Zoology Department, University of Hawaii, Honolulu.
Percino-Daniel, R., Garcia del Valle, Y., Campbell, J.A. (2014). Rediscovery and additional records for Craugastor
palenque (Anura: Craugastoridae) from the archeological Mayan site Palenque, Chiapas, Mexico. The
Southwestern Naturalist, 59: 139-141.
Stamatakis, A., Ludwig, T., Meier, H. (2005). RAxML-III: a fast program for maximum likelihood-based inference
of large phylogenetic trees. Bioinformatics, 21: 456-463.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics
Analysis Version 6.0. Molecular Biology and Evolution, 30: 2725-2729.
Thompson, J.D., Higgins, G.D., Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Research, 22: 4673-4680.
Wake, D.B. (1991). Declining amphibian populations. Science, 253: 860.
58
RESEARCH ARTICLE
2015 | VOLUME 32 | PAGES 59-65
Natural History and distribution notes on the Sreeni’s
golden frog (Indosylvirana sreeni) in the Southern
Eastern Ghats, peninsular India
S. R. Ganesh1,*, M. Arumugam1
1.
Department of Zoology, University of Madras, Guindy Campus, Chennai – 600 025, Tamilnadu, India
We studied a range-restricted, recently described, little-known endemic ranid frog Indosylvirana
sreeni from Southern Eastern Ghats, which forms a peripheral unit of its geographic range supporting
satellite populations. We provide detailed notes on the morphology, call characteristics, distribution
and a quantitative account on microhabitat associations of I. sreeni in the Southern Eastern Ghats.
We record the presence of I. sreeni in three out of four hill ranges surveyed and present an updated
distribution map for the species. Based on our field observations, we also report instances of roadkill
mortalities and malformation (anophthalmia) in this species.
INTRODUCTION
The ranid frog genus Indosylvirana Oliver, Prendini, Kraus & Raxworthy, 2015 is represented in
peninsular India by 10 species (Srinivasulu & Das, 2008; Biju et al., 2014; Oliver et al., 2015). Sreeni’s golden frog
Indosylvirana sreeni (Biju, Garg, Mahony, Wijayathilaka, Senevirathne & Meegaskumbura, 2014) is one among
the nominate congeners described from the Western Ghats, originally as Hylarana sreeni. However, a subsequent
large-scale revision by Oliver et al. (2015) involving the taxa from the entire old world previously associated with
Hylarana s. lat. revealed multiple distinct genera, including their newly erected Indosylvirana that occurs in India,
Sri Lanka, Vietnam, Thailand and Cambodia (Oliver et al., 2015).
Sreeni’s golden frog was described based on the holotype BNHS 5869 (Bombay Nat. Hist. Soc., Mumbai,
India) originating from Siruvani hills (10.970°N 76.655°E; 758 m asl). More importantly, I. sreeni is the only
congener treated by Biju et al. (2014), that also occurs in the Eastern Ghats (Srinivasulu & Das, 2008; Biju et al.,
2014). Daniels & Kumar (1998) and Vanak et al. (2001) sighted and reported this species from Kolli and Sirumalai
hills respectively. Rao et al. (2005) represented a congener as Rana temporalis complex from Nallamalais, Central
Eastern Ghats; later Srinivasulu & Das (2008) reserved the identity of that taxon as Hylarana sp. but included it in
the fauna of Nallamalai hills. Thus the presence of this genus in the Eastern Ghats has long been known, but was
almost always represented as R. temporalis complex.
A recent revision involving congeners from the Western Ghats-Srilanka biodiversity hotspot brought
about significant clarity in this group, which included the description of I. sreeni. Biju et al. (2014) in their
study, genetically tested a subadult female specimen SDBDU 2004.4553 (Delhi Univ., India) with genebank no.
KM068993 for 16S, KM069101 for COI and KM069207 for CytB (Biju et al., 2014) from Yercaud (11.775°N,
78.209°E; 1439 m a.s.l.), Shevaroys and concluded conspecificity between the Yercaud and Western Ghats
populations in this species. Biju et al. (2014) reported intraspecific genetic distance between I. sreeni populations
based on 12-14 specimens from within all their sampled localities (including Yercaud) to be: 0.8 ± 0.6 (0-1.5) %
for 16S, 2.3 ± 1.4 (0-3.6) % for COI, 3.7 ± 2.2 (0-6.1) % for CytB.
Received 03 April 2015
snakeranglerr@gmail.com
S. R. GANESH & M. ARUMUGAM
Also, I. sreeni is one of the few peninsular Indian Indosylvirana species that occurs in hill ranges on
either sides of Palghat Gap – a major biogeographic barrier, thus further supporting the conspecificity between the
Western and Eastern Ghats populations, and hence a genuinely wide distribution range. The status of the Central
Eastern Ghats population still remains unresolved (Rao et al., 2005; Srinivasulu & Das, 2008; Biju et al., 2014).
Even though Biju et al. (2014) has comprehensively provided adequate information on all the taxa, their paper only
focused on the Western Ghats-Srilanka hotspot, but not on other (Indosylvirana-occupied) regions of peninsular
India. Against this background, we here elaborate on the morphology, first in-life illustration, call characteristics,
distribution and microhabitat association of satellite or metapopulations of Sreeni’s golden frog (I. sreeni) from
the Southern Eastern Ghats.
We studied wild Indosylvirana sreeni during 2011-2015, by surveying the following massifs in the
Southern Eastern Ghats: Jawadi (=Tirupattur) hills (12.26-12.51°N, 78.60-78.76°E; 1200 m a.s.l.) in Vellore
district; Shevaroy hills (11.72-11.93°N, 78.12-78.36°E; 1620 m a.s.l.) in Salem district; Kolli hills (11.19-11.46°N,
78.28-78.47°E; 1400 m a.s.l.) in Namakkal district and Sirumalai hills (10.12-10.28°N, 77.92-78.11°E; 1400 m
a.s.l.) in Dindigul district of Tamil Nadu State (fig. 1). The dominant natural climax vegetation type in the higher
slopes (> 900 m asl) is tropical evergreen forest, interspersed with coffee plantations (except Jawadi), silver oak
estates and fruit orchards (Jayakumar et al., 2008).
We gathered primary field data from day and night surveys conducted in the region using time-constrained
visual encounter method (Crump & Scott, 1994). We noted down all the habitat details of every sighting of the
target species. We gently restrained live specimens and collected basic morphological and morphometrical data
in situ for select characters (after Dutta & Manamendra-Arachchi, 1996). Measurements were taken using vernier
calipers (least count 1 mm) and morphological features were observed using magnifying hand lenses. No voucher
specimens were collected due to want of collection permits; however, photographic vouchers were made in the
form of high resolution digital camera photographs (see fig. 2). We allocate our individuals from the study area to
I. sreeni for possessing the following combination of characters (after Biju et al., 2014): adult snout-vent length
44-80 mm; dorso-lateral skin folds well-developed; webbing on inner side of 2nd and 3rd toes at the level of 1st
subarticular tubercle; snout ovoid in dorsal and ventral views.
Figure 1. Distribution map of Indosylvirana sreeni showing the type locality (*) and the hill ranges in the Western Ghats (solid circles) where
it was reported (Biju et al., 2014, including Shevaroys); in Southern Eastern Ghats (open circles) where we recorded this species, and Jawadi
hills (dotted circle) where this species was not detected despite 500 hrs of field survey.
