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E V I O R A 



jyil 111 s e ui ma ol v^omnpaFafi ve z^oology 



us ISSN 0006-9698 



Cambridge, Mass. 



1 June 2009 



Number 517 



DESCRIPTION AND PHYLOGENETIC RELATIONSHIPS OF TWO NEW SPECIES 

OF MINIATURE ARTHROLEPTIS (ANURA: ARTHROLEPTIDAE) FROM THE 

EASTERN ARC MOUNTAINS OF TANZANIA 

David C. Blackburn'-^ 

Abstract. I describe two new species of squeaker frog (Arthroleptidae: Arthroleptis) from the Eastern Arc 
Mountains of Tanzania. These new species are distinguished from other miniature Arthroleptis in the Eastern Arc 
Mountains by the combination of very small adult body size (< 15 mm snout-vent length), a pronounced dark 
inguinal spot, and color patterns unique to each species. The new species could be the smallest frog species known 
from East Africa and among the smallest species of Arthroleptis. An estimate of phylogeny on the basis of 
mitochondrial DNA sequences reveals that these morphologically cryptic species are sister taxa that compose a basal 
lineage within a clade of miniature Arthroleptis. Their description hints at a diverse cryptic amphibian fauna in the 
Eastern Arc Mountains that awaits additional discovery through molecular methods. 

Key words: Africa; biodiversity hotspot; body size: cryptic species; evolution; molecular phylogeny 

Ninatoa maaelezo hapa kuhusu aina mbili mapya wa vyura (Familia ya Arthroleptidae: kikundi cha Arthroleptis) 
kutoka milima ya 'Tao ya Mashariki' ya Tanzania. Hawa aina mapya ni tofoauti na Arthroleptis wengine wadogo 
kwa urefu wa mwili ndogo zaidi (< 15 mm kutoka mapua hadi nyuma ya mwili), doa moja nyeusi na rangi binafsi 
kwa kila aina mpya. Kwa kutumia sayansi ya DNA inaonyeshwa hawa aina wa vyura wako chini katika kikundi cha 
Arthroleptis. Ugunduzi huu inatoa rai kuhusu aina wengine wa vyura kwa milima ya Tao ya Mashariki wanaongojea 
kugunduliwa kwa kutumia sayansi ya molekuli. 



' Museum of Comparative Zoology and Department of 
Organismic and Evolutionary Biology, Harvard Univer- 
sity, Cambridge, Massachusetts 02138, U.S.A. 
"Present address: Division of Herpetology, Natural 
History Museum and Biodiversity Research Center, 
1345 Jayhawk Boulevard, University of Kansas, Law- 
rence, Kansas 66045, U.S.A.; e-mail: david.c.blackburn@ 
gmail.com 



The systematics of miniature African 
squeaker frogs (Arthroleptis sensu Black- 
burn, 2008; Frost, 2008) has been problem- 
atic for more than half a century. Histori- 
cally, many of these small species (< 25 mm 
snout- vent length [SVL]) were placed in a 
separate genus, Schoutedenella (e.g., Laur- 
ent, 1940, 1954, 1973), but most authors have 



® The President and Fellows of Harvard College 2009. 



BREVIORA 



No. 517 



retained them in Arthroleptis (e.g., Perret, 
1991; Poynton, 1976, 2003a; Poynton and 
Broadley, 1985; Schmidt and Inger, 1959; see 
also Fretey, 2008; Zimkus and Blackburn, 
2008). Additional taxonomic problems re- 
sulted from disagreement regarding the 
validity of particular species or genera (e.g., 
Laurent, 1954, 1961, 1972; Schmidt and 
Inger, 1959). Recent molecular phylogenetic 
analysis demonstrated that these miniature 
species do not form a clade and are 
paraphyletic with respect to larger Arthro- 
leptis (Blackburn, 2008; Frost et al, 2006). 
Consequently, these two genera were synon- 
ymized (Frost et al, 2006). The most recent 
common ancestor of Arthroleptis was mini- 
ature, and the evolution of small body size 
and direct development could have contrib- 
uted to the dispersal of this lineage from the 
forests of Central Africa to diverse habitats 
throughout much of the rest of Africa 
(Blackburn, 2008). Because many Arthrolep- 
tis species are small, morphologically similar, 
or both, many species are probably unde- 
scribed and cryptic (e.g., Blackburn, 2008; 
Poynton, 2003b; Rodel and Bangoura, 
2004). Little progress has been made recently 
in describing the diversity of small Arthro- 
leptis species, at least in part because of a 
lack of genetic resources that could assist in 
delimiting species boundaries. 

A recent phylogenetic study revealed a 
cryptic lineage of Arthroleptis in the Eastern 
Arc Mountains of Tanzania (Blackburn, 
2008). This study investigates this newly 
discovered lineage, represented by only one 
specimen in the previous study, through 
analysis of additional tissue samples and 
museum specimens of miniature Arthroleptis 
from the Eastern Arc Mountains. This cryptic 
lineage was found to contain two undescribed 
species, known only from the northern part of 
the Eastern Arc Mountains in Tanzania, that 
could be the smallest frog species both in East 
Africa and within Arthroleptis. 



MATERIALS AND METHODS 

All measurements (±0.1 mm) were taken 
with digital calipers under a dissecting micro- 
scope. Measurement methodology follows 
Blackburn (2005), which is a modification of 
Matsui (1984). All limb measurements were 
made on the right side except where indicated. 
Females were identified by large body size, the 
presence of ova (visible either in dissection or 
through the wall), the lack of male secondary 
sexual characters, or a combination of fea- 
tures; males were identified by the presence of 
male secondary sexual character typical of 
Arthroleptis (Blackburn, 2009). High-magnifi- 
cation images of specimens were taken by a 
JVC 3-CCD digital camera mounted on a 
dissecting microscope with AutoMontage Pro 
5.0 (Synoptics). Museum acronyms follow 
Leviton et al (1985). 

Phylogenetic relationships were estimated 
through analysis of DNA sequences. Data 
for the mitochondrial 12S and 16S ribosomal 
RNA (rRNA) genes and the intervening 
transfer RNA for valine were collected from 
four specimens. These sequences were added 
to data collected for Arthroleptis schubotzi, 
A. xenodacty hides, and A. xenodactylus 
collected during a recent molecular phyloge- 
nefic study (Table 1; Blackburn, 2008). The 
two new species correspond to taxa that 
Blackburn and Measey (2009) refer to as 
Arthroleptis sp. nov. A and Arthroleptis sp. 
nov. B. Two distantly related species, A. 
stenodactylus and A. variabilis, were used as 
outgroup taxa (e.g., Blackburn, 2008). 

