Table Of ContentREVIORA
jyi niseiim oi Lomparafive Zoology
US ISSN 0006-9698
Cambridge, Mass. 24 July 2008 Number 513
DISTINGUISHING FEATURES OF THE SUB-SAHARAN FROG GENERA
ARTHROLEPTIS AND PHRYNOBATRACHUS: A SHORT GUIDE FOR FIELD
AND MUSEUM RESEARCHERS
Breda M. Zimkus1* and David C. Blackburn1
Abstract. Typically small body size and similar external anatomy have made it difficult for researchers to
distinguish between species oftwo distantly related, but often sympatric, African ranoid frog genera: Arthroleptis
(Arthroleptidae) and Phrynobatrachus(Phrynobatrachidae). Wedefinea suiteofexternalmorphological characters,
extendingbeyond the traditionallyused secondarysexualcharacteristics, thatcan beused to definitivelydistinguish
between adult Arthroleptis and Phrynobatrachus. Photographs comparing absence, presence, and variation of
morphological characters are included for clarification. Significant differences between body proportions (head
width/snout-ventlength)arealsoobservedbetweenthesegenera. Althoughsmallerspeciesmightbemoredifficultto
mm
identify, larger species (> 30 snout-vent length) oi Arthroleptis can be distinguished from Phrynobatrachus
becauseoftheirrelativelywiderheads.Weintendthistoserveasaheuristicguideforbothfield-andmuseum-based
researchers.
Key words: Africa; Amphibia; Arthroleptidae: identification: Ranoidea: Phrynobatrachidae
INTRODUCTION
atic research and identification in the field
and in museum collections. Within sub-
Nearly every herpetologist is familiar with
Saharan Africa, two such genera, Arthrolep-
the overwhelming diversity of "little brown
frogs." Many leaf litter anurans are small, tis Smith 1849 and Phrynobatrachus Giinther
1862, have been confused for more than a
generally drab in color, and morphologically century (i.e., Boulenger, 1882). On the basis
similar, and this has hampered both system-
of studies of skeletal morphology (e.g..
Laurent, 1940, 1941a. b; Scott, 2005) and
the results of molecular phylogenetic analy-
1Museum ofComparative Zoology, and Department of ses (e.g., Bossuyt et at.. 2006; Frost et ah.
Organismicand Evolutionary Biology. Harvard Univer-
sity, Cambridge. Massachusetts 02138. U.S.A.: 2006; Roelants et a/., 2007). it is now very
*Corresponding author. E-mail: bzimkus<Yzoeb.harvard, clear that these genera, although externally
edu. similar, are only distantly related. In our
BREVIORA No. 513
experience, many researchers are unaware of Phrynobatrachus. Whereas Dimorphognathus
the numerous external morphological char- africanus is embedded deeply within Phryno-
acters that can be used to distinguish batrachus. supporting the synonymization of
between each genus. Arthroleptis and this monotypic genus, the placement of
Phrynobatrachus continue to be confused Phrynodon sandersoni within Phrynobatra-
in the field and in museum collections, chus is more controversial because of its
which can then confound further research basal position within the lineage; the synon-
such as molecular phylogenetic analyses ymy of Phrynodon with Phrynobatrachus is
(see Discussion for an example). Thus the currently being examined as part of a
external morphological differences between phylogenetic study ofthe Phrynobatrachidae
these two genera warrant a thorough treat- (sensu Frost, 2007; Zimkus. unpublished).
ment to improve the efficacy of identifica- The long taxonomic history linking Ar-
tion. throleptis and Phrynobatrachus contributes
Squeakers (Arthroleptis sensu Frost, 2007) to the present difficulties resolving the
mm
are generally small (16-54 snout-vent identifications of these distantly related frog
length), terrestrial, leaflitter anurans. and all genera (e.g., Boulenger, 1882). Many species
of the approximately 37 species are believed originally described asArthroleptishave been
to have lost the free-living, feeding tadpole since transferred to other genera, including
stage (i.e.. direct development). The smallest 21 species that are currently considered valid
Arthroleptis species were previously placed in species of Phrynobatrachus (Frost, 2007). In
Schoutedenella (Laurent. 1954): Frost et al. part on the basis of the work of Deckert
(2006) synonymized Schoutedenella with Ar- (1938), Laurent (1940, 1941a. b) used oste-
throleptis on the basis of the results of their ological characters to separate species into
molecular phylogenetic analysis. An analysis two genera, Arthroleptis and Phrynobatra-
of the relationships within Arthroleptis and chus. which he believed were not closely
its putative sister taxon Cardioglossa is related. The morphological differentiation of
currently in preparation (Blackburn, unpub- these genera is further supported by recent
lished). molecular phylogenetic studies (e.g., Bossuyt
Puddle frogs (Phrynobatrachus) comprise a etal., 2006; Frost et al.. 2006; Roelants et al..
