Bonn zoological Bulletin 66 (1): 3-10

April 2017

Barcoding reveals the first record of Lamyctes africanus (Porath, 1871)

in Germany (Chilopoda: Lithobiomorpha)

Peter Decker", Thomas Wesener’, Jorg Spelda’, E. Norman Lindner* & Karin Voigtlander'

' Senckenberg Museum of Natural History Gérlitz, Am Museum 1, D-02826 Gorlitz, Germany

? Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute for Animal Biodiversity,

Center for Taxonomy and Evolutionary Research, Adenauerallee 160, D-53113 Bonn, Germany

> Bavarian State Collection of Zoology, Mtinchhausenstrafe 21, D-81247 Munich, Germany

* Lazarusstrape 34, D-04347 Leipzig, Germany

" peter.decker@senckenberg.de

Abstract. The henicopid centipede Lamyctes africanus (Porath, 1871) is recorded for Germany for the first time. The

species was first discovered by barcoding methods and subsequently confirmed by morphological characters, and spec-

imens were also collected from additional locations. Information about the German sites where L. africanus was found

is given and its mode of dispersal is discussed.

Keywords. Barcode, COI, Arthropoda, alien species, dispersal

INTRODUCTION

The German Barcode of Life project (GBOL) aims to

build up a DNA library of organisms occurring in Ger-

many with a corresponding voucher and DNA collection.

Further goals are to develop DNA barcode applications

with benefits for e. g. species inventory or monitoring pur-

poses. The GBOL project Myriapoda barcoded nearly all

ca. 200 indigenous Diplopoda and Chilopoda species of

Germany (Voigtlander et al. 2011) as well as specimens

and species from other European countries. While in most

cases the molecular phylogeny of the present GBOL da-

ta agrees with the current view of taxonomy, in some

groups, especially in Chilopoda, it points out taxonomic

problems and confusion (Wesener et al. 2015, 2016).

Some centipedes are not indigenous to the fauna of Ger-

many, but could become established as alien species and

disperse naturally or by human influence to synanthrop-

ic or natural habitats; additional species only occur in

greenhouses (Decker et al. 2014). While the centipede

family Lithobiidae is the dominant family of centipedes

of the order Lithobiomorpha in Europe, the family Heni-

copidae is a typical southern hemisphere group, represent-

ed in Europe mainly by a few species of the genus

Lamyctes Meinert, 1868, apart from two species of other

genera (Christian & Szeptycki 2004; Kurochkina 2007).

Currently, five species of Lamyctes are known for Europe.

Lamyctes emarginatus (Newport, 1844) is a subcos-

mopolitan, frequently recorded species from many Euro-

pean countries (Enghoff et al. 2013; Zapparoli 2010),

which is found in anthropogenic as well as natural habi-

Received: 18.04.2016

Accepted: 12.07.2016

tats. It is a typical pioneer species, combining a fast life

cycle, reaching maturity 6 to 12 weeks after hatching, with

parthenogenetic reproduction in Europe (Zerm 1997). In

Central Europe this species is mostly found in floodplains,

disturbed habitats, urban areas and in agricultural land,

with a preference for wet habitats with low vegetation cov-

er (Voigtlander 2005). The taxonomic status of L. emar-

ginatus and its synonymy with L. fulvicornis Meinert,

1868 remains doubtful (Enghoff et al. 2013). The subcos-

mopolitan Lamyctes coeculus (Brolemann, 1889) seems

to be restricted to greenhouses in Europe and is present-

ly known from Denmark, Finland, France, Germany, Great

Britain, Italy and Sweden (Barber 2009; Decker et al.

2014; Edgecombe & Giribet 2003a; Enghoff 1975). On-

ly a few records exist for Lamyctes albipes (Pocock, 1894)

from the Canary Islands (as L. mauriesi in Eason & En-

ghoff 1992), and L. inexpectatus Kurochkina, 2007 from

the European part of Russia (Kurochkina 2007). The lat-

est addition to the European alien species fauna is

Lamyctes africanus (Porath, 1871), found in outdoor habi-

tats as a pioneer species in Denmark (Enghoff et al. 2013),

and recently found in greenhouses in Edinburgh, Great

Britain (Barber, 1992) and Olomouc, Czech Republic

(Danyi & Tuf 2016) as well as in flowerpots from a gar-

den in Arles, France (Jorio 2016). Enghoff et al. (2013)

present a detailed review of L. africanus with a full re-

description and comparison with the similar L. emargina-

tus, as well as a key to all the Lamyctes species in Europe

other than L. inexpectatus.

There have been few barcoding and molecular phylo-

genetic studies on Henicopidae (Edgecombe et al. 2002;

Corresponding editor: B. Huber

4 Peter Decker et al.

Edgecombe & Giribet 2003a, b), and Enghoff et al. (2013)

have found that the only L. africanus specimen sequence

deposited on Genbank in fact represents an undetermined

species. One of the Genbank sequences from the same

original study (Edgecombe et al. 2002; Edgecombe &

Giribet 2003a, b) available for L. emarginatus was genet-

ically almost identical to the Danish L. africanus, distinct

from other L. emarginatus, and impossible to verify be-

cause the voucher specimen from Australia was damaged.

In Germany, L. emarginatus occurs frequently (Voigtlan-

der 2005) and L. coeculus has been found in the green-

houses of the Leipzig Botanical Garden and the Berlin-

Dahlem Botanical Garden (Decker et al. 2014).

The origin and native distribution area of all introduced

Lamyctes species remain doubtful due to the subcos-

mopolitan dispersal, the mostly synanthropic records and

the lack of data of natural habitats from likely areas of ori-

gin such as, for example, South Africa and Australia.

Here, we present the first record of L. africanus from

Germany, as well as a discussion of how the data on Ger-

man and Austrian Lamyctes fits into the worldwide bar-

coding data available for the genus.

MATERIAL AND METHODS

Specimen collection and preparation

Our sample included nine specimens of Lamyctes from

three localities in Germany (see Table 1). This comprises

the two species (L. emarginatus, L. coeculus) previously

known and the first record of L. africanus in Germany.

All specimens are stored as vouchers in 95% undenatured

ethanol, either at the Zoologisches Forschungsmuseum

Alexander Koenig, Bonn, Germany (ZFMK) or the Zo-

ologische Staatssammlung Miinchen, Germany (ZSM)

(see Table 1).

Specimens used in the study were collected by hand and

transferred to vials containing 95% undenatured ethanol

within days of collection. The vials contain an individual

GBOL number with which the specimens can be connect-

ed to the accompanying data. After conservation the spec-

imens were sent to the GBOL facility at the ZFMK. Up-

on arrival, all specimens were photographed (images are

or will be uploaded to BOLD,

http://www.boldsystems.org/), and a tissue sample was re-

moved for DNA extraction. For this specific GBOL sub-

project, DNA extraction was attempted for nine specimens

of Lamyctes (see Table 1). DNA extraction and sequenc-

ing protocols follow those of earlier studies (Wesener et

al. 2015, 2016).

After the first discovery of L. africanus in our GBOL-

Myriapoda project, more material of L. emarginatus from

more than 75 localities in Germany (mostly North Rhine-

Westphalia, Saxony, Saxony-Anhalt and some other fed-

eral states) with >350 specimens, all deposited in the col-

Bonn zoological Bulletin 66 (1): 3—10

lection of Senckenberg Museum of Natural History Gor-

litz (SMNG) and the private collection of N. Lindner (coll.

Lindner), were checked by P. Decker, K. Voigtlander and

N. Lindner for potential confusion with L. africanus us-

ing the redescription and key provided in Enghoff et al.

(2013).

Illustrations

The final phylogenetic tree was edited using Adobe IIlus-

trator CS4. The map was created with ArcGIS 10.

Phylogenetic analysis

Sequences were obtained for nine Lamyctes specimens.

The available sequences of ten further Lamyctes as well

as Henicops maculatus Newport, 1845, H. brevilabiatus

(Ribaut, 1923) and Lithobius forficatus (Linnaeus, 1758)

as outgroups were downloaded from GenBank and added

from previously published datasets (Edgecombe et al.

2002; Edgecombe & Giribet 2003a, b; Enghoff et al.

2013). Because of the uncertain identity of one of the L.

africanus and L. emarginatus sequences downloaded from

Genbank, as noted by other authors (Enghoff et al. 2013),

the specimen names were put in quotation marks.

Sequences were aligned by hand in Bioedit (Hall 1999).

The final dataset included 22 nucleotide sequences with

657 positions. Phylogenetic analyses were conducted in

MEGA6 (Tamura et al. 2013). Modeltest, as implement-

ed in MEGA6 (Tamura et al. 2013), was performed to find

the best fitting maximum likelihood substitution model.

Models with the lowest BIC scores (Bayesian Information

Criterion) are considered to describe the best substitution

pattern. Included codon positions were 1st+2nd+3rd+Non-

coding. Modeltest selected the General Time Reversible

model (Nei & Kumar 2000) as best fitting model. The tree

with the highest log likelihood (-4725.2866) is used here

to infer the genetic distances and evolutionary history of

the analyzed specimens. Initial tree(s) for the heuristic

search were obtained automatically by applying Neighbor-

Join and BioNJ algorithms to a matrix of pairwise dis-

tances estimated using the Maximum Composite Likeli-

hood approach, and then selecting the topology with su-

perior log likelihood value. A discrete Gamma distribu-

tion was used to model evolutionary rate differences

among sites (5 categories (+G, parameter = 0.7010)). The

rate variation model allowed for some sites to be evolu-

tionarily invariable ((+1), 45.2% sites). The bootstrap con-

sensus tree inferred from 1000 replicates (Felsenstein

1985) is taken to represent the evolutionary history of the

analyzed taxa. The tree is drawn to scale, with branch

lengths measured in the number of substitutions per site.

©ZFMK

First record of Lamyctes africanus in Germany 5

Table 1. GBOL numbers, GenBank codes and locality data. GBOL number refers to DNA extraction and BOLD registration.

ZFMK = Zoological Research Museum A. Koenig, Bonn, Germany; ZSM = Zoologische Staatssammlung Miinchen, Germany.

Species GBOL GenBank Voucher Locality

Lamyctes coeculus ZFMK-TIS-1420 KM491571 ZFMK MYR3656 Germany, Saxony, Leipzig

Botanical Garden, greenhouse

Lamyctes coeculus ZFMK-TIS-2519811 KM491619 = ZFMK MYR3807 Germany, Saxony, Leipzig

Botanical Garden, greenhouse

Lamyctes coeculus ZFMK-TIS-2538271 KX442653 ZFMK MYRS5136 Germany, Mecklenburg-Western

Pomerania, Bansin, greenhouse

Lamyctes africanus ZFMK-TIS-2538242 KX442652 ZFMK MYRS137 Germany, Saxony-Anhalt,

Gerwisch

Lamyctes emarginatus ZFMK-DNA-100410134 KX442654 = ZSM-JSP120527-016 Germany, Bavaria, Freising

Lamyctes emarginatus ZFMK-DNA-100410146 KX442655 ZSM-JSP120527-014 Germany, Bavaria, Freising

Lamyctes emarginatus ZFMK-DNA-112780067 KX442656 ZSM-JSP120530-007 Germany, Bavaria, Freising

Lamyctes emarginatus ZFMK-DNA-112780068 KX442657 ZSM-JSP120527-008 Germany, Bavaria, Freising

Lamyctes emarginatus ZFMK-DNA-112780091 KX442658 = ZSM-JSP120527-008 Germany, Bavaria, Freising

Distance analysis

The number of base differences per site between sequences

is shown. The analysis involved 22 nucleotide sequences.

Codon positions included were Ist+2nd+3rd+Noncoding.

All ambiguous positions were removed for each sequence

pair. There were a total of 657 positions in the final dataset.

Evolutionary distance analyses were conducted in MEGA6

(Tamura et al. 2013).

RESULTS

Molecular analysis

Monophyly of Lamyctes is not supported here by the mo-

lecular data (Fig. 1), as Henicops maculatus branches

within Lamyctes and forms a sister clade with L. inermipes

(Silvestri, 1897) with low bootstrap support (43%).

H. brevilabiatus, H. maculatus, L. hellyeri Edgecombe

& Giribet, 2003, L. inermipes and the Lamyctes

“africanus” specimen from Cape Town, South Africa,

form distinct lines.

The L. africanus clade is well supported (100%) and the

sequence of the German specimen from Gerwisch is iden-

tical to the Lamyctes “emarginatus” from a garden in Syd-

ney, Australia, as well as differing only in one base pair

from the Danish L. africanus specimen (Fig. 1).

The monophyly of L. coeculus 1s well supported (100%).

The German specimens from the Leipzig Botanical Gar-

Bonn zoological Bulletin 66 (1): 3-10

den have identical sequences with the specimen from Ar-

gentina and a forest northwest of Sydney, while the spec-

imen from a small greenhouse in Bansin, Germany, dif-

fers in a single base pair from the other specimens.

L. emarginatus forms a well supported branch (100%)

with two lineages, grouping as a German-Canadian clade

in opposition to a purely Canadian clade, both well sup-

ported (100%).

Intraspecific distances vary within L. coeculus

(0-0.3%), L. emarginatus (O-9.6%) and L. africanus

(0—0.8%). Distances between species ranges from 14.5%

(L. “africanus” to L. hellyeri) to 19.3% (L. emarginatus

to L. coeculus), while distances to the outgroup species

H. brevilabiatus, H. maculatus and L. forficatus ranges

from 15.6% to 24.4%.

Records of L. africanus in Germany

In Germany, L. africanus was found at seven localities

(Fig. 2), often sympatrically with L. emarginatus. No

males were found. All specimens fit the descriptions by

Porath (1893, 1894) and the redescriptions by Enghoff et

al. (2013) and Iorio (2016).

Material examined. North Rhine-Westphalia: Trup-

penubungsplatz Haltern-Borkenberge, open sandy dry

meadow with Filago minima, 51.767°N 7.299°E,

17.X1I.2006, 06.V.2007, pitfall trap, leg. K. Hannig (SM-

NG VNRO17096, 017097), 17 females; Ascheberg (West-

©ZFMK

6 Peter Decker et al.

36 |

ayn

24 |

43)

0,1

AF334314.1| L. “africanus” [ZA]

AY 214428.1| L. hellyeri [AU]

AF334316.1| Henicops maculatus [AU]

IAY214425.1| L. inermipes [AR]

AY 214426.1| L. brevilabiatus [AU]

KM491683.1| Lithobius forficatus [DE]

Fig. 1. Maximum likelihood tree under the GTR model, 1000 bootstrap replicates, for Lamyctes. Country of origin given in square

brackets: AR = Argentinia; AU = Australia; CA = Canada; DE = Germany; DK = Denmark; ZA = South Africa. For full data on

all specimens, see Table 1.

falen), railway station, fallow land, 51.783°N 7.601°E,

20.1X.2015, hand collecting, leg. K. Hannig (SMNG

VNRO17286), 2 females; Arnsberg-Bachum, bank of Ruhr

River, gravel, 51.469°N 7.932°E, 15.VIII.2008, hand col-

lecting, leg. K. Hannig (SMNG VNRO17098), 3 females;

Saxony: Leipzig, fallow land beside an abandoned railway

area; 51.342°N 12.388°E; 24.1V.2008, leg. N. Lindner

(coll. Lindner 2009-CL-3603), 1 female; Saxony-Anhalt:

Gerwisch (near Magdeburg), inland dune, 52.187°N

11.723°E, 28.VIII.2014, 28.1X.2014, 01.X.2014, hand col-

lecting (1x), leg. N. Lindner & pitfall trap (2x), leg. For-

der- und Landschaftspflegeverein Biospharenreservat

“Mittelelbe” (FOLV) (coll. Lindner 2014-CL-8325, 2014-

CL-8682, 2014-CL-8683), 4 females; Wartenburg (near

Lutherstadt Wittenberg), orchard meadow within the open

floodplain of the Elbe river, 51.809°N 12.757°E,

30.X.2012, 18.X%.2012, 4.XII.2012, 13.1X.2013,

Bonn zoological Bulletin 66 (1): 3-10

26.1X.2013, hand collection (1x), leg. N. Lindner & pit-

fall trap, leg. FOLV (coll. Lindner 2012-CL-6086, 2016-

CL-6299, 2013-CL-6695, 2014-CL-7311, 2014-CL-

7312), >10 females; Heudeber (near Wernigerode), or-

chard meadow, 51.908°N 10.854°E, 14.VHI.2013, pitfall

trap, leg. FOLV (coll. Lindner 2014-CL-7279), 1 female.

DISCUSSION

Molecular analysis

The L. “emarginatus” specimen from a garden in Sydney,

Australia, branching within L. africanus, is here confirmed

again genetically as L. africanus, showing a 100% iden-

tity with our German L. africanus. Enghoff et al. (2013)

examined the fragment of a specimen (AMS KS5760) by

©ZFMK

First record of Lamyctes africanus in Germany Z

0 2550 + 100 a :

———/§— = }

Kilometer rd eS

Baltic Sea

North Sea

Netherlands Poland

Gerwisch

Wartenburg

' Haltern-Borkenberge

5 © Heudeber

O © Ascheberg

~ Czech Republic

one en

din : ok

;

ve ,

Fig. 2. Records of Lamyctes africanus (grey circles) in Germany.

Bonn zoological Bulletin 66 (1): 3-10 ©ZFMK

8 Peter Decker et al.

mistake as the voucher of DQ201429.1. However, the orig-

inal voucher collected by G. Edgecombe at the same lo-

cality in the Australian Museum Garden on the 3rd No-

vember 2004 (MCZ 131488, DNA103924) was also ex-

amined, but as it was missing legpairs 10 to 15, it could

also not be identified as either L. emarginatus or L.

africanus (G. Edgecombe, pers. comm.). The L.

“africanus” from South Africa also forms a distinct branch

in this study, suggesting a different species. It also differs

from L. emarginatus and L. africanus in morphology (En-

ghoff et al. 2013).

Mysterious origin of Central European Lamyctes

The German L. africanus, because of its identical COI se-

quence, could have been introduced from the same source

population as the specimen from a garden in Sydney. This

species was also recorded from some localities in south-

west Australia (Attems 1911). As the European outdoor

records are scattered along the plain of the northern half

of Germany and equally scattered in Denmark (Enghoff

et al. 2013: Fig. 4) it can be assumed, that the species had

been introduced a much longer time ago and might be

widely distributed now, but until recently has been always

confused with L. emarginatus. This has already been point-

ed out by Enghoff et al. (2013).

It is not clear if the Danish specimens of L. africanus

might originate from a different source or if the slight dif-

ferences in the sequences are caused by errors during se-

quencing. There is still no evidence that this species 1s in-

digenous to Australia.

The L. coeculus from the hothouse in Leipzig are prob-

ably also from the same original source as the ones from

Australia and Argentina. Whether the Australian popula-

tion, collected in a Eucalyptus-Acacia forest in New South

Wales, represents a natural population or one originating

from elsewhere is not clear.

While the two clustering specimens of L. emarginatus

from Canada are from one original source, the German

and another L. emarginatus from Canada probably orig-

inate from the same haplotype. All German samples have

been collected from a single river bank in Freising, Bavaria

(Isar Valley), thus representing a single population. They

need to be checked with more, geographically remote sam-

ples to see if L. emarginatus shows a wider genetic vari-

ability or represents a single haplotype within Germany.

More sampling in natural habitats in Australia could

clarify whether genetic diversity is higher in this area and

whether males occur there, which could provide evidence

that both species are native to Australia. For L. emargina-

tus, specimens from the type localities in Jutland and on

islands in Denmark (L. fulvicornis) and New Zealand (L.

emarginatus) could reveal the origin and status of this sub-

cosmopolitan species.

Bonn zoological Bulletin 66 (1): 3—10

Dispersal not only by train?

In Denmark L. africanus was always recorded at aban-

doned railway areas, implying a dispersal by train. In Ger-

many there have been, up to now, two records from a sim-

ilar habitat — specimens were found in Leipzig at a site

with sparse vegetation near a railway area and in fallow

land at the railway station in Ascheberg.

The record from Gerwisch is from the edge of a dune

near the River Elbe; an additional record is from an or-

chard meadow in Wartenburg, where the River Elster flows

into the Elbe. Both sites have been flooded regularly in

the past, for example, during the last large-scale flooding

in 2013. There were no specimens of L. africanus found

in other material from numerous other eastern German

floodplains investigated, where L. emarginatus has been

recorded, on the River Elbe, River Mulde, River Zwick-

au Mulde, River Freiberger Mulde, River Havel and Riv-

er Lusatian Neisse. Possibly L. africanus has become on-

ly recently established at the site. In addition, L. africanus

was also found in river gravel on the River Ruhr in west-

ern Germany. Hence, dispersal through water and float-

ing material could be a possible source and way for dis-

persal of this species. Also a similar submersion resistance

of eggs in L. africanus to that recorded in L. emargina-

tus (Zerm 1997; Zulka 1992) may be assumed.

Another record of L. africanus is from a sparsely veg-

etated dry meadow in a military training ground. Origi-

nally all specimens were identified in error as L. emar-

ginatus (Decker et al. 2009), but both species co-occur at

this site. The Haltern-Borkenberge Training Area has been

used as a (military) training area and shooting range since

1873 and has been under the command of the British Army

from 1945 to May 2015, but was also used by the Ger-

man, Belgian, and Dutch Armies. The open sandy site with

pioneer vegetation, where L. africanus was recorded, re-

sulted from tracked and wheeled vehicles, mowing, annu-

al vegetation burning and explosive devices (see also

Olthoff et al. 2009; Zimmermann & Feuring 2009). This

training area is not accessible to the public and the most

likely source of introduction of L. africanus could be mil-

itary vehicles.

The record from Heudeber was also from an orchard

meadow, as in Wartenburg, but the site had not been flood-

ed by a river in the past.

L. africanus has not, as yet, been found in very anthro-

pogenic influenced urban habitats such as gardens, parks,

reclaimed mine sites or colliery spoil heaps in Germany.

However, this species prefers open sandy to gravel habi-

tats with sparse vegetation.

Finally, there seem to be several likely ways of disper-

sal at least for L. africanus: by train or along railways, mil-

itary vehicles, and along rivers through flooding. Enghoff

et al (2013) also mentioned a specimen of Lamyctes in a

flowerpot from Annemasse in France, which could resem-

©ZFMK

First record of Lamyctes africanus in Germany 9

ble L. africanus. Recently, L. africanus was actually con-

firmed from a flowerpot in Arles, France (Iorio 2016).

We would predict that the number of records of this

(overlooked) new alien Lamyctes species in Europe or

elsewhere will increase rapidly in the future, as also be-

cause of its current progress of dispersal, especially if it

is dispersed with flowering plants and along railways.