Genomic DNA extracts were made with 
the use of a Qiagen DNeasy Tissue Kit (Cat. 
no. 69506). Primer pairs (i.e., 12L1 and 16sh; 
12sm and 16sa; 16sc and 16sd) follow Darst 
and Cannatella (2004); polymerase chain 
reaction (PCR) and sequencing reactions 
follow Blackburn (2008). DNA sequences 
of unequal length (~ 1,900 bp) were aligned 
in ClustalX v. 1.83.1 with default parameters. 



2009 



TWO NEW MINIATURE ARTHROLEPTIS 



Table 1. Specimens included in phylogenetic analysis. 



Arthroleptis sp. 



Locality 



Coordinates 



Catalog No. GenBank No. 



Source 



fichika, n. sp. Baga II Forest Reserve, 04°48'S, 038°27'E 

West Usambara 

Mountains, Tanzania 
Mazumbai Forest 04°49'45"S, 

Reserve, West 038°30'46"E 

Usambara Mountains, 

Tanzania 
cf.Jichika Chome Forest Reserve, 04°17'S, 

South Pare Mountains, 037°55'40"E 

Tanzania 



CAS 168829 FJ 15 1064 Blackburn, 2008 



MCZ A- 138384 FJ 188697 This study 



FMNH 251864 FJ 188696 This study 



kidogo, n. sp. 


Nguru Mountains, 
■ Tanzania 


06°03'09"S, 
037°32'26"E 


MCZ A-138393 


FJ 188698 


This study 




schubotzi 


Bwindi Impenetrable 
National Park, 


00°59'34"S, 
29°36'57"E 


MCZ A- 138394 
CAS 201752 


FJ 188699-70 
FJ151061 


This study 
Blackburn, 


2008 




Uganda 




CAS 201753 


FJ151116 


Blackburn, 


2008 


xenodactyloides Amani, East Usambara 
Mountains, Tanzania 


05°05'S, 038°36'E 


CAS 168608 


FJ 15 1063 


Blackburn, 


2008 




Ruo River Gorge, 
Mulanje Massif, 
Malawi 


15°56'S, 035°37'E 


MCZ A- 137002 


FJ 15 1096 


Blackburn, 


2008 


xenodactylus 


Amani Nature Reserve, 
East Usambara 


05°05'37"S, 
038°36'00"E 


MCZ A- 138404 


FJI51156 


Blackburn, 


2008 




Mountains, Tanzania 




MCZ A-1 38405 


FJ151157 


Blackburn, 


2008 


stenodactylus 


Amani, East Usambara 
Mountains, Tanzania 


06°56'30"S, 

37°43'10"E 


CAS 168455 


FJ151054 


Blackburn, 


2008 


variabilis 


Etome, Petit Mount 
Cameroon, Cameroon 


06°56'30"S, 

37°43'I0"E 


MCZ A- 136744 


FJ151083 


Blackburn. 


2008 



However, PCR products were amplified and 
sequenced with varying success. Thus, for 
two specimens, the full sequence length could 
not be obtained: a 743 bp fragment of 12S 
rRNA was obtained from MCZ A-138393; 
two fragments (12S rRNA, 765 bp; 16S 
rRNA, 830 bp) were obtained from MCZ 
A-1 38394. After alignment, the sequences for 
the fragments amplified for MCZ A- 138394 
were merged in MacClade v.4.06 to form a 
single taxon in the analysis; the intervening 
base pairs were considered missing data. The 
alignment was trimmed to positions corre- 
sponding to 2,496-4,260 of the Xenopus 
laevis mitochondrial genome (GenBank 



NC-001573) such that the final alignment 
contained 1,902 characters. All but two 
specimens (MCZ A-138393^) have no 
missing data for these 1,902 characters; after 
trimming, 693 characters are present for all 
specimens in the analysis. 

A maximum likelihood (ML) estimate of 
phylogeny was obtained through analysis of 
sequence data in GARLI v. 0.95 (Zwickl, 
2006) with a random starting tree and a 
GTR+I+r model of evolution with all 
parameters estimated. The GARLI analysis 
was terminated 250,000 generations after the 
last topological improvement. Support for 
phylogenetic topologies was estimated by 



BREVIORA 



No. 517 



nonparametric bootstrapping in GARLI. 
One thousand bootstrap replicates were 
performed with the same model of evolution, 
with each search terminated 1,000 genera- 
tions after the last topological improvement. 
Branches present in > 70% of the bootstrap 
trees were considered well supported follow- 
ing Hillis and Bull (1993). In addition, a 
Bayesian estimate of phylogeny was obtained 
with MrBayes v. 3. 1.1 and a GTR-i-I+r model 
of evolution. Bayesian analysis was run for 5 
million generations, sampled every 1,000 
generations, with four chains, a temperature 
of 0.2, and default priors. The first 1 million 
generations were discarded as burn-in. The 
phylogeny and posterior probabilities were 
then estimated from the 4,000 post-burn-in 
trees. Topologies with posterior probabilities 
> 95% were considered well supported 
(Wilcox et al, 2002). Genetic distances were 
calculated in MEGA v.4.0.1 (Tamura et al, 
2007) with the maximum composite likeli- 
hood (MCL). The MCL calculations were 
performed with the use of data for transi- 
tions and transversions and assuming a 
heterogeneous pattern of sequence evolution 
with a r distribution of among site rate 
variation (F parameter = 1.0). 

DESCRIPTION OF NEW SPECIES 

Arthroleptis fichika^ new species 

Hidden Squeaker Frog 
Figures 1^; Table 2 

Holotype. MCZ A- 138384 (field no. Breda 
M. Zimkus [BMZ] 23104), adult female 
(gravid), Republic of Tanzania, Tanga Region, 
West Usambara Mountains, Mazumbai For- 
est Reserve, 04°49'45"S, 038°30'46"E (WGS 
datum), 1,383 m elevation, 10 March 2007, D. 
L. Mahler, L. Lawson, B. M. Zimkus. 