60
ALYTES 2015 | 32
Calls of the frogs were recorded in situ using Canon Powershot SX130IS model camera and the video files
later converted into audio (*.wav) format. A total of six calls of one individual were recorded and analysed. Detailed
waveforms and analyses were presented for the best call that was manually selected from the recordings that had
the least background noise. The analysed calls presented here were recorded in July (south-west monsoon), during
daytime (11:30 hrs, 20.8°C, 88% relative humidity) from a distance of 1.5 m from the calling frog. Call analyses
were done in Wavesurfer 1.8.8, with a sampling rate of 441000 Hz at 16-bit mono format. The audio spectrogram
was achieved at Blackman window function with 512 band resolution and peak frequency details were acquired
through Fast Fourier Transformation (FFT, width 512 points, bandwidth of 86.132 Hz) in the spectrum section plot
of the software. No filtering was done during analysis. Call terminology follows Kuramoto & Joshy (2001) who
described the call of a congener (I. indica), against which we briefly compare our present call.
Definition and terminology of amphibian malformation followed Meteyer (2000).
RESULTS
Morphological features provided here are based on 19 live, uncollected specimens of Indosylvirana sreeni.
Measurements (in mm). Snout-vent length: 46-80, (subadults 15-16), head length: 19-30, head width:
13-22, head depth: 8-15, axilla-groin distance: 26-50, (subadults 7-9), fore limb length: 21-49, hind limb length:
101-121.
Body form (fig. 2). Habitus slender; head rather long, snout narrow, ovoid when viewed dorsally or
ventrally; canthus rostralis well-defined; a rounded, white, rictal gland present at the level of jaw-angle; skin
predominantly smooth, except around limb insertions; some minute warts on mid-dorsum, especially in larger
specimens (adult females), ventrally smooth and glossy; dorso-lateral skin fold well-developed, extending from
supraorbital region to the groin; adpressed hindlimb reaches between tympanum and loreal; fingers not webbed;
digital formula: fingers 1=2<4<3; toes1<2<3<5<4. Toes 3/4 webbed; webbing on inner sides of 2nd and 3rd toes, at
the level of 1st subarticular tubercle.
Colouration in life. Dorsally golden to fawn brown, with two distinct yellow dorsolateral skin folds
extending from eye to posterior trunk; laterally of a darker shade, more so in males; adult females of a uniform
colour dorsolaterally; limbs barred with darker shades, especially in males; underside yellowish white anteriorly
with small dark speckles; iris often dark reddish, with a black circular pupil.
Sexual dimorphism. Adult females (67-80 mm) larger than adult males (46-51 mm). Adult males with
a darker, more contrasting body colouration (yellowish dorsum, bilaterally flanked with dark brown) than adult
females (uniform fawn brown dorsally and laterally). Adult males with a patch of loose-skin near corner of each
jaw-angle, indicating paired vocal sacs. During breeding season, adult males develop a thick, velvety, strawcoloured nuptial pad on each innermost finger.
Call characteristics. The call is complex, fairly long (3.87-11.30 s, 7.44 ± 3.00 (mean ± sd), n = 6 calls),
composed of short single notes (5-12 notes per call) and two types of double notes: short double note (0-5 notes
per call) and two, long, multi-pulsed notes (fig. 3). Inter call interval: 545-3117 ms (1608.17 ± 1018.49 ms, n = 6
calls); dominant frequency: 1.89 kHz (n = 6) and a peak power between 96.7-101.2 dB (n = 6).
Table 1. Microhabitat usages (frequency and percentage) of Sreeni’s golden frog (Indosylvirana sreeni) in Southern Eastern Ghats, peninsular
India.
Microhabitat
categories
Shevaroys
(n = 146)
(n = 147)
Kolli
Sirumalai
Pond
11 (7.5%)
0 (0%)
2 (3.1%)
Stream
89 (60.9%)
47 (31.9%)
50 (79.3%)
Under log
0 (0%)
3 (2%)
0 (0%)
On ground
2 (1.3%)
8 (5.4%)
0 (0%)
Leaf litter
28 (19.1%)
73 (49.6%)
5 (7.9%)
On rock
3 (2%)
3 (2%)
2 (3.1%)
Plant
4 (2.7%)
9 (6.1%)
4 (6.3%)
Tree
9 (6.1%)
4 (2.7%)
0 (0%)
(n = 63)
61
Figure 2. Sreeni’s golden frog Indosylvirana sreeni from Southern Eastern Ghats: A. adult male; B. adult female; C. ventral view profile of
an unsexed subadult; D. foot-profile of an adult female; E. dorsal head profiler of an adult female; F. ventral head profile of an adult female.
Single notes are short and single pulsed (6-69 ms, 26.60 ± 14.64 ms, n = 43 notes). Short double notes
always produced together: 30-97 ms (66.66 ± 21.70 ms, n = 10 notes); note 1: 8-30 ms (21.9 ± 7.02 ms, n = 10
notes) and note 2: 15-39 ms (27.5 ± 7.95 ms, n = 10 notes). Long multi-pulsed two notes ranged from 457-760 ms
(639.17 ± 133.89, n = 6 notes); note 1: 250-457 ms (285.67 ± 83.94 ms, n = 6 notes), 18-27 pulses; pulse rate =
0.06-0.07s-1 (0.07 ± 0.004 s-1, n = 6 notes) and note 2: 183-437 ms (349.7 ± 114.06 ms, n = 5 notes), 9-27 pulses,
pulse rate = 0.04-0.06 s-1 (21.4 ± 7.68 s-1, n = 5 notes).
Dominant frequency of short single pulsed note: 1.03-2.24 kHz (1.75 ± 0.34 kHz, n = 43 notes); peak
power : 76.1-97.4dB (89.68 ± 6.10, n = 43 notes); dominant frequency of note 1 of long multi-pulsed double notes:
1.87 ± 0.07 kHz (1.72-1.89 kHz, n = 6 notes); peak power: 99.03 ± 2.61 dB (94.7-101.2 dB, n = 6); dominant
frequency and peak power of note 2: 1.89 kHz (n = 5) and 92.64 ± 1.48 dB (90.9-94.6 dB, n = 5).
Call syllable: cruck…cruck...cru-cru-cruck.
Distribution. Among the surveyed regions, I. sreeni was encountered in Sirumalai, Kolli hills and
Shevaroys, but was not detected in Jawadi hills, despite 500 man hours of fieldwork there (see fig. 1).
Ecological notes. Sympatric stream-dwelling amphibian species include Fejervarya sp. and Fejervarya
62
ALYTES 2015 | 32
Figure 3. Waveforms depicting frequency distribution (top) and amplitude (bottom) of a selected call of Indosylvirana sreeni from Kolli hills.
cf. nilagirica in Shevaroys, Kolli and Sirumalai, and additionally, Indirana sp. in Sirumalai hills. Among these,
Fejervarya sp. is a widespread, human-commensally species that is also present in the surrounding plains.
Among the hill ranges surveyed during monsoon and post-monsoon periods (tab. 1), I. sreeni was
associated with many microhabitats. The frequency of microhabitat utilisation was as follows: stream (31.7-79.3%)
> leaf litter (7.9-49.6%) > plants (2.7-6.1%) > on rocks (1.3-3.1%) > pond (0-7.5%) > tree (0-6.2%) > on ground
(0-5.4%) > under log (0-0.7%). This species was observed to occupy mostly aquatic microhabitats, including both
lentic and lotic types. In Sirumalai 50 out of 63 (79.3%) sightings were in streams; in Kolli hills 47 out of 147
(33.5%) sightings were in streams; in Shevaroys 89 out of 146 (61.3%) sightings were in streams. Secondly, many
sightings were on leaf litter bordering such riparian vegetation. In Sirumalai 5 out of 63 (7.9%) sightings were on
leaf litter; in Kolli 73 out of 147 (49.6%) sightings were on leaf litter; and in Shevaroys 28 out of 146 (19.1%)
sightings were on leaf litter. Pond supported much lower number of these frogs compared to streams. Sightings on
other microhabitats such as on rocks, on plants, on bare ground, on trees and under fallen logs were very meager.
Potential local threats. In Kolli hills two road kills of I. sreeni were observed (fig. 4.A). They were
both adults and were observed in July 2012, in Ariyur Ashram (11.314°N, 78.355°E, 1315 m a.s.l.) covered with
evergreen forests.