Acknowledgements. For collecting material we would like

to thank K. Hannig and the Forder- und Landschaft-

spflegeverein Biospharenreservat “Mittelelbe” on behalf

of the Landesamt fiir Umweltschutz Sachsen-Anhalt.

M. Geiger, B. Rulik, J. Thormann, and L. von der Mark

form the GBOL-Team in Bonn and photographed, extract-

ed and sequenced the ZFMK specimens; their invaluable

help is greatly appreciated. Sincere thanks to H. Enghoff

for validation of L. africanus SMNG specimens, A. Bar-

ber who kindly gave useful suggestions on the draft man-

uscript, and G. Edgecombe who gave us information on

important voucher specimens used in an earlier study. The

two reviewers, H. Enghoff and G. Edgecombe, provided

useful comments which improved the quality of the arti-

cle:

This is a publication of the German Barcode of Life

(GBOL) project of the Humboldt Ring, financed by the

German Federal Ministry for Education and Research

(FKZ 01LI1101A and FKZ 01L11101B).

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©ZFMK

Bonn zoological Bulletin 66 (1): 11-14

April 2017

A tribute to Marjorie Greenwood, née George (1924—2006),

a neglected mammalian scientist

Paulina D. Jenkins' & Rainer Hutterer’

' Department of Life Sciences, The Natural History Museum, London, UK; p.jenkins@nhm.ac.uk

? Department of Vertebrates, Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, D-53113 Bonn, Germany;

rhutterer@zfmk.de

It happens sometimes that great women in science receive

little credit from their community or even none at all. The

intention of the following notes is to remember the life of

such a great lady, whom we both had the privilege to meet

in person, but who passed away unknown to many other

colleagues ten years ago (Fig. 1). As virtually nothing was

known of her life before (Beolens et al. 2009), these notes

will fill a gap.

Marjorie Greenwood was born on the 10" of August

1924 in Johannesburg, South Africa. She was the third and

youngest child of John and Vittoria George. Her father was

born in England and went to South Africa to fight in the

Boer War. At the end of the war, he decided that he liked

the country, so returned to seek a better life and settled in

South Africa. Here he worked as a lithographer in a local

printers firm. Marjorie’s mother Vittoria Regina George

(née Novelli) was the daughter of emigrants from North-

ern Italy to South Africa. Vittoria’s parents sadly died in

an influenza epidemic, leaving her at the age of eight to

be reared by nuns in a Catholic orphanage.

Marjorie was extremely fond of her brother Arthur and

elder sister, Eileen. Arthur taught her how to make and use

catapults, an activity of which she was later rather

ashamed, and both shared a passion for horses, which they

kept and rode on the smallholding where the family lived.

Eileen was a great support to Marjorie, amongst other

things, teaching her how to sew her own clothes. Eileen

became a hairdresser and emigrated to England in the

1940s, while her brother Arthur followed in his father’s

footsteps and became a printer.

Marjorie was educated at Johannesburg Girls High

School, sited in the grounds of Barnato Park, and hence

the students always referred to the school as “Barnato

Park”. The school was formerly a mansion, commissioned

by Barney Barnato (1852-1897), a diamond mining mil-

lionaire.

Marjorie was the first in her family to go to university.

She studied Zoology at the University of Witwatersrand

in Johannesburg where, after graduating, she had a job as

a ‘demonstrator’ in the Zoology Department. Here Mar-

jorie worked alongside the internationally renowned Aus-

Received: 22.08.2016

Accepted: 24.10.2016

Fig. 1. Marjorie Greenwood (undated photo provided by Nan

Greenwood).

tralian anthropologist, Raymond Dart (1893-1988) and the

South African palaeontologist, James Kitching

(1922-2003). Kitching was appointed by the Bernard Price

Institute for Palaeontological Research of the University

of Witwatersrand to collect fossils and Marjorie immense-

ly enjoyed joining him in many of his fossil-hunting trips

to the Karoo and elsewhere in southern Africa.

Marjorie also attended the Johannesburg Teachers Col-

lege (known by its former name of Normal College) to

train as a teacher. She taught for a short while after com-

pleting the course. It was while working in the Zoology

Corresponding editor: J. Decher

12 Paulina D. Jenkins & Rainer Hutterer

ty p

—— | ow ori

Fig. 2. Marjorie and Humphry Greenwood in front of their home in Uganda, with one of their daughters and her friend in the

background (photo provided by Jennifer Greenwood).

Department of the University of Witwatersrand that she

met her future husband, Peter Humphry Greenwood

(1927-1995) (known by his second name of Humphry),

who later would become a well-known specialist on

African fishes (Howes 1995). Humphry had just returned

from the Second World War, where he served in the Roy-

al Navy in the Far East and, as for all war veterans on their

return to civilian life, he was granted unconditional access

to a university education. As with Marjorie’s father,

Humphry’s parents were British, his father being a min-

ing engineer who worked in the gold mines in Johannes-

burg.

On 10" January 1950 Marjorie married Peter Humphry

Greenwood in Port Shepstone, just south of Durban and

on the south-east coast of South Africa. As Humphry had

obtained a Colonial Office Fisheries Research Stu-

dentship, they travelled to England, where Humphry spent

the first part of his studentship at the British Museum of

Natural History (BMNH) (now the Natural History Mu-

seum, London) studying ichthyology. Their first child,

Pamela, was born in November of that year and a couple

of months later, in January 1951, the small family trav-

elled to Uganda; quite a challenge with such a young ba-

by. Here Humphry had a posting with the East Africa Fish-

eries Research Organization (EAFRO) in Jinja, on the

Bonn zoological Bulletin 66 (1): 11-14

northern shores of Lake Victoria. Marjorie loved living in

Uganda and always spoke very fondly of her time there.

She learned Swahili in order to be able to converse with

local people; another language to add to her English and

Afrikaans. Her second daughter, Jennifer, was born in June

1953 and her third daughter, Nan, in March 1955 (Fig. 2).

During this time in Uganda, in addition to raising her

family, Marjorie studied material collected by Professor

Raymond Dart amongst Australopithecine deposits in the

Makapan Valley, Transvaal. In the resulting paper (Green-

wood 1955) she described a new giant species of fossil

porcupine, Hystrix major, and a new genus and species of

porcupine, Xenohystrix crassidens. The first name was pre-

occupied and subsequently replaced by Hystrix makapa-

nensis (Greenwood 1958), while the other porcupine Xeno-

hystrix crassidens has remained widely accepted (Winkler,

Denys & Avery 2010).

In July 1957 the family left Uganda, travelling by train

to South Africa to join one of the Union Castle liners to

England. Here the family remained for the remainder of

Humphry’s long career as an ichthyologist on the staff at

the Natural History Museum in London (Howes 1995). In

February 1961 their fourth daughter, Philippa, was born.

Not long after the birth of her last child, Marjorie start-

ed work with Professor Percy Butler (1912-2015) of Roy-

©OZFMK

A tribute to Marjorie Greenwood, née George (1924-2006), a neglected mammalian scientist 13

Fig. 3. Jris innominata, water colour by Marjorie Greenwood

1993 (courtesy of Jennifer Greenwood).

al Holloway College, London University (Twigg 2015),

studying the fossil shrews and bats of Olduvai Gorge in

Tanzania (Butler & Greenwood 1965a, b), as well as Pleis-

tocene hedgehogs and elephant-shrews (Butler & Green-

wood 1973, 1976). This working partnership with Profes-

sor Butler was to endure for many more years but with

only a few intermittent publications. She was based in the

Mammal Section of the BMNH, using the Recent collec-

tion of shrews and bats for comparison with the fossil

specimens. Since the bulk of the fossil shrews from Oldu-

vai consisted of mandibular fragments, the work involved

patient, painstaking care in handling, many hours spent

peering through the eyepieces of a microscope, and metic-

ulous note-taking and drawing in order to work out the

available characters to distinguish one species from anoth-

er. Her ink line drawings, which illustrated several of her

and her co-authors’ papers, were of high quality. Such

work exemplified her general attitude to life. In 1966, long

before the publication of their second paper on shrews

(Butler & Greenwood 1979) she evidently sufficiently im-

Bonn zoological Bulletin 66 (1): 11-14

pressed the French taxonomist and shrew-expert Henri

Heim de Balsac (1899-1979) for him to name a shrew,

Crocidura greenwoodi, in her honour (Heim de Balsac

1966). This extant species is endemic to the Horn of Africa

(Hutterer 2008). Their work on the Pleistocene shrews of

Olduvai not only revealed a number of species new to sci-

ence (Table 1), but also had a strong impact on subsequent

research on extant African shrews. Ten years later, they

used the subtle characters elaborated by Marjorie in her

studies of the shrew mandibles to analyse relations among

extant species of Crocidura (Butler et al. 1989).

Marjorie had her own desk in the Visitors Room of the

Mammal Section of the Zoology Department, and it was

here and in the Fossil Mammal Section of Palaeontology

that Marjorie met many of the visiting small mammal re-

searchers. Although basically a shy and retiring person,

she was nevertheless very kind, helpful and encouraging

to students and young researchers. Recognising the apti-

tude and dedication of one vacation student, she encour-

aged him to consider a medical career. Several years lat-

er, after successfully graduating and completing a Mas-

ters’ degree in Zoology, he suddenly announced a change

of course, embarked on an escalated medical degree and

qualified as a doctor.

Humphry’s work entailed visits to many parts of the

world and Marjorie greatly enjoyed accompanying him to

Sweden (for which she attempted to learn Swedish), Hol-

land and Spain, and was much impressed by China and

its people. She also attended palaeontological conferences

and returned to South Africa a number of times to meet

up with friends and family. A dedicated letter-writer, she

sent long, detailed letters back to the family during her

trips abroad and remained in regular contact with old

school and university friends in South Africa and the many

long-term friends that she had met during her travels.

A steadfast homemaker, Marjorie’s family came first

and foremost in her mind. While her shrew and bat work

was a source of great enjoyment to her and she might have

wished to have been a professional zoologist and palaeon-

tologist, she nevertheless devoted herself to providing her

four children with the best possible home-life and support.

She was extremely open-minded, with a huge enthusiasm

for learning and a life-long thirst for knowledge and was

interested in practically everything. She was always keen

to share this enthusiasm and knowledge, encouraging her

children to study hard and to enjoy learning, especially

about the natural world and the environment. A strict veg-

etarian and an inveterate saver and mender, she was ahead

of the times when it came to her concern for the environ-

ment, and the need for waste control and recycling.

Marjorie was a keen gardener, who loved birds and flow-

ers. She was an extremely accomplished water-colourist,

with a special interest in botanical illustration, her

favourite flower being irises, of which she painted many

varieties (Fig. 3). She was a perfectionist with limitless

©OZFMK

14 Paulina D. Jenkins & Rainer Hutterer

Table 1. Taxa of fossil mammals named by M. Greenwood and colleagues.

Lipotyphla

Erinaceus (Atelerix) broomi Butler & Greenwood, 1973: 8 (replacement name for Atelerix major Broom, 1937,

preoccupied by Erinaceus major Pomel, 1858)

Crocidura balsaci Butler & Greenwood, 1979: 366 (Olduvai Gorge)

Suncus varilla meesteri Butler & Greenwood, 1979: 357 (Olduvai Gorge)

Suncus leakeyi Butler & Greenwood, 1979: 363 (Olduvai Gorge)

Sylvisorex olduvaiensis Butler & Greenwood, 1979: 351 (Olduvai Gorge)

Chiroptera

Cardioderma leakeyi Gunnell, Butler, Greenwood & Simmons, 2015: 7 (Olduvai Gorge)

Scotoecus olduvensis Gunnell, Butler, Greenwood & Simmons, 2015: 10 (Olduvai Gorge)

Myzopoda africana Gunnell, Butler, Greenwood & Simmons, 2015: 3 (Olduvai Gorge)

Nycticeinops serengetiensis Gunnell, Butler, Greenwood & Simmons, 2015: 17 (Olduvai Gorge)

Hystricoidea

Hystrix major Greenwood, 1955: 79 (Makapan Valley, limeworks quarry)

Hystrix makapanensis Greenwood, 1958: 365 (replacement name for H. major Greenwood, not Gervais, 1859).

Xenohystrix Greenwood, 1955: 81

Xenohystrix crassidens Greenwood, 1955: 81 (Makapan Valley, limeworks quarry)

patience and determination, coupled with a huge capaci-

ty for observation and an extraordinary attention to detail.

Often her intermittent visits to the museum over several

months would be dedicated to a single specimen.

Lively and active to the end, Marjorie was writing up

her latest research during the last few months of her life.

The final paper to which she and Percy Butler contributed

was published posthumously (Gunnell et al. 2015). She

died in Guildford Hospital on the 4" of March 2006, af-

ter a short bout of pneumonia and was cremated at Put-

ney Vale Crematorium.

Acknowledgements. We are grateful to Pamela, Jennifer, Nan,

and Pippa Greenwood for allowing us to use their information,

memories, photographs and drawings.

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ary of mammals. Baltimore: The Johns Hopkins University

Press

Butler PM, Greenwood M (1965a) Order: Insectivora. Pp. 13—14

in: Leakey LBS (editor), Olduvai Gorge 1951-1961, vol. 1.

A preliminary report on the geology and fauna. Cambridge:

Cambridge University Press

Butler PM, Greenwood M (1965b) Order: Chiroptera. Pp. 14-15

in: Leakey LBS (editor), Olduvai Gorge 1951-1961, vol. 1.

A preliminary report on the geology and fauna. Cambridge:

Cambridge University Press

Butler PM, Greenwood M (1973) The early Pleistocene hedge-

hog from Olduvai, Tanzania. Pp. 7-42 in: Leakey LSB, Sav-

age RJG, Coryndon SC (eds), Fossil Vertebrates of Africa, vol.

3. London and New York: Academic Press

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lididae) from Olduvai and Makapansgat. Pp. 1—56 in: Savage

RJG, Coryndon SC (editors), Fossil Vertebrates of Africa, vol.

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the Early Pleistocene of Olduvai Gorge, Tanzania. Zoologi-

cal Journal of the Linnean Society 67: 329-379

Butler PM, Thorpe RS, Greenwood M (1989) Interspecific re-

lations of African crocidurine shrews (Mammalia: Soricidae)

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cal Journal of the Linnean Society 96: 373-412

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ley, Transvaal. Palaeontologia Africana 3: 77—85

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ley, Transvaal. Hystrix makapanensis nom. nov. for Hystrix

major Greenwood. Annals and Magazine of Natural History

(13) 1 (5): 365

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(Chiroptera) from Olduvai Gorge, Early Pleistocene, Bed 1

(Tanzania). American Museum Novitates (3846): 1-35

Heim de Balsac H (1966) Contribution a I’étude des Soricidae

de Somalie. Monitore Zoologico Italiano 74 (Supplement):

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ple/obituaries-humphry-greenwood-1610994.html

Hutterer R (2008) Crocidura greenwoodi. The IUCN Red List

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©OZFMK

Bonn zoological Bulletin 66 (1): 15—28

April 2017

Notes on hemipenial morphology and its phylogenetic implications

in the Pygopodidae Boulenger, 1884

Ian G. Brennan’’ & Aaron M. Bauer'

‘Department of Biology, Villanova University, 800 Lancaster Avenue, Villanova, Pennsylvania 19085, USA;

?Current address: Division of Evolution, Ecology & Genetics, Research School of Biology, The Australian National University,

Canberra, ACT 2602, Australia; corresponding author’s E-mail: Ian.Brennan@anu.edu.au

Abstract. The Pygopodidae is a near-endemic Australian family of limb-reduced, imbricately scaled gekkotans, current-

ly comprising 44 species (43 in Australia, 2 in New Guinea). Phylogenetic studies of the Pygopodidae have enlisted mo-

lecular, morphometric, and osteological methods to resolve intergeneric and interspecific relationships, however, only

one publication details the morphology of pygopodid hemipenes, providing data on four species from three genera. Here,

we augment these initial observations, by describing hemipenial condition in an additional 19 species across four gen-

era, bringing current totals to 23 pygopodid species across five of seven recognized genera. Focused sampling of the genus

Delma allows us to identify species groups based on hemipenial shape and ornamentation, and general morphology of

cloacal spurs. We identify the presence of a single-lobed hemipenis in both Aprasia and Delma, and hypothesize this mod-

ification in reproductive anatomy has occurred twice independently in pygopodid evolution.

Key words. Gekkota, Australia, hemipenes, systematics, cloacal spur, morphology.

INTRODUCTION

Diversity within a Divergent Lineage

Near-limbless geckos of the Family Pygopodidae repre-

sent a unique radiation in Australian biogeographic his-

tory. The snake-like pygopodids are characterized by an

absence of forelimbs, imbricate body scales, and reduc-

tion of hindlimbs (Cogger 2014; Wilson & Swan 2013).

Current taxonomy recognizes 44 species across seven gen-

era; Aprasia Gray 1839 (14 spp.), Delma Gray 1831 (21

spp.), Lialis Gray 1835 (2 spp.), Ophidiocephalus Lucas

& Frost 1897 (1 sp.), Paradelma Kinghorn 1926 (1 sp.),

Pletholax Cope 1864 (1 sp.), and Pygopus Merrem 1820

(6 spp.). Although limb reduction is not a novel adapta-

tion in squamate evolutionary history (Anniellidae, An-

guidae, Cordylidae, Dibamidae, Gymnopthalmidae, am-

phisbaenids and snakes), pygopodids are perhaps the most

ecologically diverse limbless squamates exclusive of

snakes (Gamble et al. 2015; Wiens et al. 2006). Current

understanding of the ecology and phylogenetics (Brennan

et al. 2016; Jennings et al. 2003; Oliver & Sanders 2009)

suggests the Pygopodidae comprises: two independent

burrowing lineages — Aprasia and Ophidiocephalus;

shrub-swimmers — Delma concinna, Pletholax; squamate-

specialist ambush predators — Lialis; a morphologically

conservative genus of arthropod generalists — Delma;

arachnid-specialists — Pygopus; and an insectivore with

nectivorous habits — Paradelma.

Received: 18.11.2015

Accepted: 18.12.2016

Despite morphometric, molecular, and osteological as-

sessments of the Pygopodidae, intergeneric and many in-

terspecific relationships remain poorly understood

(Hutchinson 1997; Jennings et al. 2003; Kluge 1974;

Kluge 1976; Maryan et al. 2007; Oliver et al. 2010; Shea

1987; Shea 1991; Underwood 1957), and no phylogenet-

ic study has included hemipenial characters. B6hme’s

(1988) description of hemipenes across squamate lineag-

es remains an invaluable contribution to the understand-

ing of the hemipenial condition, but with coverage of just

four pygopodid species across three genera, it remains too

incomplete for any phylogenetic use. Here, we present a

more inclusive look at the hemipenial morphology of the

Family Pygopodidae as a whole, with heavy sampling for

the genus Delma, and aim to elucidate the synapomorphies

shared by monophyletic groups within the family. The ad-

dition of Bohme’s descriptions to our dataset proved ex-

tremely valuable, contributing observations on species

which were inaccessible for this study.

Monophyly of the Pygopodidae and Previous

Phylogenetic Study

Externally, pygopodids differ substantially from the ple-

siomorphic gekkotan body plan, however, evidence for the

close relationship between these groups has been recog-

nized and supported for a considerable period of time

(Boulenger 1885; Greer 1989; McDowell & Bogert 1954;

Corresponding editor: Ph. Wagner

16 Ian G. Brennan & Aaron M. Bauer

Miller 1966; Shute & Bellairs 1953; Underwood 1957;

Wever 1974). Recent comprehensive morphological as-

sessment (Gauthier et al. 2012) correctly affiliated the Py-

gopodidae with the Gekkota, the only limbless squamate

lineage to be accurately phylogenetically placed. Molec-

ular studies have also solidified the position of pygopo-

dids within the Gekkota (Feng et al. 2007), as well as with-

in the Pygopodoidea (Gamble et al. 2012; Oliver &

Sanders 2009), validating Bohme’s (1988) preliminary

hemipenial assessment of the Pygopodidae. While recent

morphological (Daza & Bauer 2012) and molecular stud-

ies have elucidated the sister relationship between pygopo-

dids and carphodactylids, intergeneric relationships with-

in the Pygopodidae have varied greatly, with no single

topology receiving overwhelming support (Daza & Bauer

2012; Jackman et al. 2008; Jennings et al. 2003; Oliver

& Bauer 2011; Oliver & Sanders 2009). Morphological

methods for phylogenetic analysis have also failed to cre-

ate a single, favoured tree, and have conflicted strongly

with molecular trees. As a result of the great phenotypic

diversification between pygopodid genera, and conser-

vatism within genera, previously assessed morphological

characters are largely uninformative at the intergeneric lev-

el, and highly susceptible to homoplasy at the interspecif-

ic level (Kluge 1974; Kluge 1976; Kluge 1987). Addition-

ally, current estimates of interspecific (Jennings et al.

2003) and intergeneric (Gamble et al. 2015) relationships

within the Pygopodidae are largely influenced by poten-

tially misleading mitochondrial data (Brennan et al. 2016).

Molecular phylogenetic views within this paper follow the

results of Brennan (Brennan et al. 2016), whilst taking in-

to account the systematic results of previous pygopodid,

pygopodoidean, and gekkotan research.

Hemipenial Morphology and Systematics

Morphological characters of male squamate intromittent

organs provide sufficient systematic characteristics for in-

ferring phylogenetic relationships (Arnold 1986a; BOhme

1988; Branch 1982; Branch 1986; Kohler et al. 2012). His-

torically, numerous studies have discussed and relied up-

on the phylogenetic signal of hemipenial morphology, be-

ginning with Cope (1896) (Arnold 1986a; Arnold 1986b;

Bohme 1971; Branch 1982; Branch 1986; Cope 1895;

Dowling 1967; Dowling & Savage 1960; Keogh 1999;

Klaver & Bohme 1986; Kohler et al. 2012; McCann 1946).

Distinct morphological characters and ornamentation, pre-

sumed low intraspecific variation, and the rapid evolution-

ary trend of male genital morphology in relation to other

morphological characters, make the study of hemipenial

morphology a particularly valuable tool for systematists

(Bohme 1988; Eberhard 1985; Keogh 1999; Kohler et al.