Paratype. CAS 168829 (field no. Robert 
C. Drewes [RCD] 11039), adult female. 
Republic of Tanzania, Tanga Region, Lush- 
oto District, West Usambara Mountains, 



Baga II Forest Reserve, 04°48'S, 038°27'E 
(estimated), 1,500-1,900 m elevation (esti- 
mated from latitude and longitude), 26 April 
1988, R. C. Drewes, K. M. Howell, and J. V. 
Vindum. 

Diagnosis. A miniature Arthroleptis similar 
to other small East African Arthroleptis (i.e., 
A. schubotzi, A. stridens, A. xenochirus, A. 
xenodactyloides, A. xenodactylus, and the 
new Arthroleptis species described below), 
but distinguished from all but the other new 
species by smaller adult body size (gravid 
adult females < 1 5 mm SVL; Table 3) and a 
prominent dark brown inguinal spot that is 
darker than other prominent dorsal mark- 
ings and the dorsal base color. An inguinal 
spot is variably present in other miniature 
Arthroleptis from East Africa, but it is 
neither as prominent nor as dark relative to 
other dorsal markings as it is in these two 
new species. Arthroleptis fichika differs from 
the new species described below by less 
expanded and less pointed toe tips, a 
supratympanic band that terminates anterior 
to the arm (Fig. 4), a prominent dark brown 
spot on the anterior distal thigh, and lacking 
a reticulated pattern on the ventral surface of 
the head and body. Arthroleptis fichika 
differs in the following additional ways from 
other miniature East African Arthroleptis: 
from A. schubotzi, A. stridens, and A. 
xenochirus by darkly colored ventral thighs 
with small light gray spots; from A. schu- 
botzi, A. stridens, and A. xenodactyloides by 
a less globular and more elongate inner 
metatarsal tubercle; from A. stridens by less 
expanded toe tips, more defined dorsal and 
lateral markings, and more ventral pigmen- 
tation; from A. xenochirus by the crus longer 
than the thigh, lacking accessory metatarsal 
tubercles at the base of the second and third 
toes, and more expanded and pointed digit 
tips; from A. xenodactylus by more defined 
dorsal markings and less expanded and less 
pointed toe tips. 



2009 



TWO NEW MINIATURE ARTHROLEPTIS 




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imi 600 -1.200 m 

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j j 2,400 -5,880 m 




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Description of Holotype. Very small (SVL 
13.5 mm) gravid female; limbs relatively 
slender (Figs. 2-4; Table 2); head broad; 
head length 94% head width; snout barely 
projecting beyond lower jaw (Fig. 4A); 
rostral tip slightly rounded and nearly 
straight in dorsal view, nearly straight in 
lateral view; eyes projecting just beyond 
margins of head in dorsal view; dorsal 
surface of eyes approximately level with 
dorsal surface of head in lateral view; eye 
diameter just barely wider than interorbital 
distance; pupils small, horizontal, and ellip- 
tical in preservative; loreal region nearly flat; 
naris rounded and directed laterally with 
ventral margin visible in dorsal view; canthus 
rostralis short, slightly convex; eye diameter 
2.4 times eye-narial distance; eye diameter 
nearly 10 times distance from naris to rostral 
tip; internarial region slightly convex; inter- 
narial distance 84% interorbital distance; 
tympanum rounded, height less than half 
diameter of eye; tympanic annulus well 
defined; supratympanic region smooth with 
no fold; tongue large and posteriorly ex- 
panded with narrow anterior attachment; 
tongue with prominent rounded posterior 
notch; tongue lacking median papilla; choa- 
na completely hidden behind maxillary shelf 
in ventral view; premaxillary and maxillary 
teeth present but hidden in labial view by 
lips; vomerine teeth absent. 

Skin of limbs and dorsal, lateral, and 
ventral surfaces of head and body smooth; 
skin ventral to cloaca very weakly tubercu- 
late; median skin raphe only barely visible in 
preservative; cloacal opening horizontal. 



Figure 1. Type localities of Arthroleptis fichika n. 
sp. (black square) and A. kidogo n. sp. (black star) in the 
mountains of eastern Tanzania. Locality of specimen 
referred to A. fichika (FMNH 251864) is indicated by an 
open square. 



BREVIORA 



No. 517 




Figure 2. A, B, Arthroleptis fichika n. sp. (holotype, MCZ A-138384); C, Arthroleptis xenodactyloides (MCZ A- 
138386); and D, Arthroleptis xenodactylus (MCZ A-138401) in life. Scale bars ~ 2 mm. Photographs by D. L. Mahler. 



Limbs and digits slender; relative length of 
fingers: III > II > IV > I; finger tips neither 
swollen nor expanded; palmar and metacar- 
pal tubercles very weakly developed and 
completely flat; webbing between manual 
digits absent; fingers with somewhat distinct, 
weakly globular, and single subarticular 
tubercles; thigh length 88% crus length; 
relative length of toes: IV > III > V > II 
> I; toe tips weakly pointed, sHghtly 
expanded, and just wider than interphalan- 
geal regions; webbing between pedal digits 
absent; toes with indistinct, single subarticu- 
lar tubercles; inner metatarsal tubercle small, 
indistinct, and elongate, length 57% of first 
toe length; outer metatarsal tubercle absent. 

Measurements. See Table 2. 

Coloration in Life. From photographs of 
holotype. Dorsal base coloration ruddy 
brown with darker brown markings 



(Fig. 2A); iris golden and vermiculated with 
black; pupil black; hght gray to cream thin 
line extending from snout tip through margin 
of upper eyehd; lateral surface of body with 
dark gray base coloration; markings on 
lateral surface of body and limbs (i.e., 
supratympanic, inguinal, femoral) dark 
brown to black and darker than other dorsal 
markings; scattered small light gray spots on 
the lateral surface of body and Hmbs; ventral 
surface of head (i.e., gular region) orangey 
red; venter bright creamy yellow (Fig. 2B); 
ventral thigh with distinct red base colora- 
tion. 