lineage ofapproximately 75 species found in 2007), which show that Arthroleptis and
diverse terrestrial habitats across sub-Sa- Phrynobatrachus belong to different radia-
haran Africa (Frost, 2007). Phrynobatrachus tions within ranoid frogs.
has been a long-standing source ofconfusion The purpose of this study is to clearly
to systematists because of extensive geo- determine those external morphological
graphic and intrapopulation variation ac- characters that can be used to discrimi-
companied by only slight morphological nate between Arthroleptis and Phrynobatra-
differences among species (Largen, 2001; chus. We also document characters that
Rodel, 2000; Stewart, 1974). Molecular data could be useful for identification but vary
from mitochondrial and nuclear markers within these genera. Because we have found
indicate that both Dimorphognathus and that written descriptions of these characters
Phrynodon might be embedded within Phry- are often unclear, we provide photographs to
nobatrachus, thus rendering the genus para- illustrate the more important characters.
phyletic (Frost et a!., 2006; Scott, 2005). Although this information might not be
Both Scott (2005) and Frost et al. (2006) entirely novel for tenured African amphibian
argued for synonymizing these genera with biologists, we hope that those not familiar
.
2008 DIAGNOSING \RT1IROl.l.PIIS \\l)/7/A>) \OUATRACHUS
wilh these genera will find this to be a useful Appendix 1). We used an analysis ofcovari-
guide. ance to test for significant difference in the
slope o\~ the regression lines in which HdWd
METHODOLOGY is the dependent variable and SVL is indepen-
dent. To take into account variation in both
Using the taxonomic, systematic, and HdWd and SVL. reduced major axis regres-
fauna! literature as our guide, we examined
sion was used. Statistical significance was
museum specimens for characteristics report- evaluated for oe = 0.05.
ed!) useful in differentiating between Arthro-
leptis and Phrynobatrachus. We focused RESULTS
solely on external characteristics to make
our observations equally useful to research- The characters examined and evaluated in
ers conducting field surveys or sorting this study are summarized in Table 1. Nearly
specimens in museum collections. A total of all characters reported to differentiate Ar-
15 morphological characters were investigat- thro/eptis and Phrynobatrachus were found
ed; six of these are male secondary sexual to vary both within and between species in
characters. Characters are provided in Ap- each genus (Table 1). Only two external
pendix 1. and Appendix 2 comprises a data morphological characters definitively differ-
matrix in which all species examined are entiate these genera:
coded for these characters. 1 the presence of a tarsal tubercle and
The results of this work are based on our 2. the presence of an outer metatarsal
study ofspecimens from museum collections, tubercle.
as well as our respective experiences in the However, among African anurans. only the
field. Museum abbreviations correspond to presence of a tarsal tubercle is unique to
those of Leviton et al. (1985). with the Phrynobatrachus.
exception of MMB (Museums of Malawi. In general. Anhroleptis have relatively
Blantyre). The allocation ofspecies to either wider heads than Phrynobatrachus. The
HdWd
Anhroleptis or Phrynobatrachus follows coefficients (P) oflinear regression of
Frost (2007). High-resolution images were on SVL are significantly different between
obtained with a JVC 3-CCD digital camera these two genera {Anhroleptis: P = 0.461. SE
using AutoMontage Pro 5.0 (Synoptics). = 0.006: Phrynobatrachus: p = 0.348. SE =
Images were saved as TIFF files, cropped, 0.005: P < 0.0001, F = 64.39). Although
and contrast-adjusted with Adobe Photo- highly significant, in practice it is very
shop 7.0 for Macintosh. We gathered meris- difficult to discriminate between these genera
tiincgd2a7taspfercoimes2o8f0Aandhurlotlseppteicsimaennsd r2e4p6reasdeunltt- oarnethleessbastihsanonl3y0omfmthesSeVdLata(Fiifg.sp1e)c.imTehnes
specimens representing 31 species ofPhryno- difference in relative head width is omnlmy
obvious for specimens more than 30
batrachus (Online Supplement: Appendix 1). SVL (HdWd/SVL: 40% vs. 34%).