Secondly one adult individual (1/356, 0.28%; fig 4.B) affected by anophthalmia was observed in
Kuzhivalavu (11.331°N, 78.360°E, 1247 m as..l.), a disturbed evergreen forest site in Kolli hills. The left eye of
that individual was totally missing. Instead, the ocular region was just an empty depression merely covered by
skin without any traces of supraocular bulge and eyelid. No traumatic morphological aberrations were seen in that
individual, indicating that its eye loss was not predator-mediated, but from a malformation.
Figure 4. Potential local threats to Indosylvirana sreeni in Kolli hills: A. road killed adult individual; B. individual exhibiting anophthalmia.
63
DISCUSSION
Overall, much of the information presented here on the Eastern Ghats population of I. sreeni is consistent
with what has previously been published on this species from the Western Ghats. Although our examination of live
individuals precludes detailed morphological analyses, our individuals from Sehvaroys, Kolli and Sirumalai fully
exhibit the diagnostic features of this species (Biju et al., 2014). It is noteworthy to mention here that although Biju
et al. (2014), Kumar & Daniels (1998) and Vanak et al. (2001) have reported this taxon from Shevaroys, Kolli and
Sirumalai respectively, comprehensive information on the Southern Eastern Ghats populations is wanting. Thus
the present data on external morphology of individuals of differing maturity and sexes will serve to clarify the
confusions on the past records.
Our present examination of Indosylvirana frogs from across the Southern Eastern Ghats reveals that,
unlike in the Western Ghats where multiple congeners co-occur within an elevational band of a hill range (Biju et
al., 2014), no other congener occurs in the surveyed hills of the Southern Eastern Ghats. Indosylvirana sreeni has
a rather curious distribution (after Biju et al., 2014), with the type locality in Siruvani hills to the north of Palghat
gap, but its range continues further south to Agasthyamalai. Strangely, it is absent from Nilgiris and northerly
hills of the Central Western Ghats. In the Western Ghats adjacent to our study area, I. sreeni is sympatric with I.
flavescens in Nilgiri landscape and with the morphologically dissimilar I. doni in the Anaimalai landscape (Biju et
al., 2014). The distribution of I. montana closely borders the northern range of I. sreeni, but these two are clearly
allopatric even within the Western Ghats (Biju et al., 2014). Therefore, I. flavescens is the only morphologically
similar congener sympatric with I. sreeni in the Western Ghats.
The present study reports I. sreeni from Shevaroys, from where Biju et al. (2014) report a geneticallytested conspecific. Additionally, we confirm this species from Kolli and Sirumalai hills (Daniels & Kumar, 1998;
Vanak et al., 2001) which are much closer to the Western Ghats than Shevaroys, thus backing our identification on
biogeographic grounds, in addition to morphological similarities. Moreover, owing to geographic proximity, we
hypothesize that the Sirumalai population is arguably the metapopulation of the adjacent and much closer Western
Ghats (Palnis 30 km west) than the Kolli hills (140 km northeast).
Although the presence of multiple sympatric congeners of Indosylvirana spp. in the Western Ghats
hinders species-level ecological comparisons with pre-Biju et al. (2014) literature, it is safe to consider such
ecological information at the genus level. Thus Indosylvirana species in the Western Ghats have been consensually
considered to be highly aquatic, particularly as adults (Inger et al., 1987; Vasudevan et al., 2001; Vijayakumar et
al., 2006), while the metamorphs and subadults wandering out of streams during monsoon are often sighted on
ground vegetation (Inger et al., 1987). Our findings on the habitat use of I. sreeni observed during fieldwork that
includes the rainy season here in the Southern Eastern Ghats, concur with that reported in the Western Ghats.
An earlier study by Kuramoto & Joshy (2001; read with Biju et al., 2014 and Oliver et al., 2015) reported
calls of another congener probably Indosylvirana indica (represented as Rana temporalis sic; refined herein based
on size-class grouping and locality details) based on recordings from Madikeri and Kudremukh during July 1999.
The authors presented their individual note lengths to be 0.038-0.066 s, which makes them much shorter when
compared to I. sreeni. The dominant frequency was also higher at 22°C (2.63-2.90 kHz) than the call here described
at slightly lower temperature.
Earlier studies on herpetofaunal roadkills conducted in the Western Ghats (Vijayakumar et al., 2001;
Bhupathy et al., 2009; Seshadri et al., 2009) have reported Indosylvirana mortalities. Nevertheless, the present
report of anophthalmia is the first for this species. However, further studies on the frequency and magnitude of
such observations on this species are required to assess if these factors could become a possible threat to this
species at a local scale within the study area.
Sreeni’s golden frog (I. sreeni) is the only nominate range-restricted amphibian species in the Southern
Eastern Ghats (SRG & MA unpubl. data). Although other range-restricted genera such as Raorchestes,
Pseudophilautus and Indirana occur in this region, these taxa await proper species-level taxonomic resolution.
Therefore Indosylvirana sreeni could become a potential candidate amphibian for the conservation of Southern
Eastern Ghats.
ACKNOWLEDGEMENTS
We thank the Tamil Nadu Forest Department for granting permission to conduct field work in forest areas
and for their local logistic support provided. We are grateful to the Executive Chairman and other officers of the
Chennai Snake Park Trust for the support and encouragement provided. We record our thanks to the staff and lab
members of the Dept. of Zoology, University of Madras for all their help and encouragements provided and the
64
ALYTES 2015 | 32
anonymous reviewers for their comments on the manuscript; particularly to Dinal Samarasinghe for his help with
the call analysis.
LITERATURE CITED
Bhupathy, S., Srinivas, G., Kumar, N.S., Karthik, T., Madhivanan, A. (2009). Herpetofaunal mortality due to
vehicular traffic in the Western Ghats, India: a case study. Herpetotropicos, 5: 119-126.
Biju, S.D., Garg, S., Mahony, S., Wijayathilaka, N., Senevirathne, G., Meegaskumbura, M. (2014). DNA barcoding,
phylogeny and systematics of Golden-backed frogs (Hylarana, Ranidae) of the Western Ghats-Sri Lanka
biodiversity hotspot, with the description of seven new species. Contributions to Zoology, 83: 269-335.
Crump, M.L., Scott, N.J. Jr. (1994). Visual encounter surveys. In: Heyer, W.R., Donnelly, M.A., McDiarmid, R.W.,
Hayek, L.C., Foster, M.S. (eds). Measuring and monitoring biological diversity: standard methods for
amphibians. Smithsonian Institution Press, Washington DC: 84-92.
Daniels, R.J.R., Kumar, M.V.R. (1998). Amphibians and reptiles of Kolli Hills. Cobra, 31: 3-5.
Dutta, S.K., Manamendra-Arachchi, K. (1996). The amphibians fauna of Sri Lanka. Colombo, Wildlife Heritage
Trust of Sri Lanka.
Dutta, S.K. (1997). Amphibians of India and Sri Lanka (checklist and bibliography). Odyssey Publishing House,
Bhubaneswar, India.
Inger, R.F., Shaffer, H.B., Koshy, M. Bakde, R. (1987). Ecological structure of a herpetological assemblage in
South India. Amphibia-Reptilia, 8: 189-202.
Jayakumar, S., Ramachandran, A., Bhaskaran, G., Heo, J. (2008). Forest dynamics in the Eastern Ghats of Tamil
Nadu, India. Environmental Management, 2008: 1-20.
Kumar, M.V.R., Daniels, R.J.R. (1999). Checklist of reptiles and amphibians of Kolli hills. Cobra, 38: 21-22.
Kuramoto, M., Joshy, S.H. (2001). Advertisement call structures of frogs from southwestern India, with some
ecological and taxonomic notes. Current Herpetology, 20: 85-95.