2012). Copulatory organs are diverse in their morpholo-

gy, with various characters which can be described, count-

Bonn zoological Bulletin 66 (1): 15—28

ed, and scored, including size, shape, and ornamentation

(Dowling & Savage 1960; Keogh 1999). Despite lack of

resolution at deeper taxonomic levels, comparative phy-

logenetic study of the hemipenes is a great tool for spe-

cific and generic levels due to extremely low intraspecif-

ic variation. Ontogenetic and seasonal variation tied to re-

productive activity has been recorded from some lacertids,

iguanids, and chameleons (Bohme 1988), and more recent-

ly in the gecko genus Uroplatus (Glaw et al. 2006). How-

ever, ontogenetic change generally influences size, and not

shape or ornamentation. While various other morpholog-

ical characters or systems may be artificially influenced

by homoplasy via factors of natural history; ecology, di-

et, or locomotion, hemipenial morphology appears dis-

tanced from these pitfalls (Arnold 1986b; BOhme 1971;

Bohme 1988; Branch 1986; Dowling 1967; Keogh 1999;

Klaver & Bohme 1986).

Admittedly, intraspecific differences in hemipenial mor-

phology have been identified among several snake taxa:

Calamaria lumbricoidea (Inger & Marx 1962), Candoia

(McDowell 1979), Oxybelis aenueus (Keiser 1974), and

Siphlophis (Zaher & Prudente 1999). Perhaps due to a

dearth of studies of this kind, no published research has

included molecular phylogenetics to support instances of

intraspecific hemipenial variation. In the first three of

these, variation is strongly associated with geographic dis-

tribution, and as such, may be the result of independent-

ly evolving lineages. In fact, instances of proposed in-

traspecific hemipenial variation within Jphisa (Nunes et

al. 2012) actually highlight the value of molecular phylo-

genetics to address cryptic speciation and accurately iden-

tify species diversity.

Hemipenial diagnosis within the Gekkota began with

Cope (1896), in the description of hemipenes from five

currently recognized genera — Cyrtodactylus, Coleonyx,

Phyllodactylus, Tarentola, and Thecadactylus. Since then,

several studies have included observations and assess-

ments of gekkotan hemipenes with phylogenetic implica-

tions. Standardly, gekkotan intromittent organs are bi-

lobed, and often asymmetrical, with varying arrays of or-

namentation. The New World sphaerodactylid genus Aris-

telliger, possesses a baculum-like structure, which is

unique among gekkotans, described and illustrated first

by Kluge (1982), as a spiny ossification with a serrated

edge, which extends distally from the apex of each lobe

(Rosler & Béhme 2006). Exclusive of Bohme (1988),

however, comparative descriptions of the genital morphol-

ogy of pygopodids are lacking. Additionally, cloacal spurs,

and post-cloacal bones and sacs are often associated with

reproductive morphology, and their presence in gekkotans

was reviewed extensively by Kluge (1982), and has been

sporadically commented upon (Bastinck 1986; Russell

1977; Russell & Rosenberg 1981).

In his description of Aprasia, Delma, and Pygopus gen-

itals, BOhme interprets the position and state of calyces

©OZFMK

Pygopodidae hemipenial morphology 17

of Pygopus as a plesiomorphic character, supporting

Kluge’s (1974, 1976) placement of this genus as the basal-

most member of the family. Using Kluge’s (1976) subfa-

milial groups, the Pygopodinae (De/ma and Pygopus) and

Lialisinae (Aprasia, Lialis, Ophidiocephalus, Pletholax),

Bohme identifies conflict within this ranking by way of

similarity in ornamentation — or lack thereof — in Apra-

sia and Delma. He suggests the nude nature of Aprasia

and Delma hemipenes may reflect a close evolutionary his-

tory, or instead be the result of an ornamental-reversal, or

hemipenial simplification, as also seen in Brookesia

(Klaver & Bohme 1986). Current understanding of inter-

generic relationships based on molecular results howev-

er refutes a sister taxa relationship between Aprasia and

Delma, and phylogenetic conclusions regarding intergener-

ic relationships within the Pygopodidae by Bohme (1988)

should be viewed as an artifact of insufficient sampling.

Despite deep external morphological divergence from a

tetrapodal ancestor, Bohme does identify pygopodid

hemipenes as distinctly gekkotan. In order to continue in

the same vein as Bohme’s work, here we diagnose and de-

scribe the hemipenial morphology of a number of Aus-

tralian pygopodids, particularly of the genus Delma,

adding to current documentation of pygopodid hemipenes.

Images, descriptions, and preparation of these organs may

aid in future phylogenetic assessments of the Pygopodi-

dae and future work in this area will provide additional

discernible characteristics for recognizing, delimiting, and

describing new species.

MATERIALS & METHODS

Hemipenial Preparation

Intromittent reproductive organs have independently aris-

en in a number of vertebrate groups — Ascaphus frogs,

crocodilians, chelonians, mammals, chondrichthyes —

however paired inverted hemipenes represent a synapo-

morphy of squamate reptiles (Greer 1989). Hemipenes

themselves are paired tubular organs, which when not in

use are retracted and stored within the body in “inside-

out” fashion (Dowling & Savage 1960). When tucked in-

side the body, they are inverted like a glove, and upon stim-

ulation, are engorged and pushed outside of the body —

the inside in the stored state becoming the outer wall up-

on eversion. Upon being everted, hemipenes may protrude

laterally, anteriorly, or posteriorly from the cloaca, and may

be decorated with a number of ornaments including ca-

lyces (calyculi), flounces, spikes, hooks, and lobes. We

generally follow terminology as proposed by Dowling &

Savage (1960), however refer to Keogh (1999) for mod-

erate adjustments. Accurate morphological hemipenial as-

sessment — particularly of apical lobes — requires a fully

everted hemipenis, either preserved in situ, or manually

Bonn zoological Bulletin 66 (1): 15—28

everted as described by Pesantes (1994). We acquired spec-

imens on loan from the Western Australian Museum (Table

1) with completely everted organs, and only in the absence

of such specimens, did we select individuals with incom-

pletely everted hemipenes. We abstained from destructive

practices by excluding species for which entirely or par-

tially everted organs could not be obtained. Incomplete-

ly everted hemipenes were injected to capacity with 1%

KOH solution and left for one hour to soften tissue for

manual eversion. After an hour, KOH was removed from

organs, and liquid 1.5% agarose gel dyed with alizarin red

was injected via 30 gauge hypodermic syringe, to com-

plete volume. Dyed agarose gel set inside the hemipenes

instantly, and red coloration allowed for increased contrast

of structures, aiding observation and imaging.

Morphology of pygopodid hemipenes is severely re-

duced in complexity as well as physical size, when com-

pared to other squamate taxa (Arnold 1986a; Branch 1986;

Dowling 1967; Hoskin 2011; Kohler et al. 2012). Calcare-

ous spines, spicules, calyces, and flounces present in oth-

er squamates appear absent in sampled pygopodids, mak-

ing staining structures difficult, and imaging of pygopo-

did hemipenes a challenge. As a result, we have reduced

the number of characters addressed by Keogh (1998), and

provide brief overall descriptions of organs of each species.

Characters and Character States

Shape: Single (S), bi-lobed symmetrical (BS), bi-lobed

asymmetrical (BA), or bi-lobed with additional medi-

an lobe (T).

Ornamentation: Nude (N), undifferentiated (UD) orna-

mentation is homogeneous and uniform over the entire

surface of the hemipenis (e.g., spines only). Differenti-

ated (D) ornamentation is defined as two or more dif-

fering types of ornaments (spines and micro-ornamen-

tation).

Base: Ornamented (O) or nude (N).

Terminal sulcus: Sulcus spermaticus terminates at apex of

lobe (TSA), at lateral edge of lobe (TSL), or medially

as a Sulcal pad (TSP). In asymmetrical conditions, the

smaller lobe is listed first/followed by the larger lobe.

Spurs: General shape of the spur is rounded (R), pointed

(P), flattened (F) or terminates in comb-like projections

(C). Direction in which the spur projects is either pos-

teriorly (P), dorsally (D) or posteriodorsally (U). A

rounded spur which points dorsally would be marked

RD.

Lack of intraspecific variation in hemipenial morphol-

ogy across species sampled suggests the number of indi-

viduals examined should provide sufficient for our inter-

est (Arnold 1986a; BOhme 1988; Keogh 1999). The rel-

atively limited breadth of this study (22 spp., 5 genera)

©ZFMK

Ian G. Brennan & Aaron M. Bauer

Table 1. Taxa, locality data, and sources of specimens examined in this study. States refers to Australian states.

*UMMZ = University of Michigan Museum of Zoology; WAM = Western Australian Museum; ZFMK = Zoologisches Forschungs-

museum A. Koenig. Specimens for which coordinates are given as NA are those examined in the earlier study of Bohme (1988).

Taxon Collection* State Locality Latitude Longitude

Aprasia haroldi WAM R163615 WA Dirk Hartog Island 25°41°60"S 113°0’°0”"E

Aprasia parapulchella WAM R144181 WA 4 km S Collie 33°22°0325 116°13’49"E

Aprasia parapulchella = WAM R153920 WA Bindoon Military Training Area 31°20°31"S M6" 5? 3978

Aprasia pulchella UMMZ 131241 WA Canning Dam NA NA

Aprasia repens WAM R144255 WA Burns Beach 31°43°04’S 115°45’59"E

Aprasia repens WAM R153978 WA Bindoon Military Training Area 31°11°42”S 116°18’26”"E

Aprasia smithi WAM R120652 WA 8 km NW Mardathuna Homestead 24°25°44”S 114°30’00”E

Delma australis WAM R112667 WA Ponier Rock 32°56’00"S 123°30’00”E

Delma australis WAM R116276 WA Kalbarri 27°51°00"S 114°10’00”E

Delma australis WAM R135108 WA Bullabulling 30°51°59"S 120°54’24”°E

Delma australis WAM R140395 WA 90 km NE Wubin 29°31°23”S 117°10°10”E

Delma borea WAM R154148 WA Barrow Island 20°47°18”S 115°27°43”E

Delma borea WAM R158010 WA Koolan Island 16°08°45”S 123°44°57”°E

Delma butleri WAM R120322 WA 7 km E Cape Cuvier 24°13’26"S 113°27°41”"E

Delma butleri WAM R120819 WA 25 km SSE Peron Homestead 26°03’00"S 113°37°00"E

Delma butleri WAM R123911 WA Bulong 30°45’00”S 121°48’00”E

Delma desmosa WAM R134414 WA Lake MacKay 22°26°47"S 128°17°33”E

Delma elegans WAM R135462 WA Mount Brockman 22°28°00"S 117°18°00”"E

Delma fraseri WAM R115138 WA Ken Hearst Park 32°04’60"S 15°52 60 E

Delma fraseri WAM R135503 WA Redcliffe, Perth Suburb 31°56’00”S 115°57°00”"E

Delma fraseri WAM R141191 WA 15 Km NNE Lancelin 30°59°31"S 115°23°43”E

Delma fraseri WAM R154039 WA Muchea Air Weapons Range 31°38°16"S 115°55’°31°E

Delma grayii WAM R154364 WA Hindmarsh Nature Reserve 31°17°00"S 117°02’00”E

Delma grayii WAM R156220 WA Ballajura 31°S1711"S 115°5S’11"E

Delma haroldi WAM R138951 WA West Angelas 23°11°42”S 118°36’54”E

Delma haroldi WAM R154831 WA Goldsworthy, Shay Gap Road 20°25’45”S 1 ea ag

Delma hebesa WAM R144236 WA Bandalup Hill 33°40°29”S 120°23’54”"E

Delma hebesa WAM R172285 WA Scaddan 33°26727°S 121°43°17"E

Delma inornata UMMZ 131156 VIC Numurkah NA NA

Delma inornata UMMZ 131186 NSW Finley NA NA

Delma nasuta WAM R154288 WA Fortescue Marsh 21°48°09"S 118°54’39”"E

Delma nasuta WAM R157568 WA Robe River 21°40’26"S 11S 255221 7H

Delma pax WAM R135337 WA Cape Lambert 20°48°36"S 116°56’°31°E

Delma pax WAM R166212 WA Mount Whaleback 23°19°41S 120°01’07”"E

Delma petersoni WAM R165873 WA Queen Victoria Spring 29°19711"S 124°31’28”"E

Delma petersoni WAM R165874 WA Queen Victoria Spring 29ST OLS 124°31°28”"E

Delma tealei WAM R153811 WA Cape Range National Park 22°03’49”S 114°00°42”E

Delma tincta WAM 135487 WA Urala Station 21°46’58”S 114°52°11"E

Delma tincta WAM 141584 WA 1 km N Quobba Homestead 24°22°24"S 113°24’°19”"E

Delma tincta WAM 146589 WA 228 km SSW Port Hedland 22°20°24S 119°00’00”E

Lialis burtonis WAM 110652 WA Shay Gap Road, Goldsworthy 21°43°00"S 122°14’00”"E

Lialis burtonis WAM 154003 WA Muchea Air Weapons Range 31°38732”S 115985203"

Lialis burtonis WAM 154007 WA Muchea Air Weapons Range 31°38732”S LES255° 0325

Pletholax g. gracilis WAM 106172 WA Marangaroo 31°48’00”S 115°50°00”"E

Pletholax g. gracilis WAM 137463 WA Cervantes 30°45’03”S LIS 27117

Pygopus lepidopodus ZFMK 21290 NSW Sydney NA NA

Pygopus nigriceps UMMZ 131174 SA Innaminka NA NA

Bonn zoological Bulletin 66 (1): 15—28

©OZFMK

Pygopodidae hemipenial morphology 19

Table 2. Summary of hemipenial characters of pygopodid geckos examined. *denote taxa examined by BOhme (1988).

Symbols as follows: S=Single-lobed, BA=Bi-lobed asymmetrical, BS=Bi-lobed symmetrical, UD=Undifferentiated ornamentation,

D=Differentiated ornamentation, N=Nude of ornamentation, TSA=Sulcus terminates at apex of lobe, TSL=Sulcus terminates at

lateral edge of lobe, TSP=Sulcus terminates medially into broad sulcal pad. See materials and methods for descriptions of charac-

ters, and Table 1 for material examined.

Taxon No. Shape Ornamentation Base Terminal Length SVL Spurs

Sulcus (mm) (mm)

Aprasia haroldi 1 S UD N TSA 2.58 106 —

Aprasia parapulchella 1 BA UD N TSA/TSP 1.82—2.08 140 —

“Aprasia pulchella 1 BA UD — TSA — 120 —

Aprasia repens 2 S UD O TSA/TSP 3.41-4.28 126 —

Aprasia smithi 1 S D N TSA/TSP S61 128 —

Delma australis = S UD N TSP 4.33—5.73 88 FU

Delma borea 2 BA UD N TSP/TSA 4.38—-5.95 95 RP

Delma butleri é} BS UD N TSL/TSP 5.48-8.59 96 CP

Delma desmosa | BA UD N TSL/TSA 4.96 90 RP

Delma elegans 1 BA UD N TSL/TSP 5.5 97 RP

Delma fraseri 2 BA N N TSL/TSP 5.57-6.57 128 PU

Delma grayii 2 BS UD N TSL/TSP 5.62—7.86 121 PP.

Delma haroldi 1 BA UD N TSL/TSP 3.83-4.22 75 CP

Delma hebesa 2 S UD N TSP 3.58-3.9 85 FU

“Delma inornata 3 BA UD N — — 133 —

Delma nasuta 2 BA UD N TSL/TSA 4.28—-5.38 Dy CP

Delma pax 2 BA UD N TSL/TSA 5.55-5.96 98 RP

Delma petersoni 2 BS N N TSL/TSP 6.06—7.68 128 PU

Delma tincta 3 BA UD N TSL/TSA 3.36-4.19 92 RP

Lialis burtonis + B- D-micro and calyces N — — 290 —

Pletholax gracilis 2 BA D-micro and spines N TSA 4.07-4.88 90 —

“Pygopus lepidopodus | tT D-micro and calyces O TSA ~— 274 =

“Pygopus nigriceps 1 ila D-micro and calyces O TSA — —

allows us to describe each species independently. Close-

ly related species, or those similar in hemipenial morphol-

ogy may cross reference one another. Several species

(Aprasia parapulchella, Delma pax, Lialis burtonis), are

included despite incompletely everted hemipenes. These

examples were not everted when fixed, and could not be

manually everted, however still provide some phylogenet-

ic utility. Characters which could not be scored for these

taxa (apical characters and ornamentation) are symbolized

by “—” in Table 2.

RESULTS

Hemipenial Descriptions

*Denotes taxa examined and described by BOhme (1988)

Aprasia haroldi Storr, 1978

Fully everted; extremely minute, single lobe covered en-

tirely in micro-ornamental stippling except for the sulcus.

Bonn zoological Bulletin 66 (1): 15—28

Base nude until constriction at bottom of lobe, sulcus

broad and shallow. Hemipenis boxing-glove shaped,

highly asymmetrical, proximal lobe miniaturized in rela-

tion to distal lobe.

Aprasia parapulchella Kluge, 1974

Incompletely everted; bi-lobed. Sulcus narrow at base, un-

til constriction at base of fork, sulcus becomes broad and

shallow along lengths of lobes. Light micro-ornamenta-

tion (stippling) of asulcal side, with sulcus nude.

Aprasia pulchella Gray, 1839*

Fully everted; bi-lobed. Asymmetrical, inner lobe (as pre-

pared in-situ) larger. Sulcus spermaticus forked, with much

longer branch on inner lobe. Little ornamentation can be

observed beside the presence of a pustular epithelium, and

absence of calyx bearing surfaces.

Aprasia repens (Fry, 1914) (Figs 1—1a, b)

Fully everted; single lobe. Asulcal and lateral faces com-

©OZFMK

20 Ian G. Brennan & Aaron M. Bauer

Fig. 1. Asulcal (a) and sulcal (b) views hemipenes of eight species of pygopodids: 1) Aprasia repens WAMR144255; 2) Delma

australis WAMR112667; 3) Delma elegans WAMR135462; 4) Delma haroldi; 5) Delma nasuta WAMR154288; 6) Delma peter-

soni WAMR165873; 7) Delma pax WAMR135337; 8) Pletholax gracilis WAMR106172.

pletely covered with micro-ornamental stippling, includ-

ing base. Slipper-shaped hemipenis, recurving posterior-

ly, similar in jai alai basket, as A. smithi. Sulcus narrow

and deep until reaching lobe, becomes deep and wide,

transitioning into sulcal pad, pad clearly surrounded by

deep suclal lips. Distinct nub at posterior facing edge of

asulcal face, potential remnant of secondary lobe.

Aprasia smithi Storr, 1970

Fully everted; single lobe. Hemipenis is long, thin, and

curves dorsally and against body wall after exiting cloa-

ca, perhaps an artifact of the eversion process. Stippled

with micro-ornamentation laterally, with stronger spines

along asulcal ridge. The sulcus is wide and shallow, and

Bonn zoological Bulletin 66 (1): 15—28

reaches apex. Proportions and shape cause the hemipenis

to resemble a jai alai basket.

Delma australis Kluge, 1974 (Figs 1—2a, b)

Fully everted; single lobe. Boxing glove shape, extends lat-

erally from cloaca and curls back towards midline. Both

sulcate and asulcate surfaces covered in fine micro-orna-

mentation, with the exclusion of broad, shallow sulcus

which does not reach apex.

Delma borea Kluge, 1974

Fully everted; bi-lobed. Strongly asymmetrical,

posterior/ventral lobe elongate with truncate, nude apex,

which is reached by sulcus. Dorsal/anterior lobe hammer

©ZFMK

Pygopodidae hemipenial morphology 21

shaped, with sulcus reaching lateral face before opening

onto sulcal pad. Base and lower quarter of lobes nude, but

finely micro-ornamented across asulcal face. Terminus of

longer lobe ends in flat disc, with sulcal lips strongly fold-

ing over, nearly closing over sulcus.

Delma butleri Storr, 1987

Presentation of D. butleri includes individuals from two

geographically isolated populations suggested to represent

cryptic species. Variation in hemipenial morphology be-

tween these two groups warrants further molecular study.

WAM 120322, WAM 120819 — Fully everted; bi-lobed.

Lobes appear approximately equal in size, shallowly

forked compared to other delmas, disctinctly Y-shaped in

comparison to T shape of D. butleri WAM 123911. Base

nude until just prior to cleft, with micro-ornamentation

covering asulcal face, becoming stronger laterally. Sulcus,

buffered by deep sulcal lips, extends laterally beyond split

of lobes, opening onto sulcal pad, facing dorsally.

WAM 123911 — Fully everted; bi-lobed. T split between

lobes, with apex of lobes terminating more in points than

the lobular end of above D. butleri samples. Sulcus is deep

and narrow, forks at cleft of lobes, and extends laterally.

Medial lateral faces of lobes (closest to one another) nude.

Sulcus terminates laterally, opening out onto nude sulcal

pad.

Delma desmosa Maryan, Aplin & Adams, 2007

Fully everted; bi-lobed and highly asymmetrical, similar

in general shape to D. tincta, D. elegans, D. pax, and D.

borea. Anterior/dorsal lobe much shorter, with hammer-

head shape. Posterior/ventral lobe elongate, with truncate,

nude tip, and strongly folded sulcal lips. Both sulcal and

asulcal faces are ornamented.

Delma elegans Kluge, 1974 (Figs 1—3a, b)

Fully everted; bi-lobed. Asulcal face strongly stippled and

micro-ornamented, but restricted to the lobes. Base nude,

and lateral sides of lobes stippled. Anterior lobe much

smaller and broader, but unlike the hammerhead shape of

D. pax. Sulcus very deep and narrow on both lobes, ex-

tending laterally away from midline and not directly to-

wards apex, becoming shallow at most distal edge of sul-

cal pad;sulcal pad nude.

Delma fraseri Gray, 1831

Fully everted; bi-lobed. Smaller lobe approximately one-

third size of larger, however similar in shape, broad and

spatulate, clover leaf shaped. Entire hemipenis bare, no

strong stippling as in others. Sulcus narrow and deep, un-

til reaching cleft of lobes, then becomes extremely shal-

low channel which diverts laterally away from the mid-

line, and opens up into a shallow pad-like surface on each

lobe.

Bonn zoological Bulletin 66 (1): 15—28

Delma grayii Smith, 1849

Fully everted; bi-lobed. Both lobes approximately same

size. Asulcal side only lightly stippled, following around

to lateral edges. Sulcus deep and narrow, deepest at cleft

between lobes, and becomes extremely narrow and chan-

nel-like as bifurcates and diverts away from midline. Lobe-

sulcus-channel opens up onto broad, flat, pad covering

most of sulcal side of lobe.

Delma haroldi Storr, 1987 (Figs 1—4a, b)

Fully everted; bi-lobed. Slightly asymmetrical, with pos-

terior lobe smaller, but of similar overall shape. Sulcus is

deep and narrow, and terminates laterally on each lobe,

after splitting at lobe-fork. Sulcus opens up onto nude sul-

cal pad. Asulcal face only lightly ornamented, extending

onto base. Spur projects posteriorly, and distal tip covered

in several projects, giving it a comb-like appearance.