Coloration of Holotype (in Alcohol). Dor- 
sal base color Ught brownish gray with 
scattered small darker brown spots 
(Fig. 3A); iris dark brown with silver pupil; 
dark brown interorbital bar incomplete and 
broken into chain of small spots; snout 



2009 



TWO NEW MINIATURE ARTHROLEPTIS 




Figure 3. Holotypes in dorsal and ventral views. A, 
B, Arthroleptis flchika n. sp. (MCZ- 138384); C, D, 
Arthroleptis kidogo n. sp. (MCZ A- 138394). Scale bar = 
10 mm. 



darker gray than more posterior head; loreal 
and suborbital regions medium brown but 
not forming a mask; continuous dark brown 
supratympanic band extending from posterior 
margin of eye, over tympanum, and terminat- 
ing well anterior to forelimb (Fig. 4A); tym- 
panum brown, translucent, and distinct from 
supratympanic band; three poorly defined, 
but interconnected, gray-brown spots ar- 
ranged along dorsal midline between head 




Figure 4. Right lateral view of heads of holotypes. 
A, Arthroleptis fichika n. sp. (MCZ- 138384); B, Arthro- 
leptis kidogo n. sp. (MCZ A- 138394). Scale bar = 1 mm. 

and sacrum (i.e., typical "hour-glass" config- 
uration of Arthroleptis); margins of dorsal 
spots defined by irregularly spaced small dark 
brown spots; lateral surface of body with 
several dark brown spots between forelimb 
and hind limb; prominent, dark brown 
inguinal spot extending from base of hind 
limb over the lateral surface of the ilium 
(Fig. 3 A); dorsal surfaces of limbs similar light 
gray, grading to darker gray on distal hind 
limbs; scattered small dark brown spots on the 
dorsal surfaces of forelimb; ultimate interpha- 
langeal joints unpigmented and light gray in 
color; dark brown spot on posterodorsal 
surface of thigh; proximal anterior surface of 
thigh with small light gray spots; prominent 
dark brown spot on anterior surface of distal 
thigh; hind limbs distal to knee covered in 
brownish light and dark gray mottling; dark 
brown trapezoid centered on cloaca. 

Gular region and venter with cream base 
color and small dark brown melanocytes 



BREVIORA 



No. 517 



Table 2. Measurements (mm) of Arthroleptis fichika and A. kidogo. 





A. fii 


'hika 


A. 


kidogo 




MCZ A- 138384 


CAS 168829 


MCZ A- 138394 


MCZ A-138393 




Holotype 


Paratype 


Holotype 


Paratype 


Snout-vent length 


13.5 


14.2 


14.1 


13.9 


Head width 


4.8 


5.0 


5.0 


5.1 


Tympanum height 


0.8 


0.8 


0.8 


0.8 


Eye diameter 


1.9 


1.9 


1.7 


2.1 


Snout length 


1.2 


1.5 


1.4 


1.5 


Forearm length 


3.2 


3.0 


2.9 


2.9 


Manual digit I 


0.7 


0.7 


0.7 


0.8 


Manual digit II 


0.9 


1.1 


1.0 


1.0 


Manual digit III 


1.5 


1.9 


1.6 


1.5 


Manual digit IV 


0.8 


0.9 


0.8 


0.8 


Thigh length 


5.7 


6.2 


6.3 


6.0 


Crus length 


6.5 


6.5 


6.3 


6.3 


Pedal digit I 


0.7 (left) 


0.7 


0.8 


0.9 


Pedal digit II 


1.3 


1.3 


1.3 


1.3 


Pedal digit III 


1.9 


2.2 


2.1 


1.9 


Pedal digit IV 


2.9 


3.3 


3.2 


2.8 


Pedal digit V 


1.6 


1.4 


1.7 


1.4 


Inner metatarsal tubercle 


0.3 


0.4 


0.5 


0.5 



forming no distinct pattern (Fig. 3B); ventral 
forelimbs dark brown with irregularly sized 
and poorly defined small light gray spots; 
palmar and plantar surfaces dark gray 
brown; ventral hind limb dark brown with 
many small light gray spots at somewhat 
regular intervals. 

Variation. The dorsal surface of the 
paratype (CAS 168829) has a continuous 
wide brown band extending from between 
the eyes to just anterior to the cloaca. Light 
brown regions border this midline band 
laterally. The prominent dark spot in the 
inguinal region and anterior surface of distal 
thigh are readily apparent in both the 
paratype and the referred specimen. 

Habitat and Natural History. The holotype 
(MCZ A- 138384) was collected during a 
daytime visual survey (0800-1100 h) when 
it was active in leaf litter in dense forest 
(from field notes of BMZ). 

Conservation. The two collection localities 
are both in the forests of the West Usambara 



Mountains, which have an estimated extent 
of less than 320 km2 (Burgess et al, 2007) 
and are threatened by forest loss and 
degradation associated with population 
growth (Kaoneka and Solberg, 1994). Be- 
cause A. fichika presently appears to be 
restricted to these forests, this new species 
should be considered tentatively as Vulner- 
able according to lUCN (2001) criteria. 

Etymology. The specific epithet fichika 
should be treated as an indeclinable word. 
It is a Kiswahili word meaning "hidden" and 
refers to the fact that A. fichika is a cryptic 
species first identified through molecular 
phylogenetic analysis (Blackburn, 2008). 

Phylogenetic Relationships. See below. 

Arthroleptis kidogo^ new species 

Tiny Squeaker Frog 
Figures 1, 3, 4; Table 2 

Holotype. MCZ A- 138394 (BMZ 23288), 
adult female (gravid), Republic of Tanzania, 
Nguru South Forest Reserve, Morogoro 



2009 



TWO NEW MINIATURE ARTHROLEPTIS 



Table 3. Mean snout-vent length (SVL) and standard deviation of miniature Arthroleptis from Kenya, 

Malawi, Tanzania, and Uganda. 



Arthroleptis sp. 



fichika 

kidogo 

schubotzi 

stridens 

xenochirus 

xenodactyloides 



xenodactylus 





Mean 


SVL (mm) 


Locality 


Female 


Male 


West Usambara Mtns., Tanzania 


13.9 ± 
1-1 — 


0.5 




Nguru Mtns., Tanzania 


n — z 
14.0 ± 
n = 2 


0.1 




Bwindi Forest, Uganda' 


19.1 ± 


1.5 


19.6 ± 0.9 




n = 4 




n = 6 


East Usambara Mtns., Tanzania^ 






17.9 
n = 1 
18.5 ± 1.6 


Zambezi River Source, Zambia^ 


20.5 






n = 1 




n = 9 


Taita Hills, Kenya^ 


19.6 ± 


2.0 


17.3 




n = 4 




n = 1 


West Usambara Mtns., Tanzania^ 


18.9 ± 

n = 5 


2.2 




East Usambara Mtns., Tanzania^ 


20.3 ± 
n = 6 


2.1 




Misuku Hills, Malawi^ 


16.7 ± 


0.7 


12.8 ± 1.1 




n = 4 




n = 2 


Mulanje Massif, Malawi^ 


16.5 ± 


0.8 


15.2 ± 0.2 




n = 16 




n = 3 


Mt. Chelinda, Zimbabwe^ 


17.1 ± 


0.8 


14.5 ± 1.0 




n = 22 




n = 15 


East Usambara Mtns., Tanzania'^ 


17.5 ± 


1.3 


13.7 ± 0.1 




n = 10 




n = 2 


Nguru Mtns., Tanzania" 