Meteyer, C.U. (2000). Field guide to malformations of frogs and toads: with radiographic interpretations.
Biological Science Report. U.S. Fish and Wildlife Service, Reston, VA.
Oliver, L., Prendini E., Kraus F., Raxworthy, C.J. (2015). Systematics and biogeography of the Hylarana
frog (Anura: Ranidae) radiation across tropical Australasia, Southeast Asia, and Africa. Molecular
Phylogenetics and Evolution, 90: 176-192.
Rao, K.T., Ghate, H.V., Sudhakar, M., Javed, S.M.M., Krishna, I.S.R. (2005). Herpetofauna of Nallamala Hills
with eleven new records from the region including ten new records for Andhra Pradesh. Zoos’ Print
Journal, 20: 1737-1740.
Seshadri, K. S., Yadev, A., Gururaja, K.V. (2009). Road kills of amphibians in different land use areas from
Sharavathi river basin, Central Western Ghats India. Journal of Threatened Taxa,1: 549-552.
Srinivasulu C., Das, I. (2008). The herpetofauna of Nallamalai Hills, Eastern Ghats, India: an annotated checklist,
with remarks on nomenclature, taxonomy, habitat use, adaptive types and biogeography. Asiatic
Herpetological Research, 11: 110-131.
Vanak, A.T., Vijayakumar, S.P., Venugopal, P.D., Kapoor, V. (2001). Inventory of the flora and fauna of Khandige
estate – Sirumalai hills, Tamil Nadu, Southern India. Report submitted to the Khandige Investments Pvt.
Ltd.
Vasudevan, K., Kumar, A., Chellam, R. (2001). Structure and composition of rainforest floor amphibian
communities in Kalakad-Mundanthurai Tiger Reserve. Current Science, 80: 406-412.
Vijayakumar, S.P., Ragavendran, A., Choudhury, B.C. (2006). Herpetofaunal assemblage in a tropical dry forest
mosaic of Western Ghats: preliminary analysis of species composition and abundance during dry season.
Hamdrayad, 30: 41-54.
Vijayakumar, S.P., Vasudevan, K., Ishwar, N.M. (2001). Herpetofaunal mortality on roads in the Anamalai Hills,
Southern Western Ghats. Hamadryad, 26: 253-260.
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66
RESEARCH ARTICLE
2015 | VOLUME 32 | PAGES 67-81
Comparative osteology of anuran genera in the
Western Ghats, Peninsular India
S. R. Chandramouli1*, Sushil Kumar Dutta1
1.
Centre for Ecological Sciences, Indian Institute of Science, Bangalore
Diversity of skeletal characters in 10 genera of anurans representing nine families from the Western
Ghats of peninsular India is investigated. The osteology of each of these genera is described and
characters are compared among the genera. The analysis reveals that some pairs of genera resemble
one another osteologically and that this morphological similarity seems to be congruent with their
published phylogenetic relationships. In addition, a pattern of microhabitat-related clustering was
observed; other observed patterns are discussed and avenues for further research are advocated.
INTRODUCTION
Studies on anurans of peninsular India, particularly in the Western Ghats, where they are highly diverse,
have largely been focussed on systematics and taxonomy, and dealt with new species discoveries and descriptions
(e.g., Biju, 2001; Biju & Bossuyt, 2005; Biju & Bossuyt, 2009; Biju et al., 2011; Zacchariah et al., 2011). A few
studies have addressed anuran ecology, population, distribution, and habitat associations in the Western Ghats
(Daniels, 1992; Vasudevan et al., 2001; Gururaja et al., 2003; Krishnamurthy et al., 2008; Seshadri, 2014), but
little is known on anatomy and physiology of these frogs. Nonetheless, anatomical characters, especially osteology,
have been used in phylogenetic studies (Biju & Bossuyt, 2003; Dutta et al., 2004) and taxonomic descriptions
of some endemic taxa (Biju et al., 2014a; Modak et al., 2014). Herein, we describe and compare osteological
features of 10 different genera of anurans in the Western Ghats using the following taxa as representatives:
Duttaphrynus Frost, Grant, Faivovich, Bain, Haas, Haddad, de Sá, Channing, Wilkinson, Donnellan, Raxworthy,
Campbell, Blotto, Moler, Drewes, Nussbaum, Lynch, Green & Wheeler, 2006; Indosylvirana Oliver, Prendini,
Kraus & Raxworthy, 2015; Nyctibatrachus Boulenger, 1882; Fejervarya Bolkay, 1915; Nasikabatrachus Biju &
Bossuyt, 2003; Indirana Laurent, 1986; Micrixalus Boulenger, 1888; Raorchestes Biju, Shouche, Dubois, Dutta &
Bossuyt, 2010; Polypedates Tschudi, 1838. In addition, patterns of osteological resemblances between some taxa
are discussed in the context of published phylogenies.
At least one representative species of each genus was selected and used in this study, covering 9 families:
Duttaphrynus aff. parietalis (Bufonidae Gray, 1825); Indosylvirana aff. flavescens (Ranidae Batsch, 1796);
Nyctibatrachus aff. sylvaticus (Nyctibatrachidae Blommers-Schlösser, 1993); Fejervarya aff. sahyadris and F.
aff. mudduraja (Dicroglossidae Anderson, 1871); Nasikabatrachus sahyadrensis (Nasikabatrachidae Biju &
Bossuyt, 2003); Indirana aff. semipalmata (Ranixalidae Dubois, 1987); Micrixalus aff. saxicola (Micrixalidae
Dubois, Ohler & Biju, 2001); Uperodon mormoratus and U. taprobanicus (Microhylidae Günther, 1858 (1843));
Raorchestes sp. and Polypedates pseudocruciger (Rhacophoridae Hoffman, 1932 (1858)). However, data on the
genus Nasikabatrachus are based on Biju & Bossuyt (2003) and Dutta et al. (2004). Owing to the systematic and
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S. R. CHANDRAMOULI & SUSHIL KUMAR DUTTA
taxonomic complexity in these groups, and the presence of a large number of cryptic species (e.g., Nair et al.,
2012; Biju et al., 2014a), species-level identifications here are tentative. Specimens were selected in such a way
that at least one member of each of the families gets represented.
Specimens were collected, fixed in formalin, and preserved in 70% alcohol periods of time ranging from 3
months to about 5 yrs. The frogs were dehydrated by soaking them in absolute alcohol for 1 day, and then cleared
and stained following a protocol modified from Hanken & Wassersug (1981), as follows: (1) Removal of skin,
followed by maintenance in 100% ethanol for 1 day; (2) removal of viscera and eyes; (3) transfer of specimens to
a 7:3 solution alcian blue dye in a solvent of ethanol: glacial acetic acid to stain cartilage; (4) transfer of specimens
to 100% ethanol for 1 day; (5) transfer of specimens to a solution of 0.5 % potassium hydroxide saturated with
alizarin red S dye to stain bone; (6) clearing specimens in a solution of 0.5 % potassium hydroxide and glycerine
in the concentrations of 2:1, 1:1, and 1:2 for 7-10 days to ten days each, depending on the size and condition of the
specimen; and (7) storage in 100% glycerine.
The cleared-and-stained specimens are housed in the herpetological collections at the Centre for Ecological
Sciences, Indian Institute of Science, Bengaluru (India). Specimens were examined under a stereomicroscope and
states (tab. 1) of the following characters recorded: number of presacral vertebrae; configuration of the pectoral
girdle; phalangeal formulae of fingers and toes; structure of terminal phalange; presence/ absence and shape of
omosternum; nature of sacrococcygeal articulation; shape of sacral diapophysis; presence/ absence of sternum,
maxillary and vomerine teeth.
A matrix of character states was constructed and subjected to a hierarchical cluster analysis, with 1000
bootstrap replicates using Euclidean distance as the distance measure to construct a phenogram using the software
package PAST v. 3.0 to illustrate the similarity of osteological characters among the taxa examined.