Delma hebesa Maryan, Brennan, Adams & Aplin, 2015

Fully everted; single lobe. Apex appears more truncate and

lobe more bulbous than australis. Strongly ornamented on

both sulcal and asulcal faces, with the exclusion of the sul-

cus. Sulcus sharply edged by lips, terminating prior to

apex. Apex projects posteriorly substantially, but does not

reach point found in D. australis.

Delma inornata Kluge, 1974*

Fully everted; bi-lobed. Hemipenis is bulb-shaped, with

poorly differentiated apical lobes. The medial (in-situ) lobe

is larger than lateral lobe. Sulcus is forked, and surface ep-

ithelium is covered by strongly pustulse features, calyces

absent.

Delma nasuta Kluge, 1974 (Figs 1—Sa, b)

Fully everted; bi-lobed. Strongly asymmetrical lobes. Me-

dial lobe (generally smaller in delmas with asymmetrical

hemipenes) strongly cleft at distal end of sulcus, opening

onto laterally-facing sulcal pad. Smaller lobe balloon

shaped, much narrower at base. Lateral lobe larger, and

truncate at terminus, with sulcus reaching apex. Sulcal lips

on larger lobe strongly overlap sulcus. Asulcal face only

lightly ornamented, base nude. Spurs project dorsally and

posteriorly, and terminate in comb-like tips, not as pro-

nounced as D. haroldi.

Delma pax Kluge, 1974 (Figs 1—7a, b)

Fully everted; bi-lobed. Dorsal lobe shorter and broader,

hammerhead in shape, truncate, with sulcus reaching apex.

Ventral lobe elongate, narrow, end truncate, covered in mi-

cro-ornamentation. Asulcal surface micro-ornamented lat-

erally, nude medially. Sulcus deep and narrow, both lobes

nude until beyond fork, with finely stippled tips.

©ZFMK

pp) Ian G. Brennan & Aaron M. Bauer

Delma petersoni Shea, 1991 (Figs 1—6a,b)

Fully everted; bi-lobed. Deep cleft between approximate-

ly similar sized lobes. Surface almost entirely nude, sim-

ilar to D. fraseri. A deep, narrow sulcus bisects at lobe-

cleft, becomes narrow channels diverting away from mid-

line and opening out into broad flat surface on sulcal side.

Delma tincta De Vis, 1888

Fully everted; bi-lobed. Asymmetrical lobes, smaller of the

two hammerhead shaped. Both lobes, including asulcal

faces, but excluding sulcal pads, are covered in fine mi-

cro-ornamentation which is strongest on asulcal face of

large lobe. Sulcus narrow and deep, continues laterally

from lobe-fork, to lateral edge of sulcal pad of shorter lobe,

and to apex of elongate lobe. Smaller, hammer-shaped lobe

broad and flattened, creating large sulcal face large com-

posed of sulcal pad.

Lialis burtonis Gray, 1835

Incompletely everted; distinctly bi-lobed. Deep sulcus,

with nude base. Asulcal and sulcal faces, excluding sul-

cus, covered in fine ornamentation.

Pletholax gracilis (Cope, 1864) (Figs 1—8a, b)

Fully everted; bi-lobed. Mitten shaped, with dorsal lobe

substantially reduced, ventral lobe larger and elongate.

Asulcal surface partially nude, but densely covered in mi-

cro-spines and ornamentation laterally. Hemipenal base

nude until constriction at base of lobe division. The sul-

cus 1s narrow, but deep, and widens towards apices, sul-

cus reaches apex of each lobe.

Pygopus lepidopodus (Lacépéde, 1804)*

Incompletely everted; specimen preserved in 1864, long

before assessment by BOhme. Median lobe can be made

out, reminiscent of P nigriceps. Calyces on base are small

and end prior to apex of lateral lobes.

Pygopus nigriceps (Fischer, 1882)*

Fully everted; tri-lobed. Hemipenis generally short and

broad. Sulcus spermaticus divided into two long forks

along the length of larger, divided lobes. Sulcus is a deep

depression. A small, pear-shaped, undivided lobe sits be-

tween larger lateral main lobes. Asulcal face covered in

small calyces, base covered in transverse calyces with

beaded edges. The outer lateral lobes are covered with

small deep calyces with prickly edges. Asulcal face of

lobes smooth.

DISCUSSION

Systematic Implications of the Hemipenes of Pygopodids

Relative to other squamate groups, and even other

gekkotans, pygopodid hemipenes are markedly simplified

Bonn zoological Bulletin 66 (1): 15—28

in their overall morphology (BO6hme 1988). Morphologi-

cal reduction in hemipenial characters may be the result

of an ancestral miniaturization event at the base of the py-

gopodid tree, as seen in Brookesia chameleons (Klaver &

Bohme 1986). While in contrast, similarly small squa-

mates such as gymnopthalmids have not seen this simpli-

fication in hemipenial morphology, pygopodid hemipenial

size (length from cloaca to apex) relative to body length

(SVL) is substantially smaller than that of the

gymnopthalmid Jphisa elegans (Nunes et al. 2012). Re-

duction in hemipenial characters as a result of miniatur-

ization in fossorial limbless squamates has been observed

in the morphologically and ecologically similar ty-

phlopids (Khan 1999; Thomas & Hedges 2007). This mor-

phological simplification is most pronounced in the three

species of Aprasia (A. haroldi, A. repens, A. smithi) and

two Delma species (D. australis, D. hebesa) which exhib-

it single-lobed hemipenes. These species are among the

smallest members of their respective genera, and the dis-

junct nature of this characteristic in the pygopodid tree

suggests two independent evolutionary events. Presence

of bi-lobed hemipenes in all five examined pygopodid gen-

era, as well as the exclusive condition in carphodactylid

and diplodactylid geckos, supports the pygopodid, py-

gopodoidean, and gekkotan ancestral hemipenial condi-

tions as bi-lobed.

Mitochondrial and allozyme history suggests a basal

split within Aprasia. Aprasia haroldi, A. pulchella, A.

repens, and A. smithi, all examined here, are members of

a single clade, closely related to A. rostrataas as well as

the recently described A. clairae (Maryan et al. 2013b) and

A. litorea (Maryan et al. 2013a), for which no hemipenial

assessment has been made. Aprasia parapulchella repre-

sents the sole observed member of the alternative, mod-

erately divergent group (Fig. 2). Aprasia pulchella, which

is closely related to the single-lobed group, exhibits the

bi-lobed hemipenial condition, similar to the asymmetri-

cally bi-lobed hemipenes of 4. parapulchella, despite their

distant relatedness within Aprasia. Incomplete molecular

and hemipenial sampling of this genus necessitates addi-

tional assessment of Aprasia species to determine the an-

cestral hemipenial condition. Further study will determine

if single-lobed hemipenes occur as a synapomorphy of the

A. fusca group (A. smithi, A. litorea, A. haroldi, A. clairae,

A. repens, A. rostrata, A. fusca).

Perhaps more surprising than morphological reduction

in the diminutive fossorial Aprasia, is the occurrence of

single-lobed hemipenes in the Delma australis group.

Here, hemipenial reduction 1s noted in D. australis and the

newly described D. hebesa, however the closely related D.

torquatahas not yet been assessed (Fig. 3). Close relations

between D. australis and D. torquata suggest the poten-

tial for a similar hemipenial condition. Assessment of

hemipenial morphology of D. concinna and D. torquata

would contribute to the understanding of this group. Ob-

©OZFMK

Pygopodidae hemipenial morphology 23

—————eE Aprasia pulchella

Aprasia picturata

Aprasia rostrata

Aprasia smithi

| Aprasia repens

Aprasia clairae

Aprasia litorea

Las Aprasia haroladl

Aprasia aurita

Aprasia parapulchella

Fig. 2.

Aprasia pseudopulchella

Aprasia Striolata

Aprasia inaurita

Schematic phylogeny of Aprasia composed of mitochondrial data from Jennings et al. (2003) and allozyme data from

Maryan et al. (2013a) and Maryan et al. (2013b). Grey dotted branches indicate lineages for which hemipenial morphology has

not yet been assessed. Black branches indicate species which display bi-lobed hemipenes, and blue branches denote species with

single-lobed hemipenes.

servation of single-lobed hemipenes in D. concinna or D.

torquata would strongly support inclusion of these

species in the D. australis clade, establishing a synapo-

morphy of this group.

Within the bi-lobed pygopodids, there remains consid-

erable morphological distinction between genera, species

groups, and individual species. Although members of

Aprasia may exhibit either single- or bi-lobed hemipenes,

regardless of overall shape, the sulcus spermaticus broad-

ens out onto a sulcal pad, comprising most of the sulcal

face of the hemipenis. The sulcal lips in Aprasia also

strongly delineate the sulcus spermaticus, and the entire-

ty of the asulcal and sulcal faces, including even the sul-

cal lips, are covered in fine micro-ornamentation. The sul-

cal pad terminates at the apex in both single- and bi-lobed

species of Aprasia, and remains unornamented in all ob-

served species.

The moderate to small sized, heavily nuchal banded Del-

ma species of northern and northwestern portions of Aus-

tralia D. borea, D. desmosa, D. elegans, D. pax, and D.

tincta, can be distinguished by asymmetry not only in size,

but in general shape of the bi-lobed hemipenes. In D.

borea, D. desmosa, D. pax, and D. tincta a hammer-shaped

Bonn zoological Bulletin 66 (1): 15-28

lobe extends anteriorly or proximally, while the second

lobe, elongate, with a truncate apex and strongly folded

sulcal lips, extends dorsally or distally. Delma elegans al-

so exhibits a strongly asymmetrical design, however the

disparity in shape between the anterior/medial and

dorsal/distal lobes is less pronounced, and the dorsally pro-

jecting lobe is not elongate, nor is the apex truncate. Based

on similarity in shape, asymmetry, and ornamentation, we

suggest a close affinity among D. borea, D. desmosa, D.

pax, and D. tincta, but are unable to further hypothesize

systematics based on hemipenial morphology alone. Del-

ma elegans appears to show a much simplified asymmet-

rical condition, potentially embodying the ancestral con-

dition for this group.

Delma fraseri and D. petersoni, previously confused un-

der a single species, display a nearly identical hemipenial

condition, most likely the result of recent ancestry.

Hemipenes of these two species are either weakly asym-

metrical, or symmetrical in size and shape, both nude, with

sulci that terminate laterally on sulcal pads. Based on mi-

tochondrial data (Jennings et al. 2003), D. grayii has been

recognized as sister taxon to D. fraseri, and despite sim-

ilar hemipenial morphology — narrowly asymmetrical

©OZFMK

2A Ian G. Brennan & Aaron M. Bauer

Delma concinna

Delma torquata

Delma mitella

Delma nasuta

Delma inornata

Delma grayll

Delma butleri

Delma haroldi

Delma petersoni

Delma fraser

Delma plebeia

Delma impar

Delma molleri

Delma labialis

Delma elegans

Delma borea

Delma teale!

Delma tincta

Delma pax

Delma desmosa

Fig. 3. Species tree phylogeny of De/ma as inferred by nDNA data from Brennan et al. (2016). Grey dotted branches indicate lin-

eages for which hemipenial morphology has not yet been assessed. Black branches indicate species which display bi-lobed hemipenes,

and blue branches denote species with single-lobed hemipenes.

bulb-shaped lobes, sulcus terminating laterally in pads —

nuclear DNA data (Brennan et al. 2016) suggests this sis-

ter relationship may instead be an artifact of a historical

introgression event, the cause of such an event, or a bi-

lobed, narrowly asymmetrical, bulb-shaped, largely unor-

namented hemipenis may represent the ancestral Delma

hemipenial condition.

Moderate asulcal ornamentation, alongside a narrowly

asymmetrical bi-lobed design unites and the D. butleri

group (D. butleri, D. grayii, D. haroldi, D. inornata, D.

nasuta). Except for D. nasuta, this group is typified by

micro-ornamentation restricted to the lobes, distal to the

Bonn zoological Bulletin 66 (1): 15—28

point of bifurcation of the sulcus. The sulcus terminates

laterally onto shallow broad sulcal pads, which are also

devoid of ornamentation. Within D. butleri we recognize

two differing hemipenial conditions: large-bodied and

lightly patterned D. butleri from the Carnarvon region

(WAM 120322, WAM 120819) display hemipenes similar

to that of D. haroldi, whereas the more northern and in-

land D. butleri (WAM123911) shows a distinct shape and

lack of ornamentation. Allopatry of distinct D. butleri pop-

ulations and morphological differences highlight the po-

tential of cryptic species, or possible intraspecific varia-

tion with this broadly distributed variable species. In com-

©OZFMK

Pygopodidae hemipenial morphology ZS

Fig. 4. Lateral view of right cloacal spur of : A) Delma fraseri WAMR141191; B) Delma haroldi WAMR163615; C) Delma na-

suta WAMR154288; D) Delma australis WAMR140395. Red arrows indicate position of the spur, and black arrows indicate fine

projections on posterior-facing distal tip of the spur, a synapomorphy of the D. butleri group.

parison to other members of this group, D. nasuta exhibits

an enigmatic hemipenial condition in which the strongly

asymmetrical lobes are both truncate, and the sulcus re-

mains deep and narrow as it terminates at the apex of each

lobe. Despite disparate hemipenial morphology, D. nasu-

ta has been associated with D. butleri and D. haroldi based

on general morphology (Kluge 1974), osteology (Kluge

1976), and mitochondrial (Jennings et al. 2003) and nu-

clear DNA (Brennan et al. 2016) results. Here, we also rec-

ognize as a synapomorphy of the D. butleri clade, the

comb-like serrated edge of the cloacal spurs. The spurs

are oriented posteriorly, and small projections of the dis-

tal spur edge are most pronounced in D. haroldi, weaker

in D. butleri, and less developed but still observable in D.

nasuta and D. grayii. The generalized morphology of the

Bonn zoological Bulletin 66 (1): 15-28

hemipenes in this D. butleri group, as well as in D. fraseri

and D. petersoni, despite a non sister-taxa relationship may

suggest that an approximately symmetrical bi-lobed, and

relatively unornamented hemipenis may constitute the an-

cestral Delma hemipenial design. Although hemipenial

sampling for this genus remains incomplete, this hypoth-

esis would suggest the strongly asymmetrical hemipenis

of the northwest Australian group (D. borea, D. desmosa,

D. elegans, D. pax, D. tincta), and the single-lobed hemipe-

nis of the D. australis group represent significant morpho-

logical divergences.

The monotypic genus Pletholax displays a strongly

asymmetrical bi-lobed hemipenis which is covered by

coarse, dense spines along the lateral and apical portions

of the asulcal face, and the sulcal lips. Hemipenes of

©OZFMK

26 Ian G. Brennan & Aaron M. Bauer

Pletholax gracilis are unlike any other pygopodid hemipe-

nis in ornamentation, as well as general shape.

The morphology of Pygopus hemipenes is also unique

to pygopodids in the presence of a third, undivided, me-

dial lobe. Both observed species, Pygopus lepidopodus and

P nigriceps, display this medial lobe, as well as differen-

tiated ornamentation of the sulcal and asulcal faces, and

ornamented hemipenial bases. Assessment of Lialis bur-

tonis is limited to incompletely everted specimens, and as

such, makes phylogenetic inference difficult, however, we

observe that this species shows a bi-lobed condition, and

lobes appear covered in differentiated ornamentation sim-

ilar to that of Pygopus. Similarity in ornamentation may

suggest systematic relatedness between Pygopus and

Lialis, or may be an artifact of the much larger adult size

of species of these genera, relative to that of other pygopo-

dids. Hemipenial characteristics of Ophidiocephalus and

Paradelma remain unobserved.

Cloacal Spurs and Reproductive Behavior

Morphology of the cloacal spurs and post-cloacal bones

and sacs are often mentioned in the context of reproduc-

tive biology. In gekkotans, post-cloacal bones and sacs

have been reviewed by Kluge (1982), however little atten-

tion has been paid to cloacal spurs of geckos. Due to the

paucity of behavioral data regarding pygopodids, we draw

on the observations of other gekkotans and squamates to

address the implication of cloacal spurs and postcloacal

bones in reproductive success (Kluge 1982; Kluge 1987).

The occurrence of post-cloacal bones is a synapomorphy

of gekkotans, uniting the pygopodids with other members

of this group (Greer 1989). Although cloacal bones were

identified by Kluge (1982) in all examined pygopodid

species, cloacal sacs were absent in all Delma and Lialis;

present in both sexes of Paradelma and Pygopus; present

in males of Aprasia and Pletholax, absent in female

Pletholax, and inter- and intraspecifically variable in Apra-

sia females.

In the eublepharid gecko Coleonyx variegatus, the pres-

ence and use of spurs are important for successful mat-

ing (Greenberg 1943). Here, after positioning himself

alongside the female, and contorting himself to face vent-

to-vent, the male slides the closer spur longitudinally along

the female’s body axis, and across her cloacal opening, in

an attempt to gain purchase among the loose skin below

the vent. In doing so, the pull of the male’s spur draws back

the lower lip of the female’s cloaca, causing her cloaca to

gape, creating an opportunity for the male to evert and in-

sert his hemipenis. Although pygopodid and eublepharid

geckos differ morphologically, specifically in the presence

or absence of loose post-cloacal skin, spurs may still serve

a similar purpose.

Bonn zoological Bulletin 66 (1): 15—28

In other limb reduced squamates, such as pythonid

snakes, cloacal spurs appear as the only external vestige

of the hind limbs, where they tip the distal portion of the

femur (Greer 1997). The imbricate scales and tighter skin

of pythonids more accurately resemble the pygopodid con-

dition, and here male pythons may use the spur to stroke

and stimulate the female during courtship, gauge and en-

courage her receptivity, and as in eublepharids align the

cloaca using tactile cues and expedite mating (Greer 1997;

Hoser 1985; Murphy et al. 1981; Schouten 1985; Slip &

Shine 1988; Walsh 1985). Additionally, spurs may be used

in male-male combat, to gain purchase and scratch the op-

position (Barker et al. 1979; van der Heijden 1986). It is

important to note that the association between spurs and

femoral remnants in pythonids, and the presence of exter-

nal hindlimbs in pygopodids acknowledges the non-ho-

mology of spurs across these squamate families. This how-

ever, does not require their use to differ.

Cloacal spurs in pygopodids are small structures hid-

den behind the hindlimb flap, just dorsal and posterior to

the cloaca. Spurs are indistinguishable from hindlimb

scales in Aprasia, but in the comparatively speciose Del-

ma, spurs represent another morphological character ca-

pable of identifying species groups. As mentioned, the

large, comb-like spur of D. haroldi (Fig. 4B), is visible to

a much reduced degree in the closely related D. butleri,

D. nasuta, and D. grayii (Fig. 4C). In D. australis and D.

hebesa, the spur (Fig. 4D) is much less pronounced, round-

ed, and wider than it is long. In contrast, members of the

D. fraseri and northwest Australian groups display mod-

erate sized spurs which are rounded in the smaller mem-

bers of the northwest group D. borea, D. desmosa, D. el-

egans, D. pax, D. tincta, and are pointed in D. fraseri (Fig.

4A) and D. petersoni.

While this study adds to the current knowledge of

hemipenial structure and spur morphology and their phy-

logenetic affinities within Pygopodidae, we present this

data as a work in progress. Continued hemipenial assess-

ment of Delma species and other pygopodid genera will

contribute substantially to our understanding of reproduc-

tive evolution and isolation within the flap-footed geck-

os. The remarkable morphological divergence of pygopo-

dids when compared to their limbed gekkotan ancestors,

represents an immense leap, which may present itself in

other aspects of anatomy not yet assessed. Complete de-

scriptions of the hemipenes of monotypic Ophidio-

cephalus and Paradelma, and more complete description

of Lialis species may further provide insight into inter-

generic relationships within this unique family.

Acknowlegements. We thank Paul Doughty, Claire Steven-

son, and Ryan Ellis (Western Australian Museum) for the loan

of the material used in this paper. This research was supported

by grant DEB 0844523 and the Gerald M. Lemole endowed

Chair Funds through Villanova University.

©OZFMK

Pygopodidae hemipenial morphology 27:

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©OZFMK

Bonn zoological Bulletin 66 (1): 29-36

April 2017

New species and records of Lasioserica and Gynaecoserica from China

(Coleoptera, Scarabaeidae, Sericini)

Wan-Gang Liu’, Ming Bai’, Xingke Yang’ & Dirk Ahrens’

' Institute of Earth and Environment, Chinese Academy of Sciences, Yanxiang Road 97#,

Yanta District, Xi’an 710061 PR. China; E-mail: liuwangang@ieecas.cn

? Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Box 92, No. 1,

Beichen West Road, Chaoyang District, Beijing, 100101, PR. China

> Centre of Taxonomy and Evolutionary Research, Zoologisches Forschungsmuseum A. Koenig, Adenauerallee 160,

D-53113 Bonn, Germany; Email: ahrens.dirk_col@gmx.de, d.ahrens@leibniz-zfmk.de

Abstract. Two new species of Sericini are described from China: Gynaecoserica motuoensis Liu and Ahrens, sp. n.

and Lasioserica guangxiana Liu and Ahrens, sp. n. A checklist of the Chinese species as well as new records are given

for Lasioserica Brenske, 1896 and Gynaecoserica Brenske, 1896. The examination of the new material revealed five

new country records: Gynaecoserica nahangensis Ahrens & Fabrizi, 2009, G. namtamaiensis Ahrens & Fabrizi, 2009,

Lasioserica tricuspis Ahrens, 2000 and L. kubani Ahrens 2000 (China) as well as Lasioserica meghalayana Ahrens,

1999 (Vietnam).

Key words: Beetles, chafers, Lasioserica, Gynaecoserica, China, new species, new records.

INTRODUCTION

In the course of the revision of the material of Sericini

from China a recent series of paper was published on the

genera Gastroserica Brenske, 1897, Neoserica Brenske,

1894, and Tetraserica Ahrens, 2004 (Ahrens et al.

2014a-—c, Liu et al. 2011, 2014a—e, 2015). In continuation

of this work, we present here the results on the genera La-

sioserica and Gynaecoserica, which have been revised pre-

viously by Ahrens (1996) and Ahrens & Fabrizi (2009),

and to which a number of supplemental notes have been

released (Ahrens 1999a,b, 2000, 2004, 2005, Ahrens &

Fabrizi 2011, 2016, Liu et al. 2014b). This study compris-

es in major part the revision of the unidentified material

housed in Chinese natural history collections. Apart from

a number of new and interesting records this material con-

tains also two new species described herein.