17.0 




14.2 ± 0.8 




n = 1 




n - 3 



iCAS 104500-01, 201700, 201717-19, 201736-39 

2ZMB 66249 

3CAS 196614, 196617-18, 196620-21, 196623, 196627, 196630, 196632, 196638 

4NMK A/4538, A/4540, A/4542, A/4653/1-2 

5MCZ A- 138385-89 

6CAS 168608, FMNH 251405, MCZ A-13199, A-138390-92 

7MCZ A-137136-41 

8MCZ A-137001-15, A-137034-37, A-137074 

9MCZ A- 17038, A-23339-50, A- 19047-67; TMP 19101, 19104. 

loMCZ A-13188, A-13190-94, A-13196, A-138404-05, A-138429, A-138435, A-138437 

11 MCZ A- 138400-03 



Region, Mvomero District, Nguru Moun- 
tains, 06°03'09"S, 037°32'26"E (WGS da- 
tum), 830 m elevation, 31 March 2007, D. L. 
Mahler and B. M. Zimkus. 

Paratype. MCZ A- 139393 (BMZ 23287), 
adult female (gravid), same collection data as 
holotype. 



Diagnosis. A miniature Arthroleptis similar 
to other small East African Arthroleptis (i.e., 
A. fichika, A. schubotzi, A. stridens, A. 
xenochirus, A. xenodactyloides, A. xenodac- 
tylus) but distinguished by a prominent dark 
brown supratympanic band that continues 
posterior to the level of the arm (Fig. 4B). In 



10 



BREVIORA 



No. 517 



Other species, the supratympanic band ter- 
minates anterior to or at the level of the arm 
(Figs. 3C, D, 4A). Arthroleptis kidogo is 
differentiated from all Eastern Arc Arthro- 
leptis except A. fichika by smaller adult body 
size (gravid adult females < 15 mm SVL; 
Table 3) and a prominent dark brown 
inguinal spot that is darker than other 
prominent dorsal markings and the dorsal 
base color. Arthroleptis kidogo differs in the 
following additional ways from other mini- 
ature Arthroleptis in East Africa: from A. 
fichika by dark anterior thighs with many 
small light gray spots and a reticulated 
pattern of dark melanocytes and light gray 
spots on the ventral surface of the head and 
body; from A. schubotzi, A. stridens, and A. 
xenochirus by darkly colored ventral thighs 
with small light gray spots; from A. schubotzi, 
A. stridens, and A. xenodactyloides by a less 
globular and more elongate inner metatarsal 
tubercle and expanded digit tips with distinctly 
pointed toe tips; from A. xenochirus by a crus 
that is longer than the thigh, lacking accessory 
metatarsal tubercles at the base of the second 
and third toes, and much more expanded and 
pointed digit tips; from A. xenodactylus by 
more defined dorsal markings and digit tips 
that, although pointed, do not exhibit a 
papillate projection. 

Description of Holotype. Very small (SVL 
14.1 mm) gravid female; limbs relatively 
slender (Figs. 3, 4; Table 2); head broad; 
head length 90% head width; snout barely 
projecting beyond lower jaw; rostral tip only 
slightly rounded in dorsal view, nearly 
straight in lateral view (Fig. 4B); eyes pro- 
jecting just beyond margins of head in dorsal 
view and just above dorsal surface of head in 
lateral view; eye diameter approximately 
equal to interorbital distance; pupils hori- 
zontally elliptical in preservative; loreal 
region nearly flat; naris rounded and directed 
laterally, not visible in dorsal view; canthus 
rostralis short, slightly convex; eye diameter 



1.5 times eye-narial distance; eye diameter 

5.6 times distance from naris to rostral tip; 
internarial region slightly convex; internarial 
distance 83% interorbital distance; tympa- 
num rounded, height shghtly less than half 
diameter of eye; tympanic annulus well 
defined; supratympanic region smooth with 
no fold; tongue narrow and ovoid with 
minute posterior notch; tongue lacking 
median papilla; choana just barely visible 
behind maxillary shelf in ventral view; 
premaxillary and maxillary teeth present 
but hidden in labial view by lips; vomerine 
teeth absent. 

Skin of limbs and dorsal and lateral 
surfaces of head and body smooth; ventral 
surface of head and body smooth anteriorly, 
but very weakly tuberculate posteriorly; 
median skin raphe absent, at least following 
preservation; cloacal opening horizontal, 
surrounded by smooth skin. 

Limbs and digits slender; relative length of 
fingers: III > II > IV > I; finger tips not 
swollen or expanded but distinctly pointed; 
palmar and metacarpal tubercles present but 
weakly developed and flat; webbing between 
manual digits absent; fingers with somewhat 
indistinct, flat, and single subarticular tuber- 
cles; thigh length approximately equal to 
crus length; relative length of toes: IV > III 
> V > II > I; toe tips expanded to 
approximately twice the width of interpha- 
langeal regions; webbing between pedal 
digits absent; each toe tip with prominent 
narrow distal point; toes with prominent, 
single, flattened subarticular tubercles; inner 
metatarsal tubercle small, indistinct, and 
elongate, length 62% of first toe length; 
outer metatarsal tubercle absent. 

Measurements. See Table 2. 

Coloration in Life. No photographs or 
field notes are available to document the 
coloration of A. kidogo in life. 