Table 1. Character-states recorded from the cleared-and-stained
specimens
Characters
Pectoral girdle
Terminal phalange shape
Omosternum
Sacro-coccygeal articulation
Sacral diapophysis
Sternum
Maxillary teeth
Vomerine teeth
68
States
Codes
Firmisternal
1
Arciferal
2
Pointed
1
Expanded –T
2
Expanded –Y
3
Absent
0
Unforked
1
Slightly forked
2
Deeply forked
3
Single condyle
1
Double condyle
2
Poorly developed
0
Not expanded
1
Widely expanded
2
Absent
0
Present
1
Absent
0
Present
1
Absent
0
Present
1
Their axial and appendicular skeletal
structures are described separately, elaborating key
morphological features of each of these components.
Osteological terminologies used here followed
Noble (1931): N, Nasal; FP, frontoparietal; OC,
orbital cavity; SSC, supra scapula; MX, maxilla; SQ,
squamosal; MB, mandible; PMX, premaxilla; AT,
atlas; I-VII presacral vertebrae; S/SV, sacral vertebra;
SD, sacral diapophysis; UR, urostyle; MT, metatarsal;
PH, phalanges; CAR, carpal; OM, omosternum;
CL, clavicle; EPI, epicoracoid; COR, coracoid; ST,
sternum.
RESULTS
Osteological characters of each of the taxa
studied are described and illustrated below in detail.
The catalogue number of specimens is given in
brackets.
Bufonidae
Duttaphrynus aff. parietalis
One specimen examined here (CESF2385).
Axial skeleton. Skull large, broader than
long, with the following calcified cephalic ridges:
preorbital, postorbital, supra-orbital, supratympanic
and parietal ridges. Frontoparietals slightly longer
than broad with a conspicuous median suture; nasals
as broad as long, separated from the frontoparietals
by a thin suture. Orbit about half the length of the
skull. Skull predominantly composed of bony
elements; maxillae, mandibles and vomers edentate
ALYTES 2015 | 32
(fig. 1.A & 2.A). Vertebral column with eight procoelous presacral vertebrae; presacrals I-III with widely expanded
transverse processes oriented posterolaterally; posterior presacrals with acuminate transverse processes oriented
horizontally; sacral diapophysis well developed and expanded. Sacrococcygeal articulation with a double condyle
(fig. 3.A).
Appendicular skeleton. Pectoral girdle arciferal, with bony coracoids and clavicles; epicoracoids
cartilaginous and overlapping over one another; omosternum absent; suprascapula trapezoidal, bony with
cartilaginous outer edges; sternum cartilaginous (fig. 5.A). Phalangeal formula of the fingers: 2-2-3-3, their
relative lengths: IV>I>III>II. Pelvic girdle formed by long, curved ilia extending beyond anterior edge of sacral
diapophysis, reaching almost till last pre-sacral vertebra. Phalangeal formula of toes: 2-2-3-4-3, relative lengths:
IV>III>V>II>I. Terminal phalanges of fingers and toes pointed (without discs) (fig. 6.A & 7.A).
Dicroglossidae
Two members, each from a small-bodied and a large-bodied group in this family were studied, viz.,
Fejervarya aff. mudduraja a medium to large-bodied member and Fejervarya aff. sahyadris, a member of the
small-bodied group.
Fejervarya aff. mudduraja
Axial skeleton. Skull pointed at anterior end and streamlined in shape. Frontoparietal bones much
elongated and linear in shape, with a blunt anterior edge and well-defined median suture. Orbital cavity of skull a
little larger than half length of frontoparietal bone; nasal bones triangular in shape, rounded anteriorly and pointed
towards lateral ends; premaxillae cartilaginous. Palatine bones extending horizontally between frontoparietals
and nasals, much longer than width of nasals (fig. 1.B). Vomerine and maxillary teeth present. Vertebral column
composed of eight procoelous pre-sacral vertebrae. Elongated hypapophyses present on all vertebrae starting from
axis (second pre-sacral vertebra) till last pre-sacral vertebra; those at anterior and posterior ends (II, III and VII)
oriented laterally; IV, V and VI oriented posteriorly; on last, i.e., VIII pre-sacral vertebra, hypapophyses oriented
anteriorly. Sacral diapophysis slender and not dilated horizontally; hard calcareous substances observed towards
lateral sides of pre-sacral vertebrae. Sacro-coccygeal articulation bicondylar (fig. 3.B).
Appendicular skeleton. Firmisternal pectoral girdle architecture with non-overlapping coracoid bones
much broader than slender clavicular bones located above them. Omosternum long, deeply forked at base;
suprascapulae bony with cartilaginous interior edges. Mesosternum long and bony; episternum semi-circular in
shape and cartilaginous. Epicoracoid cartilages not visible (fig. 5.B). Ilio-sacral joint of pelvic girdle not extending
beyond anterior edge of sacral diapophysis. Phalangeal formula of fingers: 2-2-3-3 (fig. 6.B); toes: 2-2-3-4-3 (fig.
7.B); terminal phalanges pointed in both fingers and toes (lacking expanded discs). Relative length of fingers:
III>IV>I>II; toes: IV>III>V>II>I.
Fejervarya aff. sahyadris
Axial skeleton. Skull similar in shape to larger Fejervarya, but less pointed at anterior end; frontoparietal
bones shorter and broader than in larger Fejervarya with a pointed anterior edge. Nasal bones trapezoidal in shape;
much broader and shorter than in larger Fejervarya. Orbital cavity relatively smaller than in larger Fejervarya;
palatine and tympanic annulus surrounded by cartilaginous elements. Mandibles edentate; maxillary teeth well
developed (fig. 1.C & 2.B). Vertebral column composed of eight procoelous presacral vertebrae with hypapophyses
oriented posteriorly till the IV (cervical) vertebra; followed by horizontally oriented hypapophyses on V to VIII
(lumbar) vertebrae, followed by sacral diapophysis which lacks lateral expansions, and coccyx, that articulates to
sacral vertebra by a double condyle (fig. 3.C).
Appendicular skeleton. Hyoid apparatus cartilaginous; firmisternal pectoral girdle with robust, nonoverlapping coracoid bones and slender clavicles. Omosternum deeply forked at base; episternum and xyphisternum
cartilaginous, omosternum and mesosternum bony in nature. Omosternum roughly as long as mesosternum. Supra
scapula bony, bordering first two presacral vertebrae with a thick, cartilaginous outer edge. Ilia slender, ilio-sacral
joint extending slightly beyond anterior edge of sacral diapophysis (fig. 5.C). Phalangeal formula of fingers: 2-2-33; terminal phalanges with a blunt, rounded tip (fig. 6.C); toes: 2-2-3-4-3 (lacking discs). Relative length of fingers:
III>IV>I>II; toes: IV>III>V>II>I (fig. 7.C).
69
Figure 1. Skulls of different anuran species used as representatives of eight genera
studied (dorsal view): A. Duttaphrynus aff. parietalis (CESF2385); B. Fejervarya aff.
mudduraja (CESF2386); C. Fejervarya aff. sahyadris (CESF2387); D. Indosylvirana
aff. flavescens (CESF2388); E. Nyctibatrachus aff. sylvaticus (CESF2389); F. Micrixalus
saxicola (CESF2390); G. Indirana aff. semipalmata (CESF625); H. Uperodon mormoratus
(CESF2312); I. Uperodon taprobanicus (CESF2392); J. Raorchestes sp. (CESF2393). Scale
bar = 2 mm.