MATERIAL & METHODS

The terminology and methods used for measurements,

specimen dissection and genital preparation follow Ahrens

(2004). Data from specimens examined are cited in the text

with original label contents given in quotation marks, mul-

tiple labels are separated by a “/’’. Descriptions and illus-

trations of new taxa are based on the holotype specimen

if not otherwise stated, while the variation of specimens

is given separately under “variation”. Male genitalia were

glued to a small pointed card and photographed in both

Received: 02.06.2016

Accepted: 02.02.2017

lateral and dorsal view using a Zeiss AxioCam HRc

mounted on a Zeiss Stereo Discovery.V20 stereo-micro-

scope. In the Automontage software a number of single

focussed images were combined in order to obtain an en-

tirely focussed image. The resulting images were subse-

quently digitally edited.

The authors of the new names are Wan-Gang Liu and

Dirk Ahrens (art. 50.1 of ICZN).

Abbreviations used in the text for collection depositories

are as follows:

BPBM Bernice P. Bishop Museum, Honolulu, USA;

CP collection Petr Pacholatko, Brno,

Czech Republic;

CASH collection André Skale, Hof/Saale, Germany;

HBUM Museum of Hebei University,

Baoding (Hebei Province) China;

ISNB _ Institut Royal des Sciences naturelles

de Belgique, Brussels, Belgium;

IZAS _ Institute of Zoology, Chinese Academy of

Sciences, Being, China;

MZUF Museo Zoologico “La Specola’,

Universita di Firenze, Italy;

NMPC_ National Museum (Natural History),

Prague, Czech Republic;

SYUG_ Sun Yat-Sen University, Guangzhou, China;

ZFMK_ Zoologisches Forschungsmuseum A. Koenig,

Bonn, Germany.

Corresponding editor: R. S. Peters

30 Wan-Gang Liu et al.

NEW SPECIES

Gynaecoserica motuoensis Liu & Ahrens sp. n.

Type material examined. Holotype: 3 “[China] Xizang,

Motuo, 80k, 2100m, 2011-VHI-19/ LW-1134” (IZAS).

Description. Length 5.5 mm, length of elytra 4.1 mm,

width 2.9 mm. Body oblong, dorsal surface dark brown

to dark green, antenna yellowish brown, dorsal surface

dull, densely erectly setose.

Labroclypeus subrectangular, widest at base, lateral mar-

gins weakly convergent, anterior angles strongly round-

ed, lateral border and ocular canthus producing a distinct

blunt angle, margins weakly reflexed; anterior margin shal-

lowly sinuate medially; surface weakly convex medially

and shiny, finely and densely punctate, distance between

punctures equal or less than their diameter, with dense and

long, erect setae; frontoclypeal suture feebly incised, me-

dially moderately curved; smooth area in front of eye ap-

proximately 1.5 times as wide as long; ocular canthus short

and moderately slender, finely and densely punctate, with

a short terminal seta. Frons shiny, posterior half dull, with

fine, dense punctures, densely setose. Eyes moderately

large, ratio of diameter/interocular width 0.64. Antenna

yellow, with ten antennomeres; club yellow, with five an-

tennomeres, as long as remaining antennomeres combined.

Mentum weakly elevated and flattened anteriorly.

Pronotum moderately wide, widest shortly before base,

lateral margins evenly convex and weakly convergent an-

teriorly, anterior angles strongly produced and sharp, pos-

terior angles strongly rounded; anterior margin nearly

straight, with a fine marginal line; basal margin without

marginal line; surface with irregularly dense fine punc-

tures, with dense and long, erect setae; anterior and lat-

eral borders setose; hypomeron distinctly margined at base

but not ventrally produced. Scutellum moderately long and

wide, triangular, with fine dense punctures, medially nar-

rowly smooth, with short adjacent setae in punctures.

Elytra elongate, widest in apical third, striae distinctly

impressed, finely and densely punctate, intervals weakly

convex, with fine, sparse punctures concentrated along

striae, punctures with dense fine erect setae, interior api-

cal angle of elytra with robust seta; epipleural edge fine

ending at strongly curved external apical angle of elytra;

epipleura sparsely setose, apical border without short m1-

crotrichomes.

Ventral surface dull, with fine and moderately dense

punctures, densely setose; metacoxa glabrous, with a few

strong adjacent setae laterally only; each abdominal ster-

nite with indistinct transverse row of coarse punctures

bearing short setae between fine, dense punctation,

penultimate sternite apically with a very short shiny

smooth sclerotized border, last sternite medially 1.3 times

as long as penultimate one. Mesosternum between meso-

Bonn zoological Bulletin 66 (1): 29-36

coxae as wide as mesofemur, with irregularly scattered

very strong setae. Ratio of length of metepisternum/meta-

coxa: 1/1.45. Pygidium strongly convex at apex, finely and

densely punctate, with moderately broad smooth midline,

surface dull, with numerous long setae.

Legs slender and long; femora dull, with two longitu-

dinal rows of setae, finely and sparsely punctate; metafe-

mur shiny, sharply margined anteriorly and without a sub-

marginal serrate line, posterior margin weakly convex and

glabrous, its external part only weakly widened in apical

half and not serrate, internally very finely serrate, with a

few long setae. Metatibia slender and long; evenly widened

toward apex, ratio width/length: 1/3.2, dorsal margin

sharply carinate; with two groups of spines, basal group

shortly before half, apical group at three-quarters of metat-

ibial length; basally with a few single, fine setae; exter-

nal face longitudinally convex, with sparse, fine punctures,

glabrous; ventral margin carinate and serrate, with three

strong spines, the distal two more distant; medial face very

finely and sparsely punctate and smooth, apex interiorly

near tarsal articulation sharply and deeply truncate. Tar-

someres dorsally glabrous and impunctate, ventrally with

sparse, short setae; metatarsomeres ventrally with a strong-

ly serrate ridge, beside it with a subparallel, fine longitu-

dinal carina; first metatarsomere little longer than follow-

ing two tarsomeres combined and nearly twice as long as

dorsal tibial spur. Protibia moderately long, bidentate, pro-

tarsal claws symmetrical, basal tooth of interior claw nor-

mally developed.

Aedeagus: Fig. 1A—C. Habitus Fig. 1D. Female un-

known.

Diagnosis. Gynaecoserica motuoensis Liu & Ahrens sp.

n. differs from all other Gynaecoserica species by the para-

meres being strongly bent dorsally behind the middle and

widened on the left side having a baseward directed and

sharply pointed process (Fig. 1B).

Etymology. Latin adjective in the nominative singular. The

Species is named according to its type locality, Motuo.

Lasioserica guangxiana Liu & Ahrens sp. n.

Type material examined. Holotype: 4 “[China] Fulong,

Fangcheng, Guangxi, 24.V.1999, 500m, leg. Zhang Guo-

ging, Yuan Decheng/ LW-110” (IZAS). Paratype: 1 ¢

“[China] Fulong, Fangcheng, Guangxi, 24.V.1999, 500m,

leg. Zhang Guoqing, Yuan Decheng” (ZFMK).

Description. Length: 6.0 mm, length of elytra: 4.7 mm,

width: 3.5 mm. Body oblong, dorsal surface dark brown,

antenna brown, dorsal surface dull, pronotum and head

with greenish shine, densely setose, with fine long and

white, robust setae on elytra.

©ZFMK

New species and records of Lasioserica and Gynaecoserica from China 31

Labroclypeus subtrapezoidal, widest at base, lateral mar-

gins in basal half strongly convex and strongly convergent

to moderately rounded anterior angles, lateral border and

ocular canthus producing a distinct angle; anterior mar-

gin concavely sinuate, margins weakly reflexed; surface

flat and shiny, finely and densely punctate, with dense and

long erect setae; frontoclypeal suture weakly impressed

and moderately curved; smooth area anterior to eye 3 times

as wide as long; ocular canthus moderately long and nar-

row, finely and densely punctate, with a short terminal se-

ta. Frons in posterior half dull, finely and densely punc-

tate. Eyes large, ratio of diameter/interocular width: 0.8.

Antenna with ten antennomeres, club in male with four

antennomeres, twice as long as remaining antennomeres

combined, all joints of same length. Mentum elevated and

flattened anteriorly.

Pronotum widest at middle, lateral margins in basal half

straight and subparallel, anteriorly moderately curved and

convergent to weakly produced anterior and blunt anteri-

or angles, posterior angles nearly right-angled; anterior

margin weakly convex, with a fine marginal line; basal

margin without marginal line; surface with dense and fine

punctures each bearing either an short, adpressed or a

longer, white seta; anterior and lateral borders sparsely se-

tose; hypomeron carinate, basal margin of hypomeron

weakly produced ventrally. Scutellum subtriangular, apex

moderately rounded, with fine and dense punctures and

setae, smooth on basal midline.

Elytra oblong, widest shortly behind middle, striae mod-

erately impressed, with fine and dense punctures; inter-

vals moderately convex, with fine and irregularly dense

punctures concentrated along striae, impunctate areas ap-

pear darker, with sparse, short setae, on odd intervals with

a few fine white setae; epipleural edge moderately strong,

ending at strongly rounded external apical angle of ely-

tra, epipleura densely setose, apical border chitinous, with-

out a visible rim of microtrichomes (100x magnification).

Ventral surface dull, with large and dense punctures,

sparsely setose, metacoxa only laterally with a few fine,

adpressed setae. Abdominal sternites finely and densely

punctate and minutely setose, each sternite with a distinct

transverse row of coarse punctures each bearing a short,

robust seta. Penultimate abdominal sternite with two wide-

ly separated tubercles. Mesosternum between mesocoxae

as wide as mesofemur. Ratio of length of metepisternum/

metacoxa: 1/1.37. Pygidium moderately convex and dull,

with fine, dense punctures and fine, short setae, with wide

impunctate midline.

Legs moderately slender and long; femora dull on ven-

tral face, with two longitudinal rows of setae, finely and

sparsely punctate; anterior edge of metafemur acute, with

an adjacent serrate line, ventrally weakly widened ventral-

ly in apical half but not serrate, dorsally serrate. Metati-

bia moderately slender and short, widest at apex, ratio

width/length: 1/3.1, distinctly carinate dorsally, with one

Bonn zoological Bulletin 66 (1): 29-36

group of spines only at 7/8 of metatibial length, beside dor-

sal margin with a straight and continuously serrate line

convergent with dorsal margin behind apical group of

spines, between serrated line and dorsal margin finely

punctate and with a few short setae; lateral face longitu-

dinally convex, with dense and fine punctures, densely se-

tose; ventral edge serrate, with four fine and long, equi-

distant spines, medial face finely and sparsely punctate and

punctures with minute setae, apex interiorly near tarsal ar-

ticulation weakly concavely truncate. Tarsomeres dorsal-

ly sparsely punctate and finely setose, ventrally with short,

sparse setae; metatarsomeres ventrally with a strongly ser-

rate ridge, laterally not carinate, first metatarsomere as

long as the following two tarsomeres combined and near-

ly twice as long as dorsal tibial spur. Protibia short, biden-

tate, protarsal claws asymmetrical, basal tooth of inner

claw somewhat lobiform and truncate at apex.

Aedeagus: Fig. 1E—G. Female unknown.

Diagnosis. The external morphology and the shape of

parameres are similar to those of L. brevipilosa Moser,

1919. The new species differs significantly by the more

flattened and more widened left paramere, and the

straighter and longer right paramere which has external-

ly a robust, lateral, tooth-like extension (Fig. 1E, F).

Variation. Length: 6.0-6.8 mm, length of elytra: 4.7—5.2

mm, width: 3.5—3.9 mm.

Etymology. Latin adjective in the nominative singular. The

new species is named after its occurrence in the Guangxi

province.

Checklist of Chinese species and new records

of Gynaecoserica and Lasioserica

genus Gynaecoserica Brenske, 1896

Gynaecoserica Brenske, 1896: 154 (type species Gynae-

coserica pellecta Brenske, 1896 by monotypy).

Chaetoserica Brenske, 1897: 355 (type species Chaetoser-

ica cymosa Brenske, 1896 by monotypy).

Paragynaecoserica Khan & Ghai, 1982: 61 (type species

Paragynaecoserica pubescens Khan & Ghai, 1982 by

monotypy).

Gynaecoserica alma Ahrens & Fabrizi, 2009

Gynaecoserica alma Ahrens & Fabrizi, 2009: 1511.

Distribution. Yunnan.

©ZFMK

52 Wan-Gang Liu et al.

Fig. 1. A—D: Gynaecoserica motuoensis Liu & Ahrens sp. n. (holotype), E—-H: Lasioserica guangxiana Liu & Ahrens sp. n. (ho-

lotype). A, E: Aedeagus, left side lateral view; C, G: Aedeagus, right side lateral view; B, F: Parameres, dorsal view; D, H: Habi-

tus (not to scale). Scale: 0.5 mm.

Gynaecoserica amara Ahrens & Fabrizi, 2009 Distribution. Yunnan.

Gynaecoserica amara Ahrens & Fabrizi, 2009: 1522.

Gynaecoserica hani Liu & Ahrens, 2014

Distribution. Yunnan.

Gynaecoserica hani Liu & Ahrens, 2014: 160.

Gynaecoserica bocaki Ahrens & Fabrizi, 2009 Distribution. Xizang (Tibet).

Gynaecoserica bocaki Ahrens & Fabrizi, 2009: 1529.

Bonn zoological Bulletin 66 (1): 29-36 ©Z7FMK

New species and records of Lasioserica and Gynaecoserica from China 33

Gynaecoserica lohitensis Ahrens & Fabrizi, 2009

Gynaecoserica lohitensis Ahrens & Fabrizi, 2009: 1555.

Distribution. Xizang (Tibet), northeastern India.

Gynaecoserica nahangensis Ahrens & Fabrizi, 2009

Gynaecoserica nahangensis Ahrens & Fabrizi, 2009:

1567.

Material examined. 2 3'' “Mengzhe, Xishuangbanna,

Yunnan, 3.VII.1958, 891m, 1200m, leg. Meng Xuwu,

Wang Shuyong” (IZAS).

Distribution. Yunnan, Vietnam.

Remarks. This species was originally described from

northern Vietnam and is for the first time recorded for Chi-

na.

Gynaecoserica namtamaiensis Ahrens & Fabrizi, 2009

Gynaecoserica namtamaiensis Ahrens & Fabrizi, 2009:

1569.

Material examined. 1 @ “Mt. Heishan, Longxin,

Longling, Yunnan, 23-25.XII.2008, leg. Xu Jishan, Zou

Zhenhua” (HBUM).

Distribution. Yunnan, Myanmar.

Remarks. This species was originally described from

northern Myanmar (Burma) and is for the first time

recorded for China.

Gynaecoserica obliqua Ahrens & Fabrizi, 2009

Gynaecoserica obliqua Ahrens & Fabrizi, 2009: 1570.

Material examined. | 4 “X-DA1628/ X-DA1628 China

China: Yunnan prov., Mazhan env. Volcano Geological

park, 1930 m 25°13.5’N, 98°30.0’E leg. J. Hajek & J. Ru-

zicka 6.VI.2007 Gynaecoserica sp Chinal” (ZFMK).

Distribution: Yunnan.

Bonn zoological Bulletin 66 (1): 29-36

Gynaecoserica yigongensis Liu & Ahrens, 2014

Gynaecoserica yigongensis Liu & Ahrens, 2014: 160.

Distribution: Xizang (Tibet).

genus Lasioserica Brenske, 1896

Lasioserica Brenske, 1896: 155 (type species Serica no-

bilis Brenske, 1894 by subsequent designation; Arrow

1946).

Orchiserica Miyake & Yamaya, 2001: 38 (type species La-

sioserica brevipilosa Moser, 1919 by subsequent des-

ignation; Ahrens 2004).

Lasioserica antennalis Nomura, 1974

Lasioserica antennalis Nomura, 1974: 83.

Distribution. Taiwan.

Lasioserica beibengana Liu & Ahrens, 2014

Lasioserica beibengana Liu & Ahrens, 2014: 161.

Distribution. Xizang (Tibet).

Lasioserica bipilosa Ahrens, 1999

Lasioserica bipilosa Ahrens, 1999: 70.

Material examined. | ex. “N. Vietnam, 1985, Tam dao,

3.-11.6. 900-1400m J. Jelinék leg’ (NMPC), 4 ex. “N-

Vietnam: Vinh Phu Prov. Tam Dao National Park, 950m,

5-12.VI.2010, L. Bartolozzi & S. Bambi legit (n° Mag.

2894)” (MZUF), 1 ex. “N-Vietnam Vinh Phuc Prov., vic.

Tam Dao city, Tam Dao NP, 02.-5.V.2013, 21°27’N

105°38’E, 700-1000m, A. Skale” (CASH), 6 ex. “N-Viet-

nam Cao Bang Prov., vic. Tinh Tuc, Nui Pia Oac Nature

Res., 13.V.2014, 22°36’50”N, 105°52’21”E 14-1800m

leg. A. Skale” (CASH), 1 ¢ “X-DA3442 — Vietnam, N.

Vietnam: Cao Bang Prov., Mt. Pia Oac, 1600-2000m (at

light), 14-16.vi.2012, leg. L. Bartolozzi, S. Bambi, F. Fabi-

ano, E. Orbach” (ZFMK), 1 9 “X-DA3441 — Vietnam, N.

Vietnam: Cao Bang Prov., Mt. Pia Oac, 1600-2000m (at

light), 14-16.v1.2012, leg. L. Bartolozzi, S. Bambi, F. Fa-

biano, E. Orbach” (MZUF).

Distribution. Vietnam.

©ZFMK

34 Wan-Gang Liu et al.

Lasioserica brevipilosa Moser, 1919

Lasioserica brevipilosa Moser, 1919: 332.

Material examined. 2 ex. ,,Coll. R.I.Sc.N.B. Chine/ Yun-

nan Fou/ Le Moult vendit” (ISNB), 1 ex. “China- Yunnan

28.5.-9.6.1994 Dali Igt. E. Kucera” (NMPC), 1 ex. (3)

“China-Yunnan 10.-15.6.1994 Lijiang Igt. E. Kucera”

(NMPC), | ex. “China, W Yunnan prov., mts. 60Km E Ten-

gchong, 2300m, 14.-19.v.2006 S. Murzin & I. Shokin leg.”

(CP), 3 ex. “China, W Yunnan prov., mts. 20km SW Bao-

shan, 2400m, 23.-25.v.2006, S. Murzin & I. Shokin leg.”

(CP), 4 ex. “China (Yunnan) Dali Bai Aut. Pref. Wuliang

Shan, 9km SW Weishan, 2450-2500m, 25°10’14”N/

100°14’22”E (sec. Oak/ pine for., beaten from trees and

bushes) 13.VI.2007 D.W. Wrase [350]” (ZFMK), 1 ex.

“China (Yunnan) Nujiang Lisu Aut. Pref. Nu Shan, 7km

NNW Coajian 2420m 25°43’29”N/ 99°07°57”E (second.

Pine forest with shrubs, litter, moss sifted) 11.VI.2007

D.W. Wrase [30]” (ZFMK), 1 3 “[China] Mts. Yulong-

shan, Lijiang, Yunnan, 4.VII.1962, leg. Song Shimei”

(IZAS), 1 3 “[China] Mts. Yulongshan, Lijiang, Yunnan,

27,28.V1.1962, leg. Song Shimei” (IZAS), 1 4 “[China]

Institue of Agricultural Sciences, Buijie, Guizhou,

20.V1.1978, leg. Yang” (IZAS), 1 4 “[China] Moxi, Lud-

ing, Sichuan, 19.VI.1983, 1500m,1600m, leg. Chen

Yuanqing, Zhang Xuezhong, Wang Shuyong” (IZAS), 1

S “[China] Yunnan, 2010-VIH-5, Dali, Cangshan,

N:25.65140, E: 100.16907, H:2133m/ LW-1033” (IZAS),

1 6 “DA1424 China: Yunnan, Dali Bai Auton. Pref., Wu-

liang Shan, 9 km SW Weishan, 2450-2500 m/ oaks and

pines, sifted 25°10’14”N, 100°14’22”E 13.VI.2007 leg.

A. Piitz” (ZFMK), 1 6 “DA1425 China: Yunnan, Dali Bai

Auton. Pref., Wuliang Shan, 9 km SW Weishan, 2450-

2500 m/ oaks and pines, sifted 25°10’14”N, 100°14’22”E

13.VI.2007 leg. A. Piitz” (ZFMK), 1 2° “DA1423 China:

Yunnan, Dali Bai Auton. Pref., Wuliang Shan, 9 km SW

Weishan, 2450-2500 m/ oaks and pines, sifted

25°10°14”N, 100°14’°22”E 13.VI.2007 leg. A. Putz”

(ZFMK),

Distribution. Sichuan, Guizhou, Yunnan.

Lasioserica dragon Miyake & Yamaya, 2001

Lasioserica dragon Miyake & Yamaya, 2001: 36.

Material examined. | ¢ “China N-Yunnan 27°08’N

100°14’E Yulongshan mts. 2900-3500m Baishui vill. Leg.

D. Kral 7-12.V1.90” (NMPC), 1 3 “[China] Yunnan, Li-

jiang, Yulongxueshan, 2011-V-17, N: 27.013, E: 100.206,

2750m/ LW-1062” (IZAS).

Distribution. Sichuan, Yunnan.

Bonn zoological Bulletin 66 (1): 29-36

Lasioserica kuatunica Ahrens, 1996

Lasioserica kuatunica Ahrens, 1996: 25.

Material examined. 2 ex. “Kuatun, Fukien China 25.5.46

leg. (Tschung-Sen.”) (NMPC), 1 ex. “Kuatun, Fukien Chi-

na 20.6.46 leg. (Tschung-Sen.”) (NMPC), 2 ex. “Kuatun

(2300m) 27,40n.Br. 117,400.L. J. Klapperich 26.5. 1938

(Fukien)/ ex. Coll. V. Balthasar National Museum Prague,

Czech Republic” (NMPC), 1 ex. “Kuatun (2300m)

27,40n.Br. 117,400.L. J. Klapperich 3.5. 1938 (Fukien)/

ex. Coll. V. Balthasar National Museum Prague, Czech Re-

public” (NMPC), 1 ex. “Kuatun (2300m) 27,40n.Br.

117,400.L. J. Klapperich 28.5. 1938 (Fukien)/ ex. Coll. V.

Balthasar National Museum Prague, Czech Republic”

(NMPC), | ex. “Kuatun (2300m) 27,40n.Br. 117,400.L.

J. Klapperich 8.6. 1938 (Fukien)/ ex. Coll. V. Balthasar Na-

tional Museum Prague, Czech Republic” (NMPC),1 ex.