Anhroleptis and Phrynobatrachus appear
togenerallyexhibitdifferentbodyshapes,with
DISCUSSION
Anhroleptis having relatively wider heads. To
test whether this difference is significant, we In this study, we examined a suite of
took measurements (± 0.1 mm) of snout- morphological characters believed to be
vent length (SVL) and head width (HdWd) useful in distinguishing between adults of
with digital calipers (Online Supplement: Anhroleptis and Phrynobatrachus. The most
BREVIORA No. 513
Table 1. Externalmorphologicalcharactersusefulfordifferentiatingbetween Arthroleptis
AND PhRYSOBATRACHCS.
Character Arthroleptis Phrynobatrachus References
Tarsal tubercle absent present
Outer metatarsal tubercle
Hourglass or triple diadem pattern on variable
dorsum
Chevron-shapedglandsinscapularregion
Eyelid cornicle or spur
Sexually mature males
Elongate third finger
Dermal digital spines
Inguinal spines
Nuptial excrescence (thickened pad of
skin) on first finger
Femoral gland
Lateral vocal folds
2008 DIAGNOSING ARTHROLEPTISAND PHRYNOBATRACHUS
African frogs, but the presence or combina- poorly preserved specimens (Fig. 2G); how-
tion oi' particular characters can be useful in ever, the presence of this character can be
distinguishing between adult Phrynobatra- used to accurately identify Arthroleptis.
chus and Arthroleptis, Expansion o\~ the Finally, some species of Phrynobatrachus
terminal phalanx o\' manual and pedal digits exhibit circummarginal grooves on the man-
is found in both genera, but circummarginal ual or pedal digit tips; in some species, these
grooves are found only in some species of furrows are found only on the longest digits.
Phrynobatrachus (Blackburn. 2005; Scott, In addition, the presence of circummarginal
2005; Fig. 2E). Phrynobatrachus differs from grooves could be difficult to determine
Arthroleptis by the presence of pedal web- because of desiccation of preserved speci-
bing in many species of the former (Schmidt mens. However, if circummarginal grooves
and Inger. 1959). Webbing ranges from are present, this character can be used to
absent or rudimentary in numerous smaller differentiate Phrynobatrachus from Arthro-
species such as P. miliums, P. parvulus, and leptis, because it is lacking in the latter, but
P. ukingensis to extensive in the majority of not necessarily from other African frog
larger, rheophilic species such as P. acutiros- genera (Blackburn, 2005; Scott, 2005;
tris, P. krefftii, and P. versicolor (Fig. 2C, Fig. 2E).
D). Five species of Phrynobatrachus, includ- The presence of male secondary sexual
ing P. annulatus, P. calcaratus, P. cornutus, characters can lead to the most straightfor-
P. taiensis, and P. villiersi, possess a single ward identifications of specimens as Arthro-
spinelike dermal tubercle on the upper eyelid leptis or Phrynobatrachus. An elongate third
(Rodel, 2000). a character that can be used to finger is found in many male Arthroleptis, as
distinguish Phrynobatrachus from Arthrolep- well as most species of Cardioglossa, the
tis (Perret. 1988), as well as other African putative sister genus of Arthroleptis (Black-
anurans. burn, in press; Fig. 3A, B). This sexual
A number of external morphological dimorphism is not found in Phrynobatrachus
characters vary both within and between or any other anurans. Males of nearly all
species in each genus, and this variation is Arthroleptis species have dermal spines lining
due to either natural variation within or the medial surface of the elongate third
between populations or the condition of finger and are also sometimes found on the
preserved specimens. Chevron-shaped glands lateral, medial, or both surfaces of the
in the scapular region of Phrynobatrachus second finger (Fig. 3B). In some species,
and a dorsal triple diadem pattern in the presence of spines can vary seasonally
Arthroleptis can both be used to identify (e.g., Schmidt and Inger, 1959). Males of
species ofthese genera (Scott, 2005; Stewart, some species of Phrynobatrachus possess a
1974). However, these are not always prom- nuptial excrescence or thickened pad ofskin
inent in all members of a species or may be on the medial and dorsal surface ofthe first
completely lacking in some species. The size finger, which was hypothesized by Parker
and shape of the chevron-shaped glands of (1940) to be an adaptation to aquatic
Phrynobatrachus are quite variable and can amplexus. In P. africanus, this pad is greatly
originate and terminate in the scapular hypertrophied and covers much ofthe dorsal
region or instead extend almost the entire surface of the hand (Fig. 3C). The presence
length of the body (Fig. 2H). The median ofan elongate ovoid femoral gland, which is
dorsal skin raphe of Arthroleptis can be a most evident in life because it often can be
difficult character to visualize, especially in hardened and yellow in color, allows the
BREVIORA No. 513
&';'-?