Coloration of Holotype (in Alcohol). Dor- 
sal base color light gray (Fig. 3C); iris dark 



2009 



TWO NEW MINIATURE ARTHROLEPTIS 



gray to black with silvery pupil; snout and 
midline dorsal markings medium brown; 
dorsal markings extremely poorly defined, 
but three confiuent brown spots apparent 
along dorsal midline extending from poste- 
rior head to sacrum (i.e., typical "hour- 
glass" configuration of Arthroleptis); loreal 
and suborbital regions medium brown form- 
ing near continuous mask; mask broken 
posteriorly by small light gray spots between 
eye and tympanum; continuous dark brown 
supratympanic band extending from poste- 
rior margin of eye, over tympanum, and 
terminating posterior to arm on lateral 
surface of body (Fig. 4B); tympanum light 
brown, translucent, and grading into supra- 
tympanic band; lateral surface of body light 
gray with scattered, irregular unpigmented 
spots; prominent, dark inguinal spot extend- 
ing laterally over ilium and terminating over 
the sacroiliac joint; dorsal surfaces of fore- 
limbs Ught gray; dark brown band on the 
dorsal surfaces of forearm; ultimate inter- 
phalangeal joints unpigmented and light gray 
in color; dorsal surfaces of hind limbs brown 
with irregular light gray spots; anterior 
surface of thigh dark brown with well- 
defined, small, light gray spots; crus with 
prominent transverse dark brown band; feet 
medium to dark brown dorsally with scat- 
tered gray spots; poorly delimited dark 
brown circle centered on cloaca with pair 
of darker brown spots at anterodorsal 
margin. 

Gular region with reticulated pattern of 
small dark brown melanocytes and many 
light unpigmented spots (Fig. 3D); reticulat- 
ed pattern extending on ventral surface of 
proximal forelimb and terminating posteri- 
orly at level of pectoral girdle; venter mostly 
unpigmented and creamy gray; some scat- 
tered small melanocytes forming poorly 
defined reticulated pattern at lateral and 
more posterior margins of ventral surface; 
palmar and plantar surfaces dark brown 



with scattered small unpigmented spots; 
ventral hind limb dark brown with many 
small light gray spots. 

Variation. The paratype is very similar to 
the holotype with the following exceptions: 
margins of the dorsal markings are high- 
lighted in places by thin, dark spots; mask is 
unbroken posteriorly with no small gray 
spots between eye and tympanum; reticula- 
tions on the ventral head and legs are lighter 
and slightly less defined; subarticular tuber- 
cles on the pedal digits are slightly more 
globular. 

Habitat and Natural History. Both speci- 
mens were collected during daytime visual 
surveys (1300-1600 h) when they were active 
in leaf litter adjacent to a trail running 
through the forest. 

Conservation. The sole locality lies in the 
forests of the Nguru Mountains, which have 
an estimated extent of < 300 km- (Burgess et 
al, 2007) and are threatened by forest loss 
and degradation (Menegon et al, 2008). 
Because A. kidogo is only known from these 
forests, this new species should be considered 
tentatively as Vulnerable according to lUCN 
(2001) criteria. 

Etymology. The specific epithet kidogo 
should be treated as an indeclinable word. 
It is the Kiswahili word meaning "very 
small" in recognition that this is among the 
smallest species of Arthroleptis. 

Remarks. The diagnosable color patterns 
of adult A. fichika and A. kidogo are similar 
to those of juveniles of other Arthroleptis 
species in the Eastern Arc Mountains. For 
example, juvenile specimens probably refer- 
able to A. xenodactyloides (MCZ A- 1390 17- 
21) have two prominent features: a dark 
inguinal spot and a dark lateral bar extend- 
ing from the snout tip, over the eye and 
tympanum, and terminating on the posterior 
lateral surface of the body wall, sometimes 
extending nearly into the inguinal region. 
Thus, caution is needed in identifying spec- 



12 



BREVIORA 



No. 517 



imens. Arthroleptis fichika and A. kidogo are 
clearly distinct evolutionary lineages (see 
Phylogenetic Relationships below), and the 
adults are morphologically distinguishable 
from adults of other Arthroleptis in the 
Eastern Arc Mountains. Because the juvenile 
color patterns of these and other larger 
Arthroleptis are both poorly documented 
and likely very similar, molecular data may 
be required to identify small juvenile speci- 
mens. 

Phylogenetic Relationships. The ML esti- 
mate of phylogeny is well supported by both 
nonparametric bootstrap values and Bayes- 
ian posterior probabilities (Fig. 5). Arthro- 
leptis fichika and A. kidogo are divergent 
(mean: 13.0%) and sister species. These two 
new species form a clade sister to a clade 
containing A. xenodactylus, A. xenodacty- 
loides, and A. schubotzi (Fig. 5; Blackburn, 
2008). All of the species in this clade are 
found in the mountains of East Africa; A. 
xenodactyloides and A. xenodactylus are 
sometimes also found at lower elevations 
(Channing and Howell, 2006). Arthroleptis 
xenodactyloides and A. xenodactylus are 
syntopic at some localities (e.g., Amani 
Forest Reserve, East Usambara Mountains, 
Tanzania), but, as found by Blackburn 
(2008), these species are not sister taxa. 
Instead, A. xenodactyloides is the sister to 
A. schubotzi, which is restricted to the 
Albertine Rift Mountains of Burundi, 
Rwanda, Uganda, and eastern Democratic 
Republic of Congo where A. xenodactyloides 
is not known to occur (lUCN, 2008). The 
phylogenetic affinity of A. xenochirus re- 
mains unknown because of a lack of genetic 
resources. 

DISCUSSION 

Because these new taxa are morphologi- 
cally distinguishable and evolutionarily di- 
vergent, recognition of two species is war- 



ranted. Differences in color pattern clearly 
differentiate the type specimens of Arthro- 
leptis fichika and A. kidogo. Furthermore, 
the pairwise divergence between A. fichika 
and A. kidogo is on par with that between 
other species such as A. schubotzi and A. 
xenodactyloides (mean: 16.7%), A. schubotzi 
and A. xenodactylus (mean: 17.4%), and A. 
xenodactyloides and A. xenodactylus (mean: 
12.1%). 

This phylogenetic analysis also included 
an additional gravid female specimen 
(FMNH 251864) collected in a pitfall trap 
in Chome Forest Reserve (04°17'S, 
037°55'40"E [datum unavailable], 2,000 m) 
in the South Pare Mountains, just to the 
northwest of the West Usambara Moun- 
tains. Although morphologically similar to 
A. fichika, the sequence data for FMNH 
251864 demonstrates that the specimen is 
divergent (16.7%) from the two type speci- 
mens. Indeed, if this specimen is considered 
conspecific with A. fichika, then this would 
be the greatest amount of intraspecific 
sequence divergence so far observed for this 
genetic locus in Arthroleptis (Blackburn, 
2008). Thus, this specimen is only tentatively 
designated as A. cf. fichika and clearly could 
represent yet another miniature cryptic 
species. Future field research focused on 
collecting additional specimens is needed to 
evaluate further the taxonomic status of 
miniature Arthroleptis in the poorly studied 
South Pare Mountains. 