“Kuatun (2300m) 27,40 n.Br. 117,400.L. J. Klapperich

10.6. 1938 (Fukien)/ ex. Coll. V. Balthasar National Mu-

seum Prague, Czech Republic” (NMPC), | ex. “Kuatun

(2300m) 27,40n.Br. 117,400.L. J. Klapperich 16.6. 1938

(Fukien)/ ex. Coll. V. Balthasar National Museum Prague,

Czech Republic” (NMPC), 3 ex. “China, W Fujian, 3.-

4.VI. Emei Feng, 1200-1500m 27°01’N 117°04E Jaros-

lav Turna leg., 2008” (ZFMK), | 3 “[China] Datianping,

Mts. Fengyangshan, Longquan, 15.VI.1980, leg. Zeng

Xuesong” (IZAS), 1 4 “[China] Mt. Wuyanling, Taishun,

Zhejiang, 28.VII-3.VII.2005, leg. Ba Yibin” (HBUM).

Distribution. Zhejiang, Fujian.

Lasioserica kubani Ahrens, 2000

Lasioserica kubani Ahrens, 2000: 8.

Material examined. | ex. “China (Yunnan) Dali Bai Aut.

Pref. Wuliang Shan, 9km SW Weishan, 2450-2500m,

25°10°14”N/ 100°14’22”E (sec. Oak/ pine for., beaten

from trees and bushes) 13.VI.2007 D.W. Wrase [350]”

(ZFMK), 1 3 “{China] Yunnan, Caiyanghe, Yunpan, 201 1-

V-22, N:22.65119, E:101.09917, 1633m/ LW-1181”

(IZAS), 1 9 “839478 Lasioserica sp THAI_DE09_1 Thai-

land L. Dembicky 23-30.4.2009 Pha Hom Pok Mt. Chiang

Mai Prov. 20°02’35”N 99°08’45”E 1900-2000m/

839478” (ZFMK).

Distribution. Yunnan, Thailand.

Remarks. This species was originally described from

Thailand and is for the first time recorded from China.

©ZFMK

New species and records of Lasioserica and Gynaecoserica from China 35

Lasioserica meghalayana Ahrens, 1999

Lasioserica meghalayana Ahrens, 1999: 224.

Material examined. 1 ¢ “[China] Cangyuan, Yunnan,

21.V.1980, 1300m, leg. Li Hongxing” (IZAS), 1 3 “[Chi-

na] Yunnan, Caiyanghe, Yunpan, 2011-V-22, N:22.65119,

E:101.09917, 1633m/ LW-1182” (IZAS), 1 3 “[China]

Cangyuan, Yunnan, 17.V.1980, 1100m, leg. Li Hongxing

“ (IZAS), 1 & “[China] Mandian (Forest), Nabanhe Na-

ture Reserve, Jinghong, Xishuangbannna, Yunnan,

26.1V.2009, 746m, leg. Meng LZ” (IZAS), 1 &, 1 2 “[Chi-

na] Mandian (Forest), Nabanhe Nature Reserve, Jinghong,

Xishuangbannna, Yunnan, 16.V.2009, 753m, leg. Meng

LZ” (IZAS), 6 ex. “N-Vietnam — Lao Cai province, Van

Ban district: Van Ban Nature Reserve (at light) (1000m)

— 23.-26.V.2011/ L. Bartolozzi, S. Bambi, F. Fabiano, E.

Orbach leg. (Num. Magazzino 2909)” (MZUF), | & “‘Chi-

na: Yunnan Province, Tengchong, 3.-6.VI.2007 near long-

distance bus station, 25°00.3’N, 98°29.3’E, 1625m, leg.

J. Hajek & J. Ruzicka [Ch 10]/ individually collected un-

der lights of streets adjacent to mixed forest (from sunset

to midnight)/ X-DA 1629” (ZFMK).

Distribution. Yunnan, Vietnam, Laos, India, Myanmar.

Remarks. This species is for the first time recorded for

Vietnam.

Lasioserica oblita Ahrens, 1996

Lasioserica oblita Ahrens, 1996: 26.

Material examined. | ¢ “DA1411 China: Yunnan, De-

hong Dai Aut. Pref., mountain range 31 km E Luxi, 2280/

m, secnd. pine forest with old decid. trees, litter sifted,

24°29'31”N, 98°52’58”E 3.V1.2007, A. Ptitz/ X-DA1411”

(ZFMK), 1 9 “DA1410 China: Yunnan, Dehong Dai Aut.

Pref., mountain range 31 km E Luxi, 2280/ m, secnd. pine

forest with old decid. trees, litter sifted, 24°29’31”N,

98°52’58”E 3.VI.2007, A. Piitz/ X-DA1411” (ZFMK), 1

9 “China Yunnan, Nujiang Lisu Aut.Pref., Nu Shan, 7 km

NNW Coajian, 2420 m, second. pine forest with shrubs,

litter, bark sifted, 25°43’29”N, 99°07°57”E, leg. A. Piitz

11.VI.2007/ DA1413” (ZFMK).

Distribution. Yunnan, Myanmar.

Remarks. The species was already recorded for China by

Ahrens (2005).

Bonn zoological Bulletin 66 (1): 29-36

Lasioserica pacholatkoi Ahrens, 2000

Lasioserica pacholatkoi Ahrens, 2000: 17.

Distribution. Xizang (Tibet), Bhutan.

Lasioserica tricuspis Ahrens, 2000

Lasioserica tricuspis Ahrens, 2000: 11.

Material examined. 1 3 “Defu, Napo, Guangxi,

18.V1.2000, 1350m, leg. Chen Jun” (IZAS)

Distribution. Guangxi, Laos, Thailand.

Remarks. This species was originally described from

northern Thailand and Laos and is for the first time record-

ed for China.

Lasioserica tuberculiventris Moser, 1915

Lasioserica tuberculiventris Moser, 1915: 118.

Material examined. 1 ex. (3) “China W Sichuan

Kangding 2500 m Rejsek 16.6.1995” (ZFMK), 1 ex. (3)

“Mt. Omei Szechuan, China VII-13-32 Franck coll./ EC.

Hadden Collection” (BPBM), 3 ex. “China: N-Yunnan

Baiyungshan (Bai Railing Mts.) 2400 m Yong Ren, VII-

2003 leg. Ying et al.” (ZFMK), 2 63,2 9° “[China] Shi-

ping, Fengdu, Sichuan, 2,3.V1I.1994, 610m, leg. Zhang

Youwei” (IZAS), 1 @, 1 2 “[China] Moxi, Luding, Si-

chuan, 19.VI.1983, 1600m, leg. Chen Yuanqing, Wang

Shuyong” (IZAS), 2 ¢@, 2 99 “[China] Shiping,

Fengdu, Sichuan, 2.V1I.1994, 610m, leg. Zhang Youwei”

(IZAS), 1 3 “[China] Mt. Leigongshan, Guizhou,

15.VII.1983, leg. Chen Zhenguang, No. En-045848” (SY-

UG).

Distribution. Shandong, Sichuan, Guizhou, Yunnan.

Acknowledgements. Part of this research was supported by the

National Natural Science Foundation of China (No. 31501889),

the National Science Fund for Fostering Talents in Basic Re-

search (Special Subjects in Animal Taxonomy, NSFC-J1210002),

Research Equipment Development Project of Chinese Academy

of Sciences (YZ201509). We are thankful to Ms Hong Pang

(SYUG), Prof. Guodong Ren (HBUM), L. Bartolozzi, D. Wrase,

and A. Skale for providing additional unidentified specimens for

this study and to AleS Bezdék and Alberto Ballerio for the help-

ful comments on the manuscript.

©ZFMK

36 Wan-Gang Liu et al.

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©ZFMK

Bonn zoological Bulletin 66 (1): 37—53

April 2017

First report on the amphibian fauna of Ha Lang karst forest,

Cao Bang Province, Vietnam

Cuong The Pham™, Hang Thi An’, Sebastian Herbst’", Michael Bonkowski’,

Thomas Ziegler*& Truong Quang Nguyen'***

' Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi,

Vietnam. E-mail: nqt2@yahoo.com; cuongiebr@gmail.com

? AG Zoologischer Garten Koln, Riehler Strasse 173, D-50735 Cologne, Germany. E-mail: ziegler@koelnerzoo.de

* Institute of Zoology, Department of Terrestrial Ecology, University of Cologne, Ziilpicher Strasse 47b,

D-50674 Cologne, Germany. E-mail: m.bonkowski@uni-koeln.de

* Graduate University of Science and Technology, Vietnam Academy of Science and Technology,

18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam

> Corresponding author. E-mail: nqt2@yahoo.com

Abstract. A total of 21 species of amphibians was documented on the basis of a new herpetological collection from the

karst forest of Ha Lang District, Cao Bang Province. Three species, Odorrana bacboensis, O. graminea, and Rhacopho-

rus maximus, are recorded for the first time from Cao Bang Province. The amphibian fauna of Ha Lang District also con-

tains a high level of species of conservation concern with one globally and two nationally threatened species and three

species, Odorrana mutschmanni, Gracixalus waza, and Theloderma corticale, which are endemic to Vietnam.

Keywords: Amphibians, karst forest, distribution, diversity, new records, Cao Bang Province.

INTRODUCTION

Recent herpetological research has underscored the spe-

cial role of karst habitats in promoting speciation of rep-

tiles and amphibians in the border areas between Vietnam

and China (Pham et al. 2016c). Numerous new species and

new country records have been recently discovered in the

border region of both countries, for example Odorrana

geminata Bain, Stuart, Nguyen, Che & Rao, 2009; Pro-

tobothrops trungkhanhensis Orlov, Ryabov & Nguyen,

2009; Oligodon nagao David, Nguyen, Nguyen, Jiang,

Chen, Teynié & Ziegler, 2012; Gekko adleri Nguyen,

Wang, Yang, Lehmann, Le, Ziegler & Bonkowski, 2013

(Bain et al. 2009; Orlov et al. 2009; David et al. 2012;

Nguyen et al. 2013c; Guo et al. 2016). In addition, some

other new species, that were recently described from Ha

Lang forest in Cao Bang Province, are also expected to

be found in the karst formations in Guangxi Province of

China, viz. Gracixalus waza Nguyen, Le, Pham, Nguyen,

Bonkowski & Ziegler, Hemiphyllodactylus zugi Nguyen,

Lehmann, Le, Duong, Bonkowski & Ziegler, and Odor-

rana mutschmanni Pham, Nguyen, Le, Bonkowski &

Ziegler (Nguyen et al. 2013a, 2013b; Pham et al. 2016c).

These new discoveries of reptiles and amphibians from

Cao Bang Province underline the still significantly under-

estimated biodiversity of northeastern Vietnam. Based on

the results of our recent field surveys in the period between

2011 and 2015, we herein provide the first list of amphib-

ians recorded from Ha Lang karst forest in Cao Bang

Province.

Received: 22.06.2016

Accepted: 09.02.2017

MATERIAL & METHODS

Field surveys were conducted in the Ha Lang forest of Cao

Bang Province (Fig. 1) in October 2011, in April and May

2012, in July 2014, and in April 2015 by T.Q. Nguyen, C.T.

Pham, D.T. Le, H.T. An, M. Bonkowski, S. Herbst, T.

Lehmann, M. Bernardes, and M. van Schingen. Survey

transects were set up along streams, ponds, cave entrances,

limestone cliffs, in the valleys, and forest paths. The main

habitat type was secondary karst forest of medium and

small hardwoods mixed with shrubs and vines at eleva-

tions between 400 to 700 m above sea level (Fig. 2). Af-

ter taking photographs, the specimens were anaesthetized

with ethylacetate, fixed in 80% ethanol for 4-6 hours and

subsequently stored in 70% ethanol. Voucher specimens

have been deposited in the collections of the Institute of

Ecology and Biological Resources (IEBR), Hanoi, Viet-

nam, and the Zoologisches Forschungsmuseum Alexan-

der Koenig (ZFMK), Bonn, Germany.

Morphological identification generally followed Bour-

ret (1942), Taylor (1962), Inger et al. (1999), Ziegler

(2002), Bain & Nguyen (2004), Bain et al. (2003), Nguyen

et al. (2013a), and Mo et al. (2015). Measurements were

taken with a digital caliper to the nearest 0.1 mm. Abbre-

viations are as follows: ED: horizontal eye diameter (eye

length), EN: distance between anterior corner of eye and

nostril, HL: head length, HW: head width, IND: internar-

ial distance, IOD: interorbital distance, SN: distance be-

tween nostril and tip of snout, SL: snout length, SVL:

snout-vent length (from tip of snout to cloaca), TD: max-

Corresponding editor: Ph. Wagner

38 Cuong The Pham et al.

Elevation (m)

Im 1200+ inte A

0 75 150 et ee

i pet tO, 0 et ¢

Kilometers o 150, _ 300 Kilometers ‘ OK

Fig. 1. Map showing the survey site (red spot) in Ha Lang District in Cao Bang Province, Vietnam.

Bonn zoological Bulletin 66 (1): 37-53 ©Z7FMK

Amphibians of Ha Lang Forest, Vietnam 39

Fig. 2. Limestone karst forest in Ha Lang District, Cao Bang Province, Vietnam. Photo: T.Q. Nguyen.

imum tympanum diameter, TED: distance between ante-

rior margin of tympanum and posterior corner of eye,

UEW: width of upper eyelid, and asl.: above sea level. Sex

was determined by the presence of internal vocal sac open-

ing or gonadal inspection.

RESULTS

Taxonomic accounts

Bufonidae

Duttaphrynus melanostictus (Schneider, 1799) (Fig. 3a)

Identification was based on direct observations in the field

and photographs: Cranial crests conspicuous, black and

more distinct on supraorbital region; parietal crest absent;

parotoid glands prominent, elongated; tympanum distinct,

round; dorsum and upper surface of limbs with conical,

spiny warts; warts smaller on flanks; ventral surface gran-

ular; fingers free of webbing, toes with webbing at base.

Coloration in life: Upper head and dorsum yellowish

grey to dark brown with black spines; ventral cream (de-

termination after Bourret 1942; Ziegler 2002).

Bonn zoological Bulletin 66 (1): 37—53

Ecological notes. Several individuals were found in the

evening in rice fields and forest paths.

Distribution. This is a common species in Vietnam. Else-

where, the species has been known from Sri Lanka, In-

dia, Pakistan, Nepal, China, Myanmar, Laos, Thailand,

Cambodia, Malaysia and the Philippines (Nguyen et al.

2009).

Microhylidae

Microhyla fissipes Boulenger, 1884 (Fig. 3b)

Specimens examined. Three adult females EBR 3828:

SVL 19.0 mm, IEBR 3829: SVL 19.9 mm, IEBR 3830:

SVL 16.0 mm) collected by T.Q. Nguyen et al. on 6 April

2012, at elevations between 492 and 631 m asl.

Morphological characters. Body flattened, triangle

shaped; head small, longer than wide (HL 5.5—6.2 mm,

HW 4.8—5.2 mm); snout length greater than eye diame-

ter (SL 2.1—2.4 mm, ED 1.7—2.0 mm); nostril closer to tip

of snout than to eye (SN 1.0—1.2 mm, EN 1.5—1.8 mm);

interorbital distance broader than internarial distance and

upper eyelid (OD 1.7—1.8 mm, IND 1.5—1.6 mm, UEW

1.1—1.3 mm); tympanum hidden; vomerine teeth absent;

tongue notched posteriorly. Fingers free of webbing, with

©ZFMK

40 Cuong The Pham et al.

haat Ve , LL. ae oe 4 Fa at

Pas fle . i, ce ee

= i -

Fig. 3. a) Duttaphrynus melanostictus, b) Microhyla fissipes, c) M. heymonsi d), M. pulchra, ¢) Fejervarya limnocharis,

f) Hoplobatrachus rugulosus, g and h) Limnonectes bannaensis. Photos: C.T. Pham and H.T. An.

Bonn zoological Bulletin 66 (1): 37-53 ©Z7FMK

Amphibians of Ha Lang Forest, Vietnam 41

small discs; relative length of fingers I<II<IV<HI; toes

with rudimental webbing, without discs; relative length of

toes I<II<V<IN<IV; subarticular tubercles well developed;

inner and outer metatarsal tubercles small; tibio-tarsal ar-

ticulation reaching to the eye. Dorsal and ventral skin

smooth; supratympanic fold indistinct.

Coloration in life: Dorsal head and body brown, with a

grayish brown patch from orbit to hind body, a dark patch

on dorsolateral fold; ventral surface cream (determination

after Bourret 1942; Manthey & Grossmann 1997; Ziegler

2002; Bain & Nguyen 2004).

Ecological notes. The specimens were found between

19:00 and 23:00 in corn fields.

Distribution. This is a widespread species in Vietnam.

Elsewhere, the species is known from China, Taiwan,

Myanmar, Laos, Thailand, Cambodia, Malaysia and Sin-

gapore (Nguyen et al. 2009).

Microhyla heymonsi Vogt, 1911 (Fig. 3c)

Specimen examined. One adult male (EBR 3831: SVL

22 mm) collected by T.Q. Nguyen et al. on 3 May 2012,

at an elevation of 487 m asl.

Morphological characters. Body flattened, triangle

shaped; head longer than wide (HL 8.3 mm, HW 6.4 mm);

snout obtusely pointed, pronounced, somewhat longer than

eye (SL 2.9 mm, ED 2.3 mm); nostrils closer to tip of

snout than to eye (SN 1.1 mm, EN 1.5 mm); interorbital

distance broader than internarial distance and upper eye-

lid GOD 2.3 mm, IND 2.1 mm, UEW 1.5 mm); tympa-

num hidden; vomerine teeth absent; tongue round poste-

riorly; male with vocal sacs.

Fingers free of webbing, with small discs; relative length

of fingers I<U<IV<HI; toes with rudimental webbing; rel-

ative length of toes I<II<V<II<IV; subarticular tubercles

well developed; inner and outer metatarsal tubercles small;

tibio-tarsal articulation reaching nearby tip of snout. Dor-

sal and ventral skin smooth; supratympanic fold indistinct.

Coloration in life: Dorsal head and body light brown,

with a white stripe from tip of snout to cloaca, and a small

dark spot in the middle of the back; lateral sides of head

and flanks dark brown; anterior part of thighs, cloacal re-

gion and lower parts of feet black; limbs with thin trans-

verse bars; ventral surface white to grey; male with black

vocal sacs (determination after Bourret 1942; Ziegler

2002; Bain & Nguyen 2004)

Ecological notes. The specimen was collected at 19:00

in a small rivulet. The surrounding habitat was mixed sec-

ondary forest, bamboo, small hardwoods, liane and shrub.

Distribution. This is a widespread species in Vietnam.

Elsewhere, the species is known from India, China, Laos,

Thailand, Cambodia, Malaysia and Indonesia (Nguyen et

al. 2009).

Bonn zoological Bulletin 66 (1): 37—53

Microhyla pulchra (Hallowell, 1861) (Fig. 3d)

Specimens examined. One adult female (IEBR 3832: SVL

24.8 mm) and one adult male TEBR 3833: SVL 29 mm)

collected by T.Q. Nguyen et al. in April 2012, at elevations

between 492 and 594 m asl.; one adult female (IEBR 3834:

SVL 28.6 mm) collected by C.T. Pham et al. on 14 June

2014, at an elevation of 537 m asl.

Morphological characters. Body flattened, triangle

shaped; head longer than wide (HL 8.4-9.4 mm, HW

6.9—7.4 mm); snout obtusely pointed, slightly pronounced,

longer than eye (SL 3.3—3.6 mm, ED 2.7—3.1 mm); pupil

round; interorbital distance broader than upper eyelid JOD

2.1-2.5 mm, UEW 1.7—1.9 mm); tympanum indistinct;

vomerine teeth absent; tongue round posteriorly; male with

vocal sacs. Fingers free of webbing; tips of fingers not en-

larged; relative length of fingers I<II<IV<II; toes

webbed, webbing formula [2/3-11I1/3-11/21II1-21V21/2-

1V; relative length of toes I<U<V<IN<IV; subarticular tu-

bercles well developed; inner metatarsal tubercles present,

outer metatarsal tubercles round; tibio-tarsal articulation

reaching between eye and tip of snout. Dorsal skin smooth;

a distinct fold present between posterior edges of the eyes;

ventral skin smooth; cloacal region granular.

Coloration in life: Dorsum brown with a dark band be-

tween eyes; several V-shaped dark and lighter alternating

bands on dorsum pointing to the head; dark transversal

band from posterior corner of the eye towards upper

flanks; rear of flanks and inner parts of legs yellow; limbs

with transverse bars; ventral surface whitish yellow; throat

and chest speckled with dark (determination after Bour-

ret 1942; Ziegler 2002; Bain & Nguyen 2004).

Ecological notes. The specimens were collected between

19:00 and 22:00. One specimen was found in a corn field.

Other specimens were found on forest paths, the surround-

ing habitat was mixed secondary forest of bamboo, small

hardwoods, liane and shrub.

Distribution. This is a common species in Vietnam. Else-

where, this species in known from northeastern India to

southern China, Cambodia, Guam, Laos and Thailand

(Frost 2017).

Dicroglossidae

Fejervarya limnocharis (Gravenhorst, 1829) (Fig. 3e)

Specimens examined. One adult female (IEBR 3835: SVL

41 mm) and one adult male (IEBR 3836: SVL 33 mm)

collected by T.Q. Nguyen et al. on 6 April 2012, at eleva-

tions between 492 and 516 m asl.

Morphological characters. Head longer than wide (HL

13.2-15.9 mm, HW 12.7—14.6 mm); snout pointed, longer

©ZFMK

42 Cuong The Pham et al.

than eye (SL5.4—6.9 mm, ED 4.1—5.6 mm); canthus ros-

tralis obtuse; loreal region oblique; nostrils closer to tip

of snout than to eye (NS 2.6—3.2 mm, EN 3.2—3.6 mm);

internarial distance and upper eyelid broader than interor-

bital distance (IND 3.0-3.6mm, UEW 2.9—3.4mm, IOD

2.4-2.5mm); tympanum distinct (TD 2.0-2.6 mm);

vomerine teeth present; tongue bifid; male with vocal sacs.

Fingers free of webbing; tips of fingers not enlarged;

relative length of fingers H<IV<I<III; toes webbed, web-

bing formula I1/4-1II1/4-11/4013/4-21V2-2/3V; relative

length of toes I<H<HI<V<IV; subarticular tubercles well

developed; inner metatarsal tubercles present, outer

metatarsal tubercles small; tibio-tarsal articulation reach-

ing to eye. Dorsal skin granular with several irregular der-

mal folds; ventral surface smooth; supratympanic fold dis-

tinct; males with nuptial pads.

Coloration in life: Dorsal head and body greyish green

with camouflage-pattern; light vertebral stripe present or

absent; lips with dark vertical bars; limbs with transverse

bars or spots; ventral surface white; throat marbled with

black in males (determination after Bourret 1942; Man-

they & Grossmann 1997; Ziegler 2002).