70
ALYTES 2015 | 32
Figure 2. Skulls of different anuran species used as representatives of eight genera studied (in lateral view): A. Duttaphrynus aff. parietalis
(CESF2385); B. Fejervarya aff. sahyadris (CESF2387); C. Indosylvirana aff. flavescens (CESF2388); D. Nyctibatrachus aff. sylvaticus
(CESF2389); E. Micrixalus saxicola (CESF2390); F. Indirana aff. semipalmata (CESF625); G. Uperodon mormoratus (CESF2312); H.
Raorchestes sp. (CESF2393). Scale bar = 2 mm.
Ranidae
Indosylvirana aff. flavescens
The specimen described here (CESF2388) belongs to the Indosylvirana flavescens group, but has not been
identified to the species level owing to the complexity and cryptic diversity within the genus (Biju et al., 2014b).
Oliver et al. (2015) provided a finer taxonomic resolution and identified several cryptic genera which were earlier
referred to as Hylarana.
Axial skeleton. Skull about as long as broad, with a rounded and blunt anterior end. Frontoparietals
71
Figure 3. Vertebral column of different anuran species used as representatives of seven genera studied: A. Duttaphrynus aff. parietalis
(CESF2385); B. Fejervarya aff. mudduraja (CESF2386); C. Fejervarya aff. sahyadris (CESF2387); D. Indosylvirana aff. flavescens
(CESF2388); E. Nyctibatrachus aff. sylvaticus (CESF2389); F. Micrixalus saxicola (CESF2390); G. Indirana aff. semipalmata (CESF625);
H. Uperodon mormoratus (CESF2312); I. Uperodon taprobanicus (CESF2392). Scale bar = 2 mm.
long and flat; separated from nasal bones. Nasals triangular in shape, with broadest side towards posterior end
and slightly curved towards lateral sides. Orbital cavity about half as long as frontoparietals (fig. 1.D). Palatine
bones separating frontoparietals and nasals to a considerable extent. Tympanic annulus about 3/4th of orbital
cavity and situated towards quadrato-mandibular apex. Maxillary teeth present; small and projecting vertically
downwards from maxilla; vomerine teeth in two oblique rows (fig. 2.C). Vertebral column composed of eight
procoelous presacral vertebrae, which are bordered by a thick lining of calcareous substances; hypapohyses of
anterior vertebrae oriented posteriorly, while those of posterior end oriented laterally (fig. 3.D). Sacrococcygeal
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ALYTES 2015 | 32
articulation bicondylar.
Appendicular
skeleton.
Firmisternal pectoral girdle architecture
with
non-overlapping
coracoids.
Omosternum forked at base; mesosternum
much longer than omosternum, extending
downwards from junction of coracoid
bones; xyphisternum poorly developed
and bony in nature (fig. 5.D). Phalangeal
formula of fingers 2-2-3-3; their relative
lengths: III>IV>I>II (fig. 6.D); terminal
phalanges of fingers and toes rounded
and lacking ossification of discs. Sacral
diapophysis not dilated. Phalangeal
formula of toes: 2-2-3-4-3; relative lengths:
IV>III=V>II>I (fig. 7.D).
Figure 4. Vertebral column of Rhacophorids: (A) Raorchestes sp. (CESF2393)
and (B) Polypedates pseudocruciger (CESF 2394). Scale bar = 2 mm.
Nyctibatrachidae
Nyctibatrachus aff. sylvaticus
Axial skeleton. Skull broader than long and rounded in shape with a blunt snout. Frontoparietal elements
much broader at base than at anterior end. Nasals trapezoidal, with broadest side oriented towards nostrils and
pointed lateral edges. Posterior and anterior regions of nasals and palatine bones surrounded by cartilaginous
elements (fig. 1.E). Vomerine and maxillary teeth present. Tympanic annulus much smaller, less than 1/4th of
orbital cavity (fig. 2.D). Exoccipitals articulated to cartilaginous elements posteriorly; which surround atlas
(first vertebra). Vertebral column composed of eight procoelous vertebrae; all of which bearing well developed,
horizontally expanded hypapophyses. Hypapophyses of first three vertebrae dilated and expanded laterally; IV to
VI oriented posteriorly and last presacral vertebra bearing hypapophyses oriented anteriorly. Sacral diapophysis
not dilated, articulating with coccyx by a double condyle (fig. 3.E).
Appendicular skeleton. Firmisternal pectoral girdle with well-developed, non-overlapping coracoid
bones and slender clavicles. Supra scapula relatively slender, bordered by cartilaginous elements surrounding
atlas. Mesosternum short and bony, xyphisternum cartilaginous. Episternum bony and forked, followed by a long
omosternum with a deeply forked base (fig. 5.E). Phalangeal formula of fingers: 2-2-3-3; their relative lengths:
III>IV>I>II; terminal phalanges trapezoidal in shape with a broader base and a flat, narrow anterior end (fig. 6.E).
Toes: 2-2-3-4-3; their relative lengths: IV>III>V>II>I; terminal phalanges lacking dilations (fig. 7.E).
Micrixalidae
Micrixalus saxicola
Biju et al. (2014a) provided a description of the type species of the genus, M. fuscus. The specimen
examined here (CESF2390) belongs to Micrixalus saxicola, which belongs to a different species-group. A specimen
of Micrixalus herrei CESF 023 (a member of the M. fuscus group) was also examined but is not described here in
detail (fig. A1 in Appendix 1).
Axial skeleton. Skull elongate in shape with a pointed snout tip. Frontoparietals extending from anterior
end of orbital cavity till a little beyond its posterior end, with an ill-defined median suture. Nasals triangular, with
cartilaginous elements covering top and snout tip, above premaxillae (fig. 1.F). Maxillary teeth well-developed,
vomerine teeth absent; palatine bones located about midway through length of maxillae. Mandibles edentate;
tympanic annulus much smaller than orbital cavity (fig. 2.E). Presacral vertebrae procoelous; eight in number, II to
VIII bearing horizontally elongate hypapohyses. Urostyle extending posteriorly from sacral vertebra to length of
about four presacral vertebrae, articulating with sacrum by a double condyle (fig. 3.F).
Appendicular skeleton. Firmisternal pectoral girdle formed primarily of bony elements. Episternum
73
Figure 5. Structure of the pectoral girdle in the different anuran species used as representatives eight genera studied: A. Duttaphrynus aff.
parietalis (CESF2385), B. Fejervarya aff. mudduraja (CESF2386); C. Fejervarya aff. sahyadris (CESF2387); D. Indosylvirana aff. flavescens
H. Uperodon mormoratus (CESF2312); I. Raorchestes sp. (CESF2393). Scale bar = 2 mm.
cartilaginous, triangular in shape, omosternum bony, unforked at base; coracoids broader medially, not overlapping
over one another and narrow distally; clavicles slender. Mesosternum short and bony, as long as omosternum;
xyphisternum cartilaginous, with bifurcated terminal ends (fig. 5.F). Suprascapula broad and dilated, with
cartilaginous outer margins, bordering first three presacral vertebrae. Sacral diapophysis slightly dilated laterally;
ilia extending a little beyond anterior border of sacral diapophysis. Phalangeal formula of fingers: 2-2-3-3 (fig.
6.F); toes: 2-2-3-4-3 (fig. 7.F); terminal phalanges of both fingers and toes modified to ‘Y’ shaped structure. Hyoid
apparatus cartilaginous.
Ranixalidae
Indirana aff. semipalmata
Axial skeleton. Skull about as long as broad; frontoparietal bones slender and elongate, with a prominent
median suture; orbital cavity large, about 3/4th of the length of frontoparietals; nasals broader than long, with
a broad medial edge and pointed laterally; region between frontoparietals and nasals and premaxillary region
with cartilaginous elements (fig. 1.G). Tymapnic annulus cartilaginous; maxillary and vomerine teeth present
(fig. 2.F). Vertebral column composed of eight procoelous presacral vertebrae; first three and VIII vertebrae with
horizontally elongate and dilated hypapophyses; IV to VII vertebrae bearing posteriorly oriented hypapophyses.