The Eastern Arc Mountains of Tanzania 
and Kenya constitute a global hotspot of 
biodiversity (Burgess et al, 2007; Myers et 
al, 2000). The climate of these mountains is 
under direct influence of the climatic regime 
in the Indian Ocean (Marchant et al, 2006) 
and is believed to have been relatively stable 
since the uplift of these mountains approx- 
imately 30 million years ago (Burgess et al , 
2004). It is unclear whether the high levels of 
endemic biodiversity in these mountains are 



2009 



TWO NEW MINIATURE ARTHROLEPTIS 



13 



1.0 



100 



1 A. stenodactylus (CAS 1 68455) 
— A. variabilis (MCZ A-1 36744) 

1.0 
1.0 



1.0 



100 



100 



100 



100 i.o-|sr' 

99l, 



\A. kidogo n. sp. (MCZ A-1 38393) 
I A /f/dogfo n. sp. (MCZ A-1 38394) 

A. cf. ficliika (FMNH 251864) 



1.0 



1.0 



99 



100 



1.0 



0.10 substitutions/site 



1.0 
80 



100 



1.0 



100 



I A. fichika n. sp. (CAS 168829) 
A. fichika n. sp. (MCZ A-1 38384) 
I A. xenodactylus (MCZ A-1 38404) 
\a. xenodactylus (MCZ A-1 38405) 
I A. schubotzi (CAS 201 752) 
\a. schubotzi (CAS 201 753) 
A. xenodactyloides (CAS 1 68608) 
A. xenodactyloides (MCZ A-1 37002) 



Figure 5. Phylogram estimated from mitochondrial DNA sequences depicting relationships of miniature 
Arthroleptis in the Eastern Arc Mountains. Numbers above branches are Bayesian posterior probabilities and below 
branches are nonparametric bootstrap proportions. 



the result of higher speciation rates, lower 
extinction rates, or some combination of 
both (Lovett et al, 2005). Biodiversity 
research has been carried out in these 
mountains for more than a century, but 
both cryptic and surprisingly distinct verte- 
brate taxa continue to be described (e.g., 
Channing and Stanley, 2002; Davenport et 
al, 2006; Fjeldsa et al, 2006). The number of 
new amphibian species recently described 
from the Eastern Arc Mountains is truly 
remarkable, and this trend shows no sign of 
abating (e.g., Channing and Stanley, 2002; 
Loader et al, 2006; Menegon et al, 2004, 
2007; Miiller et al, 2005; Pickersgill, 2007; 
Poynton, 2003b; de Sa et al, 2004). These 
descriptions contribute to making this region 
one of the hotspots of global amphibian 
diversity (Stuart et al, 2004). However, to 
date, molecular phylogenetic study has 
played a relatively small role in describing 



this diversity. Undoubtedly, many more 
cryptic amphibian species are yet to be 
described from East Africa and molecular 
analysis will play an important role in this 
work. 

Recently, Pickersgill (2007) described Ar- 
throleptis stridens from Kambai and Long- 
uza Forest Reserves in the East Usambara 
Mountains. The taxonomic status of A. 
stridens is difficult to evaluate because 
molecular data are unavailable, an audio- 
spectrogram was not published, it is known 
from only one adult specimen (the holotype), 
and morphological characters that differen- 
tiate this species from the very similar A. 
xenodactyloides were not presented. Pickers- 
gill (2007) differentiated A. stridens from A. 
xenodactyloides by its call, but did not 
compare it to the call of A. xenodactylus. 
My study of the holotype of A. stridens 
indicates that, although morphologically sim- 



14 



BREVIORA 



No. 517 



ilar to these two species, A. stridens can be 
differentiated from A. xenodactyloides and A. 
xenodactylus by a greater degree of sexual 
dimorphism. Males of many Arthroleptis 
species have third fingers that are relatively 
longer than those of conspecific females 
(Blackburn, 2009). Notably, A. xenodacty- 
loides and A. xenodactylus are two of the few 
Arthroleptis species with little sexual dimor- 
phism in third finger length (Blackburn, 
2009). The holotype of A. stridens (ZMB 
66249) is a male and has a notably elongate 
third finger (21.8% SVL) compared with A. 
xenodactyloides (mean from type locality, Mt. 
Selinda, Zimbabwe: 16.7% SVL; n = 16) and 
A. xenodactylus (mean from Nguru Moun- 
tains, Tanzania: 12.7% SVL; n = 3). Relative 
male third finger length is much greater in two 
other East African miniature Arthroleptis, A. 
schubotzi (mean from Bwindi Impenetrable 
Forest, Uganda, 30.8%; n = 6) and A. 
xenochirus (mean from Zambezi River 
Source, Zambia, 27.5%; n = 9), but these 
species do not co-occur with A. stridens. 
Additional specimens are needed to confirm 
whether relatively longer male third fingers is 
diagnostic of A. stridens relative to A. 
xenodactylus and A. xenodactyloides. More 
generally, specimens with associated tissue 
samples and recorded calls are critically 
needed in the study of African frog diversity. 
Without these data, African frog systematics 
will continue to be plagued by potentially 
unresolvable taxonomic problems, such as the 
status of Arthroleptis stridens. 

The diversity of miniature Arthroleptis 
species remains poorly described and little 
studied. Because of the morphological simi- 
larities among these species, molecular data 
can be used to recognize species boundaries 
and thus enable better morphological diagno- 
ses. The two new Arthroleptis species de- 
scribed here might be both the smallest 
Arthroleptis species (e.g., Blackburn, 2008; 
Laurent, 1954) and the smallest frog species 



in Tanzania (e.g., Channing and Howell, 
2006). The maximum snout- vent length of 
gravid females of both A. fichika and A. 
kidogo is < 15 mm. Males of either species are 
currently unknown. However, because mean 
male body size is less than that of females for 
all known Arthroleptis, it is probable that 
males, when found, will also be smaller than 
15 mm SVL. It is possible that these species 
have been overlooked previously because their 
small size is similar to that of small juveniles of 
other Arthroleptis. Furthermore, it is likely 
that other undescribed miniature Arthroleptis 
species from East Africa are present in existing 
museum collections but are misidentified as 
juveniles of larger species or confused with 
other miniature species. Maximum and mean 
snout-vent length of A. fichika and A. kidogo 
are clearly less than those of other miniature 
Arthroleptis in East Africa (Table 3; Material 
Examined). Interestingly, northern popula- 
tions of the widespread Arthroleptis xenodac- 
tyloides are larger than southern populations. 
A recent investigation of body size evolution 
in Arthroleptis found that body size has both 
decreased and increased several times across 
the phylogeny (Blackburn, 2008). For in- 
stance, the two largest Arthroleptis species, 
both from the Eastern Arc Mountains {A. 
tanneri, Grandison, 1983; A. nikeae, Poynton, 
2003b), are not closely related, which indicates 
that large body size has been attained at least 
twice independently (Blackburn, 2008). The 
diversity of cryptic miniature and large 
Arthroleptis within East Africa awaits further 
molecular and anatomical study. 