Ecological notes. The specimens were found between

19:00 and 20:00 in the rice field.

Distribution. In Vietnam, this species has been report-

ed from the entire country. Elsewhere, this species is

known from China, Myanmar, Laos, Cambodia, Thailand,

Malaysia and Indonesia (Frost 2017).

Hoplobatrachus rugulosus (Wiegmann, 1834) (Fig. 3f)

Specimen examined. One adult female (IEBR 3837: SVL

62.5 mm) collected by T.Q. Nguyen et al. on 17 October

2011, at an elevation of 620 m asl.

Morphological characters. Head broader than long (HW

27.8 mm, HL 25.3 mm); nostril oval, closer to eye than

to tip of snout (NS 5.1 mm, EN 4.4 mm); interorbital dis-

tance and internarial distance smaller than upper eyelid

(OD 3.2 mm, IND 4 mm, UEW 5.6 mm); tympanum

round, smaller than eye diameter (TD 4.5 mm, ED 8.3

mm); vomerine teeth present; tongue bifid. Arms short;

fingers without webbing; tips of fingers obtusely point-

ed; relative length of fingers H<I<IV<II; outer metatarsal

tubercle absent; hind limbs short; toes fully webbed, tips

of toes pointed, without discs, subarticular tubercles small;

relative length of toes I<II<III<V<IV; tibio-tarsal articu-

lation reaching to the eye. Skin granulate on dorsum and

flanks containing 10 irregular rows of elongate ridges; ven-

tral side of body smooth; supratympanic fold distinct.

Coloration in life: Dorsum olive green with dark blotch-

es; upper lip with irregular dark bars; dorsal surface of

limbs with dark bands; ventral surface cream; gular region

with dark marbling; lower part of flanks with dark mar-

bling (determination after Bourret 1942; Taylor 1962;

Ziegler 2002).

Bonn zoological Bulletin 66 (1): 37—53

Ecological notes. The specimen was found at ca. 21:00

on the bank of a small stream. The surrounding habitat was

mixed secondary karst forest, composed of medium and

small hardwoods, shrubs and vines.

Distribution. In Vietnam, this species has been report-

ed from the entire country. Elsewhere, this species is

known from China, Tatwan, Myanmar, Laos, Thailand,

Cambodia and Malaysia (Nguyen et al. 2009).

Limnonectes bannaensis Ye, Fei & Xie, 2007

(Fig. 3g, h)

Specimens examined. One female IEBR 3859: SVL 80.6

mm), and two adult males (IEBR 3861: SVL 66.2 mm,

IEBR 3867: SVL 69.5 mm) collected by T.Q. Nguyen et

al. on 15 October 2011, at an elevation of 476 m asl.; and

four adult males TEBR 3888: SVL 65.5 mm, IEBR 3889:

SVL 60 mm, IEBR 3890: SVL 64 mm, IEBR 3899: SVL

92 mm) collected by T.Q. Nguyen et al. on 13 April 2012,

at an elevation of 544 m asl.

Morphological characters. Head large, flattened, wider

than long (HL 21.5—36.0 mm, HW 22.8—37.5 mm); snout

round, longer than eye diameter (SL 7.9-10.3 mm, ED

7.1-8.4 mm); canthus rostralis indistinct; loreal region

oblique and slightly concave; nostril closer to tip of snout

than to eye (EN 4.6—6.6 mm, NS 4.1—4.9 mm); internar-

ial distance broader than interorbital distance and upper

eyelid (IND 4.2-6.0 mm, IOD 4.0-5.2 mm, UEW

3.8—5.0 mm); tympanum hidden; vomerine teeth present;

tongue bifurcated posteriorly; lower jaw with two tooth-

like processes; males without vocal sacs. Arms short, fin-

gers free of webbing, tips of fingers obtuse or slightly

swollen, relative length of fingers II<IV<I<II; sub-

articular tubercles on fingers and toes large; hind limbs

short; toes short, fully webbed; tips of toes dilated into

small discs; relative length of toes I<I<HI<V<IV; subar-

ticular tubercles large; inner metatarsal tubercle large, out-

er metatarsal tubercle absent; tibio-tarsal articulation

reaching to the eye. Skin above and below smooth; small

and homogenous tubercules on the leg and foot; supratym-

panic fold present.

Coloration in life: Dorsum reddish brown to greyish

brown, mostly with dark mottling or marbling; sometimes

with light vertebral stripe; lower region of flanks lighter;

limbs with dark transversal bands; ventral surface cream,

ventral side of limbs darker; throat, pectoral region, bel-

ly and outer edges of limbs with dark mottles (determi-

nation after Ye et al. 2007; McLeod 2010; McLeod et al.

2015).

Ecological notes. The specimens were found between

19:30 and 23:00 on the bank of a small stream. The sur-

rounding habitat was mixed secondary karst forest, com-

posed of medium and small hardwoods, shrubs and vines.

Distribution. In Vietnam, this species was reported from

Lao Cai Province in the North to Dong Nai and Kien Gi-

©ZFMK

Amphibians of Ha Lang Forest, Vietnam 43

ang provinces in the South. Elsewhere, this species is

known from China, Myanmar and Laos (Nguyen et al.

2009; McLeod 2010).

Remarks: McLeod (2010) stated that Limnonectes kuh-

lii (Tschudi, 1838) is a complex of cryptic species. The

“true” L. kuhlii is known only from the type locality in

Java, Indonesia, and specimens identified as L. Awhlii from

China and Vietnam should be assigned to L. bannaensis.

Ranidae

Hylarana guentheri (Boulenger, 1882) (Fig. 4a)

Specimens examined. One adult male (IEBR 3838: SVL

49.7 mm) collected by T.Q. Nguyen et al. on 17 October

2011, at an elevation of 544 m asl.; and one adult male

CEBR 3939: SVL 76.6 mm) collected by T.Q. Nguyen et

al. on 10 April 2012, at an elevation of 459 m asl.

Morphological characters. Head longer than wide (HL

19.2-29.1 mm, HW 16.3—24.2 mm); snout pointed, strong-

ly projecting; canthus rostralis distinct; loreal region mod-

erately oblique, concave; nostril closer to tip of snout than

to eye (EN 4.5—6.4 mm, NS 3.9—5.3 mm); internarial dis-

tance broader than interorbital distance and upper eyelid

(IND 4.9-6.9 mm, [OD 4.4—6.2 mm, UEW 3.6—4.6 mm);

tympanum distinct (TD 4.0—5.8 mm); vomerine teeth pres-

ent; tongue notched posteriorly; males with vocal sacs.

Fingers slender and rather long, free of webbing, finger

tips swollen; relative length of fingers II<IV<I<III; sub-

articular tubercles large; tips of toes dilated into small

discs, with a median groove; relative length of toes

I<II<IN<V<IV; webbing formula I1/2-2111/2-21111-31V3-

1V; subarticular tubercles small; inner metatarsal tuber-

cle present, outer metatarsal tubercle very small; tibio-

tarsal articulation reaching nearby tip of snout. Skin above

and below smooth; dorsolateral fold distinct; supratym-

panic fold prominent.

Coloration in life: Dorsal surface of head and body light

brown to reddish brown, uniform or with dark brown

spots; lateral head and flanks with a dark line, bordering

the dorsolateral fold; tympanum dark brown or reddish;

limbs with brown crossbars, back of thighs yellow with

black mottles; ventral surface white or yellow, throat or

chest speckled with brown (determination after Bourret

1942; Ziegler 2002).

Ecological notes. One specimen was found between

19:00 and 21:00 on the bank of a small stream, another

one was found near the cave entrance. The surrounding

habitat was mixed secondary karst forest, composed of

medium and small hardwoods, shrubs and vines.

Distribution. This is a common species in lowland ar-

eas of Vietnam. Elsewhere, the species has been reported

from China, Taiwan, Myanmar and Laos (Nguyen et al.

2009).

Bonn zoological Bulletin 66 (1): 37—53

Odorrana bacboensis Bain, Lathrop, Murphy, Orlov &

Ho, 2003 (Fig. 4b)

Specimens examined: Three adult females (IEBR

A.2015.82: SVL 101.1 mm, IEBR 3886: SVL 103 mm,

TEBR A.2015.83: SVL 93.2 mm) collected by T.Q. Nguyen

et al. on 10 April 2012, at an elevation of 459 m asl.

Morphological characters. Head longer than wide (HL

37.1-39.7 mm, HW 32.5—35.9 mm); snout obtusely round

in dorsal view, projecting beyond lower jaw, round in pro-

file; canthus rostralis distinct; nostril lateral, closer to tip

of snout than to eye (NS 7.3—7.6 mm, EN 8.8—9.2 mm);

snout length greater than eye diameter longer than eye (SL

16.1-17.4 mm, ED 10.6—12.0 mm); interorbital distance

narrower than internarial distance but broader than upper

eyelid (IOD 10.5—11.3 mm, IND 11.1—12.3 mm, UEW

6.9-8.6 mm); tympanum distinct, half of eye diameter (TD

5.4-6.1 mm, ED 10.6—12.0 mm); vomerine teeth present;

tongue notched posteriorly. Fingers free of webbing; rel-

ative length of fingers Il<I<IV<II; finger discs larger than

those of toes, with circummarginal groove; toes fully

webbed; relative length of toes I<H<HI<V<IV; outer

metatarsal tubercle absent; inner metatarsal tubercle

large, flat; tibio-tarsal articulation reaching to tip of snout.

Dorsal skin smooth; flanks with tubercles; supratympan-

ic fold present; dorsolateral fold absent; dorsal surface of

limbs smooth; throat, chest, belly and ventral surface of

thighs smooth.

Coloration in life: Dorsum brown to dark brown, with

tiny, irregular, black blotches; flanks brown, with some

large black spots; throat, chest and belly white (determi-

nation after Bain et al. 2003; Wang et al. 2015).

Ecological notes. The specimens were found between

19:00 and 23:00 on the bank of a rocky stream inside a

cave. The surrounding habitat was mixed secondary karst

forest, composed of medium and small hardwoods,

shrubs and vines.

Distribution. In Vietnam, this species has been report-

ed from Lao Cai, Tuyen Quang, Bac Kan, Nghe An

provinces (Nguyen et al. 2009). This is the first record of

O. bacboensis from Cao Bang Province. Elsewhere, the

species has been reported from China (Wang et al. 2015).

Odorrana graminea (Boulenger, 1900) (Fig. 4c, d)

Specimens examined. Five adult females (IEBR

A.2015.80: SVL 85 mm, IEBR 3887: SVL 90 mm, IEBR

3891: SVL 91 mm, IEBR A 2015.81: SVL 94 mm, IEBR

3892: SVL 92 mm) and eight adult males (IEBR 3893:

SVL 51.1 mm, IEBR 3894: SVL 47.3 mm, IEBR A

2015.79: SVL 50 mm, IEBR 2015.78: SVL 45 mm, IEBR

3895: SVL 45 mm, IEBR 3896: SVL 43 mm, IEBR 3898:

SVL 46 mm, IEBR 3897: SVL 48 mm) collected by T.Q.

Nguyen et al. in April and May 2012, at elevations bet-

ween 428 and 594 m asl.

©ZFMK

44 Cuong The Pham et al.

ae oe ee , : al

tS Ps a pn .

ene. Bro ee

Fig. 4. a) Hylarana guentheri, b) Odorrana bacboensis, c and d) O. graminea, e) O. lipuensis, f and g) O. mutschmanni, h) Gracix-

alus waza. Photos: C.T. Pham and H.T. An.

Bonn zoological Bulletin 66 (1): 37-53 ©Z7FMK

Amphibians of Ha Lang Forest, Vietnam 45

Morphological characters. Males smaller than females

(SVL 42-53 mm in males, SVL 78-100 mm in females);

head longer than wide (HL 30.2—38.1 mm, HW 27.7—34.1

mm in females, HW 14.8-16.4 mm, HL 16.7—-18.7 mm

in males); snout round, longer than eye (SL 12.5—15.7 mm,

ED 9.1—10.8 mm in females, SL 7.0—7.8 mm, ED 5.9-6.8

mm in males); canthus rostralis distinct; interorbital dis-

tance wider than internarial distance and upper eyelid

(OD 8.3—9.8 mm, IN 9.5—11.1 mm, UEW 6.5-—7.3 mm

in females, IOD 4.5—-4.8 mm, IN 5.1-5.8 mm, UEW

3.8—4.4 mm in males); pupil round; tympanum distinct,

approximately half of eye diameter (TD 4.5—5.6 mm, ED

9.1-10.5 mm in females, TD 3.7—3.9 mm, ED 5.9-6.8 in

males); vomerine teeth present; tongue cordiform, deeply

notched posteriorly; males with vocal sacs. Fingers free

of webbing; relative length of fingers II<I<IV<I]; finger

discs larger than those of toes, with circummarginal

groove; toes fully webbed; relative length of toes

I<II<III<V<IV; outer metatarsal tubercle absent; inner

metatarsal tubercle elongate; tibio-tarsal articulation

reaching beyond tip of snout. Dorsal skin smooth, flank

with tubercles; supratympanic fold distinct; dorsolateral

fold absent.

Coloration in life: Dorsum green with or without black

spots; lateral sides of head and flanks brownish grey; lips

white; hind limbs with distinct dark bars; webbing dark

grey; ventral surface whitish (determination after Bour-

ret 1942; Bain et al. 2003).

Ecological notes. The specimens were found between

19:00 and 23:00 on the bank of a rocky stream. The sur-

rounding habitat was mixed secondary karst forest com-

posed of medium and small hardwoods, shrubs and vines.

Distribution. In Vietnam, this species has been report-

ed from Bac Kan, Lang Son, Vinh Phuc and Lam Dong

provinces (Nguyen et al. 2009). This is the first record of

O. graminea from Cao Bang Province. Elsewhere, the

species has been reported from China (Nguyen et al.

2009).

Odorrana lipuensis Mo, Chen, Wu, Zhang &

Zhou, 2015 (Fig. 4e)

Specimens examined. Two adult males (IEBR A.2015.63:

SVL 48.1 mm, IEBR A.2015.64: SVL 49.8 mm) collect-

ed by T.Q. Nguyen et al. on 21 May 2012, at an elevation

of 669 m asl.; three adult females TEBR A.2015.65: SVL

60.1 mm, IEBR A.2015.66: SVL 58.5 mm, IEBR

A.2015.67: SVL 59.5 mm) collected by C.T. Pham et al.

on 7 June 2014, at an elevation of 532 m asl.

Morphological characters. Males smaller than females

(SVL 48.1—49.8 mm in males, 58.5—60.1 mm in females);

head longer than wide (HL 18.9-19.5 mm, HW 16.8—17.3

mm in males; HL 21.8—23.2 mm, HW 20.2—21.5 mm in

females); snout obtusely rounded in dorsal view, project-

ing beyond lower jaw, round in profile; nostril lateral, clos-

Bonn zoological Bulletin 66 (1): 37—53

er to the tip of snout than to eye (NS 3.5—3.6 mm, EN

4,.2-4.4 mm in males; NS 4.0-4.3 mm, EN 5.4—-5.7 mm

in females); canthus rostralis distinct; pupil horizontally

oval; loreal region slightly concave and oblique; snout

length greater than eye diameter (SL 7.6—7.7 mm, ED

6.8-6.9 mm, in males; SL 9.2—-9.5 mm, ED 7.3—7.5 mm

in females); interorbital distance wider than internarial dis-

tance and upper eyelid TOD 5.6—5.6 mm, IND 5.2—5.3

mm, UEW 4.5—-4.7 mm in males; IOD 7.0—7.2 mm, IND

6.3-6.6 mm, UEW 4.8—5.2 mm in females); tympanum

distinct, round, smaller than eye diameter (TD 4.2-4.3 mm

in males; TD 5.4—5.7 mm in females); vomerine teeth in

two oblique ridges; tongue deeply notched posteriorly;

males without vocal sacs; females contained creamy yel-

low eggs, without black poles. Forelimbs robust; tips of

fingers expanded into discs with circummarginal grooves;

relative length of fingers Il<I<IV<III; inner metatarsal tu-

bercle oval, elongate; outer metatarsal tubercle small; nup-

tial pads present in males. Hindlimbs long; tips of toes ex-

panded into discs with circummarginal grooves; width of

disc of toe [TV smaller than that of finger III; relative length

of toes I<H<III<V<IV; webbing formula 10-1/4II0-

1/21110-3/41V3/4-0V; inner metatarsal tubercle elongate;

outer metatarsal tubercle absent; tibio-tarsal articulation

reaching to tip of snout. Dorsal surface of head and body

smooth; tiny spinules on flanks, upper edge of eyelid; an-

terior and posterior edge of tympanum; supratympanic

fold weakly developed; dorsolateral fold absent; throat,

chest, belly and ventral surface of thigh smooth.

Coloration in life: Iris black, surrounded by golden net-

work; dorsum and upper part of flanks moss-green with

brown mottles; dorsal surface of fore and hindlimbs moss-

green with dark brown cross bars; upper lip with dark

brown bars; throat, chest and belly cream with dark brown

mottles (determination after Mo et al. 2015).

Ecological notes. The specimens were found between

19:30 and 23:00. Two juveniles were found on trees, about

0.2—0.5 m above the ground, near the entrance of caves,

on October 18", 2011. Two males and five juveniles were

found on trees, ca. 1.0-1.5 m above the ground, near cave

entrances, in April and May 2012. Three females were

found on trees, ca. 0.3-0.5 m above the water surface, near

a pool from the waterfall of a rocky stream, in June 2014.

The surrounding habitat was secondary karst forest, com-

posed of medium and small hardwoods, shrubs and vines

Distribution. This species was originally described from

China by Mo et al. (2015) and recently recorded from Viet-

nam by Pham et al. (2016a).

Remarks: The Vietnamese specimens of O. lipuensis dif-

fer from the type specimens from China in having larger

sized females (SVL 58.5—60.1 mm versus 51.1—55.4 mm

in the Chinese specimens) and a smaller ratio of TD/ED

(0.68 in males and 0.63 in females vs. 0.73 in males and

0.69 in females from China, respectively) (see Mo et al.

201'5).

©ZFMK

46 Cuong The Pham et al.

Odorrana mutschmanni Pham, Nguyen, Le, Bonkowski

& Ziegler, 2016 (Fig. 4 f, g)

Specimens examined. One adult male IEBR 3724: SVL

91.6 mm) collected by T.Q. Nguyen et al. on 15 April 2012,

one adult female (EBR 3725: SVL 108.7 mm) and four

adult males TEBR 3726: SVL 85.8 mm, IEBR 3727: SVL

89.0 mm, IEBR 3728: SVL 90.1 mm, IEBR 3729: SVL

86.9 mm) collected by T.Q. Nguyen et al. on 16 April 2012,

two adult males (ZFMK 97329: SVL 90.4 mm, ZFMK

97330: SVL 91.6 mm) collected by H.T. An et al. on 3

May 2012, one adult female (IEBR 3730: SVL 109.6 mm)

collected by C.T. Pham et al. on 10 June 2014, one adult

male (IEBR 3723: SVL 85.9 mm) and one adult female

(EBR 3731: SVL 110.1 mm) collected by T.Q. Nguyen

on 22 April 2015, at an elevation of 447 m asl.

Morphological characters. Size large (SVL 85.9-91.6

mm in males, 108.7—110.1 mm in females); head longer

than wide (HL 33.5—-36.5 mm, HW 28.9-31.7 mm in

males, HL 41.2-43.5 mm, HW 38.7-41.1 mm in females);

snout round anteriorly in dorsal view; projecting beyond

lower jaw; nostril lateral, closer to the snout tip than to eye

(NS 6.2—7.2 mm, EN 7.1—8.1 mm in males, NS 8.4—8.8

mm, EN 9.1—9.7 mm in females); canthus rostralis distinct;

loreal region slightly concave and oblique; snout length

greater than eye diameter (SL 13.3-15.8 mm, ED

9.9-11.1 mm in males, SL 17.2-17.9 mm, ED 12.1—12.8

mm in females); internarial distance wider than interor-

bital distance and upper eyelid (IND 9.9-11.5 mm, IOD

7.8-10.3 mm, UEW 6.6—7.3 mm in males, IND 12.4—12.7

mm, IOD 10.7—11.5 mm, UEW 8.4—8.7 mm in females);

tympanum distinct, round, 70% of eye diameter; vomer-

ine teeth in two oblique ridges; tongue cordiform, deeply

notched posteriorly; males without vocal sacs. Tips of fin-

gers expanded into discs, with circummarginal grooves;

relative length of fingers Il<I<I V<UI; finger webbing rudi-

mental; outer metatarsal tubercle small; finger I with nup-

tial pad, elongate in males; tips of toes expanded into discs,

with circummarginal grooves; width disc of toe IV nar-

rower than that of finger III; relative length of toes

I<II<IN<V<IV; webbing formula I0-0II0-OI0-1/2TV 1/2-

OV; inner metatarsal tubercle elongate; outer metatarsal tu-

bercle absent; tibio-tarsal articulation reaching to tip of

snout. Dorsal surface of head and anterior part of body

smooth; posterior part of body and flanks with tubercles;

spinules present on lateral sides of body, anterior and pos-

terior edge of tympanum; supratympanic fold present; dor-

solateral fold absent; dorsal surface of limbs granular;

throat, chest, belly and ventral surface of thigh smooth.

Coloration in life: Iris black; dorsum green with dark

brown spots; lateral side of head and flanks greyish brown

with dark brown spots; lips with dark bars; tympanum dark

brown; spinules on flank ivory; dorsal surface of fore and

hindlimbs greyish brown with dark crossbars; throat and

chest grey; ventral surface of fore and hindlimbs and bel-

Bonn zoological Bulletin 66 (1): 37—53

ly with large dark brown spots, edged in white, forming

a network; toe webbing dark brown (determination after

Pham et al. 2016c).

Ecological notes. The specimens were found between

19:00 and 23:00 around a water pool. Most of specimens

were found on rock boulders, ca. 0.5—1.0 m above the

ground, few frogs were in the water. The surrounding habi-

tat was secondary karst forest of medium and small hard-

woods mixed with shrubs and vines.

Distribution. The species was recently described from

Cao Bang Province and is currently known only from its

type locality in Vietnam (Pham et al. 2016c).

Rhacophoridae

Gracixalus waza Nguyen, Le, Pham, Nguyen, Bonkows-

ki & Ziegler, “2012” 2013 (Fig. 4h)

Specimens examined. Five adult females (IEBR 3840:

SVL 34 mm, IEBR 3841: SVL 32 mm, IEBR 3842: SVL

34 mm, IEBR 3843: SVL 35 mm, IEBR 3844: SVL 31

mm); seven adult males JEBR 3845: SVL 33 mm, IEBR

3846: SVL 32.5 mm, IEBR 3847: SVL 32 mm, IEBR

3848: SVL 33 mm, IEBR 3849: SVL 31 mm, IEBR 3850:

SVL 34 mm, IEBR 3851: SVL 30 mm), and two subadults

(IEBR 3852: SVL 24.2 mm, IEBR 3853: SVL 25 mm)

collected by T.Q. Nguyen et al. in April and May 2012, at

elevations between 462 and 655 m asl.