"J'
:ik)n DIAGNOSING [RTHROLEPTIS AND PHRYNOBATRACHl S
identification of breeding Phrynobatrachus froglets hatch from a small clutch of
males (Blackburn, 2005; Stewart. 1967). terrestrially deposited eggs. In contrast, most
However, in most species, this gland might Phrynobatrachus species deposit hundreds to
be difficult to identify after preservation. thousands of eggs in ponds, streams, or
Breeding Phrynobatrachus males have a pools, and a small number ofspecies deposit
single SLibgular vocal sac, which, when not small clutches of eggs in stagnant water
distended, forms one or multiple folds, found in tree holes, in empty fruit capsules,
roughly parallel to the lower jaw, on the within snail shells, or terrestrially (Rodel,
lateral margins of the throat (Stewart, 1967; 1998; Rodel and Ernst, 2002). Species
Fig. 3E). Although the gular region of exhibiting these alternative reproductive
Arthroleptis can be distended and wrinkled, modes include P. dendrobates, P. guineensis.
prominent gular folds are not present P. krefftii, P. phyllophilus, P. sandersoni, and
(Fig. 3D). P. tokba, although all have free-living
For many field biologists that regularly tadpoles (Amiet, 1981; Rodel, 1998; Rodel
collect Arthroleptis and Phrynobatrachus, it is and Ernst, 2002). Although few advertise-
obvious that the body proportions of these ment calls of these genera are published,
frogs generally differ. Indeed, we find that those currently available will undoubtedly
Arthroleptis have heads that are relatively assist in distinguishing Phrynobatrachus from
wider than Phrynobatrachus. Meristic data Arthroleptis (e.g., Drewes and Perret, 2000;
collected in this study illustrate that the Rodel, 2000; Schiotz, 1964).
linear regression coefficients differ signifi- Although we demonstrate in this study
cantly between these two genera. However, that it is possible to distinguish adult
in general, the difference in body proportions Phrynobatrachus and Arthroleptis with the
mm
is only obvious in large specimens (> 30 use of morphological characters, the identi-
SVL). Thus, at least for larger specimens, the fication ofjuvenile and subadult Arthroleptis
relative width of the head could be a useful and Phrynobatrachus continues to be chal-
quick diagnostic feature, especially ifused in lenging, even occasionally for the authors of
concert with other morphological characters this study. Fortunately, the identification of
discussed herein (Table 1). numerous taxa has been greatly facilitated by
DNA
It is also important to note that numerous the recent use of barcoding (e.g.,
nonmorphological characters such as call Hebert et a/., 2003; Moritz and Cicero,
structure, breeding biology, and habitat 2004; Vences et a!., 2005). The use of the
preference can also be used in the field to mitochondrial 16S ribosomal RNA (rRNA)
assist in the identification ofArthroleptis and gene has proven to be particularly successful
DNA
Phrynobatrachus. All Arthroleptis are be- in amplification ofamphibian (Vences
lieved to have direct development, in which et al, 2005). We have found that the
illustrating extensive webbing. E, Tips of left pedal digits III and IV of P. tokba (MCZ A-26905) illustrating
expansion of digit tip and circummarginal grooves. F, Dorsal surface of A. stenodactylus (MCZ A-137060)
illustrating the hourglass (i.e.. triple diadem) pattern. G. Dorsal surface ofA. sp. nov. (MCZ A-137978), in life,
illustrating the median dorsal skin raphe. H, Dorsal surface of P. steindachneri (MCZ A-136907) on left and P.
auritus(MCZ A-138095) on right illustrating variability in chevron-shaped glands. Scale bar 1 mm in 2A-D, F-H;
mm
0.1 in 2E.
BREVIORA So. 513
Figure 3. Secondary sexual characters of males useful in distinguishing between Arthroleptis and Phrynoba-
trachus.A. RighthandofA. stenodactylus(MCZ A-137060)inventralviewshowingelongatedigit III. B. FingersII
and III ofmale A. schubotzi (CAS 201717) in ventral view illustrating dermal spines. C. Right hand ofmale P.
natalensis(MCZ A-138084). above, and left hand ofmaleP. (Dimorphognathas)afrkanus (MCZ A-136757). below.
indorsalview illustratingnuptialexcrescence(pad)ondigitI. D.ThroatofmaleA. schuborzHCAS201717).left,and
A. stenodaaylus (MCZ A-137061), right, illustrating lack ofvocal folds. E. Throat ofmale P. auritus (MCZ A-
138095) and P. (Phrynodon) sanderso?iH\\CZ A-136790) illustrating visible vocal folds. Scale bar 1 mm.