MATERIAL EXAMINED 

Type specimens and reference samples, 
sex, and snout-vent length (in mm) indicated 
in parentheses. 

Arthroleptis schubotzi. Burundi: ZMB 
21774 (holotype; female, 20.4); Uganda: 
CAS 104500-01 (females, 17.2, 19.3), 201700 



2009 



TWO NEW MINIATURE ARTHROLEPTIS 



15 



(female, 18.8), 201717 (male, 19.2), 201718 
(female, 20.9), 201719 (male, 18.5), 201736-39 
(males, 20.0, 19.6, 18.9,21.1). 

Arthroleptis stridens. Tanzania: ZMB 
66249 (holotype; male, 17.9). 

Arthroleptis xenochirus. Angola: BMNH 
1947.2.30.54 (holotype; male, 17.8); Demo- 
cratic Republic of Congo: MCZ A-21794 
(male, 20.8), A-21799 (female, 20.7); Zambia: 
CAS 196614 (male, 17.9), 196617-18 (males, 
19.0, 18.8), 196620-21 (males, 18.1, 15.9), 
196623 (male, 19.0), 196627 (male, 16.9), 
196630 (male, 20.8), 196632 (male, 20.5), 
196638 (female, 20.5), MCZ A-37418 (fe- 
male, 19.8). 

Arthroleptis xenodactyloides. Kenya: NMK 
A/4653/1 (female, 15.5), A/4538 (female, 20.5), 
A/4542 (female, 20.7), A/4540 (female, 20.6), 
A/4653/2 (female, 16.7); Malawi: MCZ A- 
137001 (male, 15.4), A-1 37002 (female, 15.5), 
A-137003 (male, 15.3), A-1 37004 (female, 
16.8), A-137006-13 (females, 18.0, 16.8, 16.4, 

15.8, 15.8, 16.9, 16.7, 14.8), A-137014 (male, 
15.0), A- 1370 15 (female, 16.2), A- 13703^37 
(females, 16.5, 17.6, 15.7, 17.3), A-137074 
(female, 17.0), A-1 37 136-37 (females, 17.4, 
17.3), A-137138-39 (males, 12.0, 13.5), A- 
137140-41 (females, 16.2, 16.0), TMP 84805 
(female, 16.7); Tanzania: CAS 168608 (female, 
23.4), FMNH 251405 (female, 18.2), MCZ A- 
13199 (female, 18.2), A-13210 Guvenile, 14.1), 
A-25403 (female, 19.2), A-25404-05 Guveniles, 

13.9, 12.9), A-138383 (female, 16.6), A- 
138385-92 (females, 19.8, 19.4, 14.9, 20.1, 

20.2, 19.6, 22.2, 20.3); Zimbabwe: A-17038 
(female, 16.3), A-19038 (female, 17.4), A- 
139047 (female, 17.3), A-1 39048^9 (females, 

16.3, 16.3), A-139050 (female, 18.0), A-139051 
(female, 16.8), A-139052 (female, 16.5), A- 
139053 (female, 17.5), A-139054 (male, 16.1), 
A- 139055-56 (female, 18.1, 15.9), A-1 39057 
(female, 16.4), A- 139058 (female, 16.8), A- 
139059 (female, 16.5), A-1 39060-61 (males, 
14.3, 16.5), A-1 39062-63 (females, 16.1, 16.9), 
A-139064 (female, 17.1), A-1 39065 (female. 



17.7), A-1 39066-67 (females, 17.2, 16.9), A- 
23339-50 (males, 14.4, 13.9, 14.4, 14.0, 14.5, 
13.1, 14.5, 13.7, 15.7, 13.6, 14.8, 13.7), TMP 
19101 (female, 18.4), 19104 (female, 19.0). 

Arthroleptis xenodactylus. Tanzania: 
BMNH 1947.2.6.92 (holotype; unknown, 
15.3), MCZ A-138188 (female, 17.5), A- 
13190 (male, 13.8), A- 13 19 1-94 (females, 
18.0, 16.0, 19.6, 15.7), A-13196 (male, 13.6), 
A- 13400 (male, 14.5), A-1 38401 (female, 
17.0), A-138402-03 (males, 14.8, 13.3), A- 
138404-05 (females, 17.4, 16.5), A-138435 
(female, 17.1), A-138437 (female, 19.2), A- 
138429 (female, 18.3). 

ACKNOWLEDGMENTS 

B. Zimkus provided access to field notes 
and D. Mahler provided photographs of 
specimens in life. D. Martins provided a 
translation of the abstract into Kiswahili. J. 
Hanken, D. Kizirian, and C. Schaefer 
provided useful comments on previous ver- 
sions of this manuscript; A. Bauer, E. 
Greenbaum, and S. Loader provided insight- 
ful and helpful peer reviews. B. Clarke 
(Natural History Museum, London), R. 
Drewes and J. Vindum (California Academy 
of Sciences, San Francisco), A. Resetar 
(Field Museum of Natural History, Chicago, 
Illinois), M.-O. Rodel (Museum fiir Natur- 
kunde, Berlin), P. Malonza and G. J. Measey 
(National Museums of Kenya, Nairobi), and 
E. Prendini (Transvaal Museum, Pretoria, 
South Africa) loaned specimens in their care. 
S. Walker (Harvard Map Collection) made 
Figure 1 . Research support during this study 
came from the Department of Organismic 
and Evolutionary Biology (Harvard Univer- 
sity) and NSF grant EF-0334939 (Amphi- 
biaTree) to J. Hanken. 

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