Sacral diapophysis not dilated laterally and with a posteriorly oriented curvature. Coccyx long, slightly shorter
than rest of vertebral column (fig. 3.G).
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ALYTES 2015 | 32
Figure 6. Structures of the palmar elements in the different anuran species used as representatives of eight genera studied: A. Duttaphrynus aff.
parietalis (CESF2385), B. Fejervarya aff. mudduraja (CESF2386); C. Fejervarya aff. sahyadris (CESF2387); D. Indosylvirana aff. flavescens
Appendicular skeleton. Firmisternal pectoral girdle with non-overlapping coracoid bones being much
broader towards medial end; clavicles slender and long. Episternum absent; omosternum short with a mildly
forked base; sternum about as long as omosternum; xyphisternum absent (fig. 5.G). Suprascapula blade shaped,
with cartilaginous inner margins, bordering first two presacral vertebrae. Sacrococcygeal articulation bicondylar.
Phalangeal formula of fingers: 2-2-3-3 (fig. 6.G); toes: 2-2-3-4-3 (fig. 7.G); terminal phalange modified to ‘Y’shaped structure.
Microhylidae
Uperodon mormoratus
Axial skeleton. Skull much broader than long; frontoparietal elements relatively shorter and broader than
75
Figure 7. Structures of the foot in the different anuran species used as representatives of eight genera studied: A. Duttaphrynus aff. parietalis (CESF2385), B. Fejervarya aff. mudduraja (CESF2386); C. Fejervarya aff. sahyadris (CESF2387); D. Indosylvirana aff. flavescens
in other taxa, with a well-defined median suture in between; nasal bones separated from frontoparietals; orbital
cavity longer than frontoparietals; premaxillae small, located below nasals; quadratojugal bones articulating
with ventral ramus of squamosal near base of tympanic annulus; maxillae and vomers edentate (fig. 1.H &
2.G). Vertebral column composed of six presacral vertebrae; first two vertebrae bearing posteriorly oriented and
elongated hypapophyses; following ones poorly developed and rudimentary. Sacral diapophysis laterally dilated;
coccyx slightly expanded anteriorly, tapering posteriorly; sacrum articulating with coccyx by a single condyle (fig.
3.H).
Appendicular skeleton. Firmisternal pectoral girdle. Coracoids well separated from each other; slightly
broader in the medial than distal ends; omosternum short and bony; sternum well developed, with a rudimentary
mesosternum and a well-developed, widely expanded xyphisternum. Suprascapula bordering atlas of vertebral
column (fig. 5.H). Sacral diapophysis short, extending slightly on lateral edges with wide expansions. Ilia as long
76
ALYTES 2015 | 32
Figure 8. Phenogram showing similarity in osteological configuration between the different genera of anurans studied.
as the presacral vertebral column. Fingers with widely expanded to T-shaped terminal phalanges; phalangeal
formula: 2-2-3-3 (fig. 6.H); toes with less expanded terminal ends; phalangeal formula: 2-2-3-4-3 (fig. 7.H).
Uperodon taprobanicus
Axial skeleton. Overall structural conformation very similar to that of Uperodon mormoratus. Skull
broader than long with a blunt, rounded snout tip. Maxillary and vomerine teeth absent. Frontoparietal elements
short and much broader than long; nasals large, about 3/4th of the length of frontoparietals; about as broad as long,
with laterally pointed edges. Median suture more conspicuous between nasals than between frontoparietals. Orbital
cavity nearly as long as frontoparietals (fig. 1.J). Six procoelous presacral vertebrae, with very short hypapophyses
extending laterally. Sacral vertebra articulating to coccyx by a single condyle (fig. 3.I).
Appendicular skeleton. Firmisternal pectoral girdle; coracoids robust; omosternum short and forked at
base; sternum widely expanded and similar in structure to that of Uperodon mormoratus. Suprascapula petal
shaped, with broad, interior edges bordering first two presacral vertebrae. Sacral diapophyses short but widely
expanded laterally. Ilio-sacral joint not exceeding the level of coccyx, which areticulates with sacral vertebra by a
single condyle. Terminal phalanges of fingers expanded to ‘T’-shape; formula: 2-2-3-3; terminal phalanges in toes
expanded to ‘Y’-shaped structures with phalangeal formula: 2-2-3-4-3 (fig. 7.I).
Rhacophoridae
Raorchestes sp.
The specimen examined here (CESF2393) has not been identified owing to the taxonomic complexity of
this group (e.g. Biju & Bossuyt, 2009; Zaccharia et al., 2011; Vijayakumar et al., 2014).
Axial skeleton. Skull as broad as long; snout tip obtusely pointed in dorsal view. Frontoparietal elements of
skull longer than broad; with a well-defined median suture; nasals not much broader than frontoparietals, preceded
by premaxillary rows separated from each other. Orbital cavity about 3/4th as long as frontoparietals; maxillary
and vomerine teeth present (fig. 1.J & 2.H). Vertebral column composed of eight procoelous presacral vertebrae;
I-III with horizontally oriented, elongate, and expanded hypapophyses; transverse process on IV presacral vertebra
oriented posteriorly; V-VIII oriented horizontally. Sacrococcygeal articulation bicondylar (fig. 4.A).
77
Appendicular skeleton. Firmisternal pectoral girdle, with non-overlapping coracoid bones. Omosternum
short and unforked at base. Sternum bony and much longer; projecting posteriorly till almost midbody; xyphisternum
short and cartilaginous (fig. 5.I). Sacral diapophysis not expanded laterally; ilia extending beyond the level of
sacral vertebra. Phalangeal formula of fingers: 3-3-4-4; relative lengths: III>IV>II>I (fig. 6.I); toes: 3-3-4-5-4;
relative lengths: IV>V=III>II>I (fig. 7.I); terminal phalanges of fingers and toes with slight lateral expansions
forming a ‘T’-shaped structure.
Polypedates pseudocruciger
Axial skeleton. Skull similar in structure to that of Raorchestes. Vertebral column composed of eight
procoelous presacral vertebrae; II and III with widely expanded, horizontally elongate hypapophyses; vertebra IV
with posteriorly oriented hypapophysis. Coccyx articulating with sacral vertebra by a double condyle (fig. 4.B).
Appendicular skeleton. Firmisternal pectoral girdle; omosternum mildly forked at base; sternum bony.
Sacral diapophysis slender and not expanded laterally; ilia extending beyond anterior edge of sacral diapophyses.
Phalangeal formula of fingers: 3-3-4-4; toes: 3-3-4-5-4. Terminal phalange of fingers with a slight, ‘T’-shaped
expansion; toe tips modified into ‘V’-shaped structures at terminal end.
Nasikabatrachidae
Nasikabatrachus sahyadrensis
As some osteological characters of this family have already been published with illustrations of specimens
by Biju & Bossuyt (2003) and Dutta et al. (2004), details of osteological characters for this taxon were used in
the analysis based on the above published information and their illustrations. This taxon was identified to possess
certain unique osteological features when compared to other taxa. Specimen WII uncatalogued.
Axial skeleton. Skull much broader than long; frontoparietals of skull long; nasals broader than long;
vertebral column composed of seven procoelous presacral vertebrae, a short transverse process (hypapophyses)
present on atlas (first vertebra); vertebrae II and III with large hypapophyses; IV to VII bearing short lateral
process; hard, calcareous substances present beside presacral vertebrae; urostyle about as long as preceding
section (presacral) of vertebral column; sacral vertebra with rudimentary dispophysis; sacrococcygeal articulation
bicondylar.
Appendicular skeleton. Firmisternal pectoral girdle; unique in configuration of the coracoids, which are
slender bones with wider distal than proximal ends; sternum absent. Ilia as long as coccyx, ilio-sacral joint not
exceeding beyond the anterior level of sacral diapophysis; terminal phalanges of fingers and toes pointed; a large
calcified inner metatarsal tubercle present at base of foot.