)
2IMIS DI.\ti\()M\ii IRTIIROLEPIIS AND PHRYNOBATRACHl V
amplification of this gene is effective in APPENDIX 1. MORPHOLOGICAL
identifying tadpoles,juveniles, and subadults CHARACTERS EXAMINED.
of numerous African ranoid frogs, including
External Morphology
Phrynobatrachus and Arthroleptis. However,
molecular data and analyses can be con- 1. Tarsal tubercle: (0) absent: ( 1 ) present.
founded b\ the misidentificatioD of voucher 2. Outermetatarsal tubercle: (0) absent: ( 1
specimens. For instance, nucleotide BLAST present.
searches against GenBank sequence data for 3. Heel tubercle (located at the proximal
the Arthroleptis specimens for which Yences end of the tarsus): (0) absent: present.
( 1 )
et al (2003) amplified the 16S rRNA gene 4. Circummarginal groove at manual and/
reveals that these specimens are misidentified or pedal digit tips: (0) absent: (1) present. In
(GenBank sequences AF215139^10). Al- some species, this groove is present only on
though Vences et al. (2003) attribute the the tips of the longest digits. Variation in
unusual paraphyly ofArthroleptis 16S genes museum specimens might be the result of
to uncertainties in their sequence data, this preservation, desiccation, or both.
result is easily explained: These sequences 5. Pedal webbing: (0) absent or rudimen-
have high similarity to Phrynobatrachus and tary with more than two distalmost phalan-
thus are misidentified at the genus level. We ges of digit IV unwebbed: (1 ) moderate to
DNA
advocate identification of specimens extensive with no more than the two distal-
only to the level of genus because of the most phalanges of digit IV unwebbed.
difficulty in accurately identifying species of 6. Median dorsal skin raphe: (0) absent:
either genus. Thorough taxonomic study in present.
(1)
combination with molecular data will be 7. Hourglass or triple diadem pattern on
DNA
necessary before identification can be dorsum: (0) absent: (1) present.
used confidently to identify species of these 8. Chevron-shaped glands in scapular
two genera. region: (0) absent: (1) present. These ridges
ofskin can be short or can extend the entire
ACKNOWLEDGMENTS length of the body when present.
9. Eyelid cornicle or spur: (0) absent: (1)
This study was supported by the Depart-
present.
ment of Organismic and Evolutionary Biol-
ogy and Museum of Comparative Zoology
Secondary Sexual Characteristics
(Harvard Lniversity) and NSF grant EF-
0334939 (AmphibiaTree) to J. Hanken. 10. Sexually mature males, third finger
Many thanks to the following individuals relatively longer than in females: (0) absent:
who made specimens under their care avail- (1) present.
able for study: M. Burger. J. Campbell. D. 11. Sexually mature males, dermal spines
Cannatella. B. Clarke. R. Drewes. R. Gun- on fingers: (0) absent: ( 1 ) present.
ther. V. Gvodzik. T. Harstell. T. LaDuc. L. 12. Sexually mature males, inguinal spines:
Lawson. S. Loader. P. Malonza. L. Mazi- (0) absent: ( 1 ) present.
buko. J. McGuire. J. Measey. A. Ohler. A. 13. Sexually mature males, nuptial excres-
Resetar. J. Rosado. A. Schmitz. E. Scott. J. cence (thickened pad ofskin that can appear
Simmons. L. Trueb. J. Vindum, D. Wake. A. velvety) on first finger: (0) absent: ( 1 ) present.
Wynn. J. Hanken provided useful comments 14. Sexually mature males, femoral glands:
on a draft of the manuscript. (0) absent: (1) present. Most often the
10 BREVIOR-4 \o. 513
femoral glands of Phrynobatrachus are lo- 15. Sexually mature males, lateral vocal
on the posterior thigh. However. folds: (0) absent: (1) present. When pres-
.
glands can be situated closer to either the ent, folds run roughly parallel to the
_ s or vent. These glands are most easily lower jaw. at the lateral margins of
seen in living specimens because they are the throat, and form one or multiple
usually bright yellow. crease>.
APPENDIX
2.
DISTRIBUTION OF MORPHOLOGICAL CHARACTERS AMONG SPECIES OF
ARTHROLEPTIS AND PHRY\OBATH4CHlS. ALL CHARACTER STATES ARE
BINARY (0, 1). MISSING DATA ARE CODED AS "?"; 0.1 DENOTES POLYMOR-
PHISM. REFER TO APPENDIX FOR CHARACTER DESCRIPTIONS.
1