Similarity in osteological conformation
A hierarchical cluster analysis carried out based on the coded matrix of skeletal characters detailed above,
yielded a phenogram showing the following (fig. 8):
(i)
A well supported (bootstrap: 81) cluster comprising the genera Indosylvirana, Nyctibatrachus, Fejervarya, Indirana and Micrixalus.
(ii)
A close osteological similarity between the following pairs of genera: Indosylvirana – Nyctibatrachus
(Euclidean distance: 0); small and large bodied members of the genus Fejervarya (Euclidean distance: 0);
Indirana – Micrixalus (Euclidean distance: 1).
(iii)
Duttaphrynus was found to be the basal taxon to the above cluster, although, with a weak bootstrap support (40).
(iv)
A close osteological similarity between the microhylids of the genus Uperodon (Euclidean distance: 0).
(v)
Nasikabatrachus forming the basal taxon to the above clusters with a strong bootstrap support (77).
(vi)
A cluster comprising the rhacophorid genera Polypedates and Raorchestes (Euclidean distance: 0), which
forms the base of the entire phenogram.
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ALYTES 2015 | 32
DISCUSSION
The data presented herein, forms the baseline for osteology of Anurans in peninsular India comprising
details of several genera. A comparison of the phenogram of osteological characters of the above genera has
been made with their respective phylogenetic relationships published (Pyron & Wiens, 2011; Bocxlaer et al.,
2006). This reveals the following pattern: the structural similarity between Micrixalus (Micrixalidae) and
Indirana (Ranixalidae) corresponds to the sister relationship identified between these taxa by Bocxlaer et al.
(2006). These two genera differ only in the condition of their omosternum. The similarity in skeletal structures
between the large and small bodied Fejervarya spp. (the latter referred to Minervarya earlier) reflects the close
phylogenetic affinities of these taxa, reported by Kuramoto et al. (2007), who synonymised Minervarya with
Fejervarya. Nyctibatrachus, an endemic lineage of Nyctibatrachidae from the Western Ghats, was found to show a
considerable similarity in osteology to Indosylvirana, a member of the family Ranidae. This pattern, however, does
not reflect any phylogenetic signatures and rather just points at the ecological similarity of these taxa. Both these
taxa being associated with streams seem to possess similar osteological characteristics. Duttaphrynus of family
Bufonidae is quite unique osteologically and does not resemble any other taxa compared herein. Conditions such
as the absence of omosternum and arciferal configuration of pectoral girdle are unique to Duttaphrynus among the
taxa studied here. Microhylids are known to possess the most diverse osteological structures when compared to
any other anuran families (Trueb et al., 2011). The two members of Microhylidae examined here (earlier attributed
to Ramanella and Kaloula respectively) share great number of characters. Though attributed to different genera
earlier, these two species were found to show a considerable similarity in their osteology. Peloso et al. (in press)
reassessed the generic placement of the taxa from peninsular India and referred them to the genus Uperodon
Duméril & Bibron, 1841.
Apart from the abovementioned osteological similarities corresponding to phylogenetic signatures,
certain similarities related to ecological (microhabitat) guilds of these anurans were also observed. The genera
Indosylvirana and Nyctibatrachus, which are stream associated; Micrixalus and Indirana which occur along
rocky streams and cascades; and Fejervarya, that are associated with stagnant water pools, together, comprise a
cluster (the terrestrial / aquatic guild), which also includes the terrestrial Bufonid Duttaphrynus. The semi-arboreal
microhylids of the genus Uperodon form a guild. The enigmatic and largely fossorial Nasikabatrachus stands
unique in its position. Tree-dwelling frogs of the family Rhacophoridae, Raorchestes and Polypedates cluster
together in the arboreal guild. This pattern could be because of their highly specialized osteological features.
Unlike all other members, rhacophorids are equipped with a small additional phalanx in both fingers and toes,
thereby making their digits well adapted for an arboreal lifestyle.
Final considerations
This paper is just an initial attempt towards exploration of diversity in osteological characteristics
among a group of broadly syntopic anurans. Further studies at a finer scale with more number of taxa as well as
characters included are essential for a complete understanding of the evolution of these traits. Several families
examined here comprise multiple genera that show a considerable variation in morphology and adaptation. One
such example might be from the family Bufonidae comprising other genera (not examined in this study) such as
Pedostibes and Ghatophryne, both of which are highly specialized in their adaptations (arboreal and torrential
forms respectively). Likewise, in the family Microhylidae, ample avenues await investigation, especially in taxa
with different microhabitat associations such as Microhyla and Melanobatrachus. Thus, an extension of this study
with an examination of more number of taxa along with additional characters might yield a better understanding
of their evolution.
ACKNOWLEDGEMENTS
We thank Dr. K. Shanker (CES, IISc) and members of the lab for facilities and infrastructure provided;
Sneha Dharwadkar for the photograph (icon) of Nasikabatrachus.
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APPENDIX 1
Figure A1. A cleared and stained specimen of Micrixalus herrei (CESF023) showing key components of the skeleton described in the
manuscript: FP, frontoparietal; AT, atlas; SSC, suprascapula.
81
CONTENTS
Obituary
Krzysztof Kolenda, Mikołaj Kaczmarski
Leszek Berger: 1925-2012....................................................................................................................
3-5
Research Article
Jeanne Soamiarimampionona, Sidonie Samina Sam, Rainer Dolch, Katy Klymus, Falitiana Rabemananjara,
Eric Robsomanitrandrasana, Justin Claude Rakotoarisoa, Devin Edmonds
Effects of three diets on development of Mantidactylus betsileanus larvae in captivity........................
7-15
Research Article
James I. Menzies, Awal Riyanto
On the generic status of “Nyctimystes rueppelli” (Anura: Hylidae), a tree frog of Halmahera Island,
Indonesia...............................................................................................................................................
17-22
Research Article
Rocco Tiberti
The increase of an amphibian population: 11 years of Rana temporaria egg-mass monitoring in 30
mountain ponds....................................................................................................................................
23-29
Review Article
Heike Pröhl, Beatriz Willink
Ecología y comportamiento de las ranas venenosas del género Oophaga en Costa Rica y Panamá.
31-45
Research Article
S. R. Chandramouli, Tasneem Khan, Roshni Yathiraj, Nayantara Deshpande, Shreya Yadav, Cara Tejpal,
Sanne de Groot, Isabelle Lammes
Diversity of amphibians in Wandoor, South Andaman, Andaman and Nicobar Islands, India.............
47-54
Scientific Note
Josiah H. Townsend, Thomas J. Firneno, Jr., Dorian L. Escoto, Einstein A. Flores-Girón, Melissa
Medina-Flores, Olvin Wilfredo Oyuela
The first record of the streamside frog Craugastor rupinius (Anura: Craugastoridae) in Honduras,
confirmed by 16S DNA barcoding........................................................................................................
55-58
Research Article
S. R. Ganesh, M. Arumugam
Natural History and distribution notes on the Sreeni’s golden frog (Indosylvirana sreeni) in the
southern eastern Ghats, peninsular India............................................................................................
59-65
Research Article
S. R. Chandramouli, Sushil Kumar Dutta
Comparative osteology of anuran genera in the Western Ghats, Peninsular India.............................
67-81
This journal is available online at www.amphibians.org/alytes
Cover photo: first row: Fejervarya andamanensis (© S.R. Chandramouli); Mantidactylus betsileanus (© D. Edmonds); Rana temporaria (© R. Tiberti);
second row: Craugastor rupinius (© M. Medina); Litoria rueppelli (© A. Riyanto); Oophaga pumilio orange morph (© S. Hagemann); Oophaga pumilio
yellow-green morph (© C. Dreher)

Alytes_2015_32 - Amphibian Survival Alliance

Transcription

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