Morphological characters. Head as long as wide (HL

10.5—13.9 mm, HW 10.9—14.2 mm), convex above; snout

round anteriorly from dorsal view, slightly protruding, its

length (SL 4.8—6.4 mm) longer than horizontal diameter

of eye (ED 4.0—5.5 mm); canthus rostralis round, loreal

region oblique, concave; interorbital region flat, broader

than upper eyelid OD 3.1—4.2 mm, UEW 2.7—3.8 mm),

as broad as internarial distance (IND 3.2—4.1 mm); nos-

tril round, closer to tip of snout than to the eye (NS 2.2—3.0

mm, EN 3.2-4.0 mm); pupil oval, horizontal; tympanum

distinct (TD 1.8—2.6 mm), round, half of the eye diame-

ter; pineal ocellus absent; spinules on upper eyelid absent;

vomerine teeth absent; choanae small, oval; tongue cor-

date, deeply notched posteriorly; males with a pair of vo-

cal sacs at base of jaw. Arm short, about half of hand

length; fingers free of webbing; relative length of fingers

I<II<IV<III; tips of fingers with well-developed discs with

distinct circum-marginal grooves; subarticular tubercles

distinct, blunt, round; nuptial pads prominent, oval; out-

er palmar tubercle divided into two; relative length of toes

I<II<IN<V<IV; tips of toes with well-developed discs with

distinct circum-marginal grooves; webbing formula I1-

11/31/2-21111-21V2-1V; subarticular tubercles distinct;

inner metatarsal tubercle small; outer metatarsal absent;

tibio-tarsal articulation reaching to tip of snout. Dorsal sur-

face of head and body smooth; posterior part of tympa-

©ZFMK

Amphibians of Ha Lang Forest, Vietnam 47

num, flank and lateral sides of limbs with small, flattened

granules; dorsolateral folds absent; supratympanic fold

distinct; throat and chest smooth, belly and ventral sur-

face of thigh granular; dermal appendage at vent absent.

Coloration in life: Background of dorsal surface of head,

body and limbs greyish green to moss-green; a dark

brown, blotched pattern between eyes bifurcating into two

bands continuing posteriorly on the back; a dark stripe

present in the middle of posterior part of dorsum. Fore-

limb, dorsal parts of thigh, tibia, and foot moss-green with

some dark brown bands; throat and chest white with dark

brown marbling; belly immaculate white (determination

after Nguyen et al. 2013a).

Ecological notes. The specimens were found between

19:00—23:30 on leaves and a limestone cliff, about 0.3—1.2

m above the ground, near the cave entrances and in the

valleys surround by limestone cliffs, relatively far from

water sources. The surrounding habitat was secondary for-

est consisting of medium and large hardwoods, shrub and

liane.

Distribution. In Vietnam, this species has been only re-

ported from Cao Bang Province (Nguyen et al. 2013a).

Kurixalus bisacculus (Taylor, 1962) (Fig. 5a)

Specimens examined. One adult female (IEBR 3854: SVL

34 mm), and eight adult males TEBR 3855: SVL 28 mm,

IEBR 3856: SVL 29 mm, IEBR 3857: SVL 28.5 mm,

IEBR 3858: SVL 30 mm, IEBR 3860: SVL 31 mm, IEBR

3862: SVL 31 mm, IEBR 3863: SVL 31 mm, IEBR 3864:

SVL 31 mm) collected by T.Q. Nguyen in April and May

2012, at elevations between 352 and 669 m asl.

Morphological characters. Head longer than wide (HL

10.0-14.8 mm, HW 9.4—14.1 mm); snout pointed anteri-

orly, longer than eye (SL 4.5—6.0 mm, ED 3.6—4.4 mm);

nostril closer to tip of snout than to eye (NS 2.0—2.8 mm,

EN2.6—3.1 mm); interorbital distance wider than internar-

ial distance (IOD 3.4—4.4 mm, IND 2.7—3.6, mm); tym-

panum distinct, smaller than eye (TD 1.9-2.6 mm, ED

3.64.4 mm); vomerine teeth in two ridges, arising near

inner edges of choanae; males with vocal sacs. Fingers free

of webbing, tips of fingers with enlarged discs; relative

length of fingers I<H<IV<II; toes webbed, webbing for-

mula [2/3-1111/4-11111/4-11V1-1/4V; relative length of toes

I<II<III<V<IV; inner metatarsal tubercle small; outer

metatarsal absent; tibio-tarsal articulation reaching be-

tween eye and tip of snout. Some scattered flat tubercles

on the head, eyelids, and occiput, sparse on dorsum, more

dense and larger on flanks, fine granules on rump; chin

granular, chest nearly smooth; venter and lower part of

sides granular.

Coloration in life: Dorsal head and body light or red-

dish brown with green marking, occiput with a dark green

marking in triangular shape; tympanum brownish; chin

cream with dark spots; throat, chest, venter, and underside

Bonn zoological Bulletin 66 (1): 37—53

of limbs cream (determination after Taylor 1962; Yu et al.

2010).

Ecological notes. The specimens were found between

19:00 and 23:30 on leaves, about 0.5—1.5 m above the

ground, near cave entrances and water rivulets. The sur-

rounding habitat was secondary forest consisting of medi-

um and large hardwoods, shrub and liane.

Distribution. Yu et al. (2010) stated that the distribution

of K. bisacculus extends from Vietnam over Thailand,

Cambodia, North and Central Laos to the South of Chi-

na.

Polypedates megacephalus Hallowell, 1861 (Fig. 5b)

Specimens examined. One adult male (IEBR 3865: SVL

62.0 mm) and one adult female (EBR 3866: SVL 80.0

mm) collected by T.Q. Nguyen et al. in April and May

2012, at elevations between 461 and 669 m asl.

Morphological characters. Head longer than wide (HL

21.7—30.8 mm, HW 19.8—28.6 mm); vomerine teeth pres-

ent; snout pointed, longer than eye (SL 10.5—13.9 mm, ED

8.3—10.2 mm); canthus rostralis distinct, slightly concave;

loreal region vertical; nostrils closer to tip of snout than

to eye (EN 6.89.1 mm, NS 3.24.4 mm); interorbital dis-

tance wider than internarial distance and upper eyelid

(lOD 7.4—-8.8 mm, IND 5.4—6.4 mm, UEW 6.4—7.3 mm);

tympanum distinct, approximately half of eye diameter

(TD 4.4-5.3 mm); vomerine teeth present; tongue notched

posteriorly; external vocal sacs absent. Fingers free of web-

bing; relative length of fingers I<H<IV<III; tips of finger

with enlarged discs; relative length of toes I<II<III<V<IV;

webbing formula I1-1111/4-11/21I11/2-21V2-1/4V; subar-

ticular tubercles present; outer metatarsal tubercle pres-

ent; tibio-tarsal articulation reaching to tip of snout. Dor-

sal skin and upper part of flanks smooth; lower part of

flank, belly and ventral surface thighs granular; supratym-

panic fold present.

Coloration in life: Dorsum beige yellow or brown, with

dark blotches; an X-shaped pattern present on neck; limbs

with dark transverse bars; dark brown stripe bordering

supratympanic fold from eye beyond arm; flanks some-

times with few dark brown spots; back of thighs with large

white spots; belly yellowish white (determination after

Ziegler et al. 2006; Kuraishi et al. 2012).

Ecological notes. The specimens were found between

19:00 and 21:00, on tree branches near a water pool, about

1.5—3 m above the ground. The surrounding habitat was

secondary karst forest of small hardwoods mixed with

shrubs and vines.

Distribution. In Vietnam, this species was reported from

Lang Son Province in the North southwards to Quang Binh

Province. Elsewhere, the species is known from China,

Myanmar, Laos, Cambodia and Thailand (Nguyen et al.

2009; Kuraishi et al. 2012).

©ZFMK

48 Cuong The Pham et al.

Fig. 5. a) Kurixalus bisacculus, b) Polypedates megacephalus, c) PR mutus, d) Rhacophorus dennysi, e) R. kio, f) R. maximus,

g) Theloderma asperum, h) T: corticale. Photos: C.T. Pham, T.Q. Nguyen, and H.T. An.

Bonn zoological Bulletin 66 (1): 37-53 ©Z7FMK

Amphibians of Ha Lang Forest, Vietnam 49

Polypedates mutus (Smith, 1940) (Fig. 5c)

Specimens examined. One adult female (IEBR 3868: SVL

63.9 mm) collected by T.Q. Nguyen et al. on 18 October

2011, at an elevation of 620 m asl.; and one adult female

(IEBR 3869: SVL 68.4 mm) collected by T.Q. Nguyen et

al. on 8 April 2012, at an elevation of 606 m asl.

Morphological characters. Head longer than wide (HL

23.3—24.3 mm, HW 22.0—22.8 mm); snout pointed, longer

than eye (SL 10.2-10.7 mm, ED 7.8—8.1 mm); canthus

rostralis distinct, slightly concave; loreal region vertical;

nostrils closer to tip of snout than to eye (EN 6.5—6.8 mm,

NS 3.3-3.8 mm); interorbital distance wider than internar-

ial distance and upper eyelid (IOD 7.8-8.1 mm, IND

5.3-5.6 mm, UEW 5.8—6.0 mm); tympanum distinct, ap-

proximately half of eye diameter (TD 4.5—4.8 mm);

vomerine teeth present; tongue notched posteriorly. Fin-

gers free of webbing; relative length of fingers

I<II<IV<III; tips of finger with enlarged discs; relative

length of toes I<II<IN<V<IV; webbing formula I1-11I1/4-

11/21111/2-21V2-1/4V; subarticular tubercles present; out-

er metatarsal tubercle present; tibio-tarsal articulation

reaching to tip of snout. Dorsal skin and upper part of

flanks smooth; lower part of flanks, belly and ventral sur-

face of thighs granular; supratympanic fold present.

Coloration in life: Dorsum beige yellow or brown, with-

out dark blotches; limbs with dark transverse bars; brown

stripe bordering supratympanic fold from eye to arm; back

of thigh with small white spots; belly yellowish white (de-

termination after Ziegler et al. 2006; Kuraishi et al. 2012).

Ecological notes. The specimens were found between

19:00 and 20:00, on tree branches near a water pool, about

1.5—3 m above the ground. The surrounding habitat was

secondary karst forest of small hardwoods mixed with

shrubs and vines.

Distribution. This is a widespread species in Vietnam.

Elsewhere, the species has been reported from China,

Myanmar, Laos, Cambodia and Thailand (Nguyen et al.

2009; Kuraishi et al. 2012).

Rhacophorus dennysi (Blanford, 1881) (Fig. 5d)

Specimens examined. One adult female (IEBR 3870: SVL

94 mm) collected by T.Q. Nguyen et al. on 5 May 2012,

at an elevation of 481 m asl.; one adult male (IEBR 387:

SVL 63.9 mm) collected by T.Q. Nguyen et al. on 9 May

2012, at an elevation of 497 m asl.

Morphological characters. Head broader than long (HW

22.6—31.5 mm, HL 21.5—30.8 mm); nostril oval, closer to

tip of snout than to eye (NS 5.1—6.1 mm, EN 6.4—8.3 mm);

interorbital distance wider than internarial distance and up-

per eyelid (OD 7.7—10.7 mm, IND 6.7—8.5 mm, UEW

5.7—7.5 mm); tympanum round, smaller than eye diame-

ter (TD 3.9-5.5 mm, ED 7.6—9.5 mm); vomerine teeth

present; tongue notched behind; the male without exter-

Bonn zoological Bulletin 66 (1): 37—53

nal vocal sacs. Tips of fingers with enlarged discs; rela-

tive length of fingers I<H<IV<II; webbing formula I1-

1111/4-11/41111/2-1/21V; tips of toes with enlarged discs;

relative length of toes I<I<III<V<IV; toes completely

webbed; subarticular tubercles present; inner metatarsal

tubercle present; outer metatarsal tubercle absent; nuptial

pad present in male; tibio-tarsal articulation reaching be-

tween eye and tip of snout. Dorsum and flanks smooth;

belly and ventral surface of thighs granular; supratympan-

ic fold distinct.

Coloration in life: Dorsum green with white-gray blotch-

es; fringe on outer edge of outer finger, forearm and foot

brightly colored; lower part of flanks and ventral surface

whitish, in part dark speckled; gular region green anteri-

orly; webbing of fingers and toes grayish (determination

after Ziegler 2002; Fei et al. 2010).

Ecological notes. The specimens were found between

19:00 and 20:00, on tree branches near a water pool in a

rice field, about 1—2 m above the ground. The surround-

ing habitat was secondary karst forest of small hardwoods

mixed with shrubs and vines.

Distribution. In Vietnam, this species has been report-

ed from Cao Bang Province in the North southward to

Quang Binh Province. Elsewhere, the species has been re-

ported from China, Myanmar and Laos (Nguyen et al.

2009).

Rhacophorus kio Ohler & Delorme, 2006 (Fig. Se)

Specimens examined. Five adult males (IEBR 3872: SVL

74 mm, IEBR 3873: SVL 69 mm, IEBR 3874: SVL 72

mm, IEBR 3875: SVL 67 mm, IEBR 3876: SVL 69 mm),

and one adult female (EBR 3877: SVL 85 mm) collect-

ed by T.Q. Nguyen et al. in April and May 2012, at ele-

vations between 447 and 532 m asl.

Morphological characters. Head broader than long (HW

22.8—28.5 mm, HL 24.1—30.1 mm); nostril oval, closer to

tip of snout than to eye (NS 5.2—5.9 mm, EN 6.3—7.1 mm);

interorbital distance wider than internarial distance and up-

per eyelid JOD 7.8—-11.4 mm, IND 6.1—8.0 mm, UEW

5.1—6.0 mm); tympanum round, smaller than eye diame-

ter (TD 4.3-5.2 mm, ED 7.9-8.9 mm); vomerine teeth

present; tongue notched posteriorly; males without exter-

nal vocal sacs. Tips of fingers and toes enlarged into discs,

relative length of fingers I<II<IV<III; webbing formula

I1-1110-OIII0-O1V; relative length of toes I<II<III<V<IV;

toes fully webbed; inner metatarsal tubercle present; out-

er metatarsal tubercle absent; tibio-tarsal articulation

reaching to the eye. Dorsum smooth; ventral side of bel-

ly and thighs and lower part of flanks ganular; fringes on

outer edge of forearm well developed; supratympanic fold

distinct; dermal appendage above vent present.

Coloration in life: Dorsal surface of head, body and up-

per part of flanks green with small white spots; lower part

of flanks dark brown with yellow spots; a distinct black

©ZFMK

50 Cuong The Pham et al.

spot in armpit; throat, chest, belly and lower part of thighs

yellow (determination after Ohler & Delorme 2006).

Ecological notes. The specimens were found between

20:00 and 22:00, on tree branches near a water pool, about

2—4 m above the ground. The surrounding habitat was sec-

ondary karst forest of small, medium and large hardwoods

mixed with bamboo, shrub and liane.

Distribution. In Vietnam, this species has been report-

ed from Lao Cai Province in the North southwards to Gia

Lai Province. Elsewhere, the species has been reported

from India, China, Laos, Thailand and Cambodia (Nguyen

et al. 2009).

Rhacophorus maximus Ginther, 1858 (Fig. 5f)

Specimen examined. One adult male JEBR 3878: SVL

73.1 mm) collected by T.Q. Nguyen on 22 April 2015, at

an elevation of 526 m asl.

Morphological characters. Head broader than long (HW

26.5 mm, HL 25.4 mm); nostril oval, closer to eye than

to tip of snout (NS 6.3 mm, EN 5.7 mm); interorbital dis-

tance wider than internarial distance and upper eyelid

(OD 8.5 mm, IND 7.9 mm, UEW 5.9 mm); tympanum

round, smaller than eye diameter (TD 4.4 mm, ED 8.3

mm); vomerine teeth present; tongue notched behind; the

male without external vocal sacs. Tips of fingers with en-

larged discs; relative length of fingers I<II<IV<III; web-

bing formula I1-1111/4-1II11/2-1/21V; tips of toes with en-

larged discs; relative length of toes I<II<III<V<IV; toes

fully webbed; subarticular tubercles present; inner

metatarsal tubercle present; outer metatarsal tubercle ab-

sent; nuptial pad present; tibio-tarsal articulation reach-

ing between eye and tip of snout. Dorsum smooth; belly,

ventral surface of thighs, and lower part of flank ganular;

supratympanic fold distinct.

Coloration in life: Dorsal surface of head and body uni-

formly green; a narrow white stripe present along the

flanks; ventral surface cream (determination after Anders

& Rai 2002; Hecht et al. 2013).

Ecological notes. The specimen was found at ca. 20:00,

on tree branches near a water pool, about 4 m above

ground. The surrounding habitat was secondary karst for-

est of medium and large hardwoods, shrub and liane.

Distribution. In Vietnam, this species has been report-

ed from Bac Giang, Dien Bien, and Thanh Hoa provinces

(Nguyen et al. 2009; Nguyen et al. 2015; Pham et al.

2016a). This is the first record of R. maximus from Cao

Bang Province. Elsewhere, the species has been reported

from India, China and Thailand (Nguyen et al. 2009; Luu

et al. 2014).

Theloderma asperum (Boulenger, 1886) (Fig. 5g)

Specimens examined. One adult female (IEBR 3879: SVL

28.5 mm) collected by T.Q. Nguyen et al. on 16 October

Bonn zoological Bulletin 66 (1): 37—53

2011, at an elevation of 544 m asl.; one adult male IEBR

3880: SVL 29.5 mm) collected by T.Q. Nguyen on 12

April 2012, at an elevation of 526 m asl.; one adult male

CEBR 3881: SVL 26 mm) collected by T.Q. Nguyen et

al., on 2 May 2012, at an elevation of 601 m asl.; and one

adult male TEBR 3882: SVL 29.8 mm) collected by C.T.

Pham et al., on 8 June 2014, at an elevation of 586 m asl.

Morphological characters: Head longer than wide (HL

10.59-11.81 mm, HW 11.73—12.01 mm); snout round,

longer than eye (SL 4.1—5.1 mm, ED 3.9-4.6 mm); can-

thus rostralis indistinct; loreal region slightly concave; nos-

tril closer to tip of snout than to eye (NS 1.05—1.48 mm,

EN 2.61—2.71 mm); interorbital distance wider than in-

ternarial distance and upper eyelid JOD 3.1—3.7 mm, IND

2.5—2.8 mm, UEW 2.3—2.4 mm); tympanum distinct (TD

2.42.5 mm); vomerine teeth absent; tongue notched be-

hind; males without external vocal sacs. Fingers free of

webbing, tips of fingers with enlarged discs; relative length

of fingers I<H< IV<III; tips of toes enlarged into round

discs; webbing formula I0-1110-1111/4-11/41TV1-0V; rel-

ative length of toes I<II<IH<V<IV; inner metatarsal tu-

bercle present, small; tibio-tarsal articulation reaching to

tip of snout. Skin on dorsum and flanks with granular tu-

bercles; throat and chest smooth; venter granular;

supratympanic fold absent.

Coloration in life: Dorsal surface greyish brown with

large white blotches on head, loreal region, anterior part

of dorsum, upper part of flanks and hip; hind limbs with

dark transverse bars; head with short brown stripe between

eyes; ventral surface blackish with white marbling; iris

pinkish brown (determination after Bourret 1942; Taylor

1962; Neang & Holden 2008).

Ecological notes. Two specimens were found between

19:00 and 23:00 in a tree hole, about 0.5-1.0 m above the

ground, and two other ones were found on leaves near a

cave entrance. The surrounding habitat was secondary

karst forest of small, medium and large hardwoods, shrub

and liane.

Distribution. In Vietnam, this species has been report-

ed from Lai Chau and Lao Cai provinces in the North

southwards to Lam Dong and Dong Nai provinces. Else-

where, the species has been reported from India, China,

Myanmar, Laos, Thailand, Cambodia and Malaysia

(Nguyen et al. 2009).

Theloderma corticale (Boulenger, 1903) (Fig. 5h)

Specimens examined: One adult female (IEBR 3883: SVL

54.1 mm) collected by T.Q. Nguyen et al. on 1 May 2012,

at an elevation of 484 m asl.; one adult female IEBR

3884: SVL 67 mm), and one adult male TEBR 3885: SVL

69 mm) collected by T.Q. Nguyen et al. on 6 May 2012,

at an elevation of 537 m asl.

Morphological characters: Head wider than long (HL

20.8—28.1 mm, HW 21.5—28.9 mm); snout longer than eye

©ZFMK

Amphibians of Ha Lang Forest, Vietnam 51

Corn WwW UD ~ oO WO

Fig. 6. Species richness of the amphibian families from Ha Lang forests, Cao Bang Province.

diameter (SL 18.8—12.3 mm; ED 6.4—7.3 mm); canthus

rostralis round; loreal region concave; interorbital distance

wider than internarial distance and upper eyelid (IOD

7.2—9.2 mm, IND 4.2—5.3 mm, UEW 4.5—5.8 mm); nos-

tril closer to tip of snout than to eye (NS 2.7—3.5 mm; EN

6.5—8.2 mm); tympanum oval (TD 4.2—5.6mm); vomer-

ine teeth present; tongue notched posteriorly; the male

without external vocal sacs. Fingers free of webbing; rel-

ative length of fingers I<II<IV<III; tips of fingers and toes

enlarged into round discs; webbing formula I0-1/2II0-

III10-11V1-0V; relative length of toes I<XH<III<V<IV; sub-

articular tubercles present; inner metatarsal tubercle

present; outer metatarsal tubercle absent; tibio-tarsal ar-

ticulation reaching between eye and tip of snout. Dorsal

surface of head, body and limbs covered with tubercles or

warts of different sizes; ventral skin with small tubercles;

supratympanic fold absent; nuptial pad present in the male.

Coloration in life: Dorsum green marbled with reddish

brown spots; flanks yellow, mottled or marbled with black;

dark brown bars present on upper surface of fore and hind

limbs; ventral surface yellow with green marbling (deter-

mination after Inger et al. 1999; Orlov et al. 2006).

Ecological notes. The specimens were found between

21:00 and 23:00, on leaves next to a forest path. The sur-

rounding habitat was secondary karst forest of small,

medium and large hardwoods, shrub and liane.

Distribution. This species is currently known only from

Vietnam, from Ha Giang and Cao Bang provinces in the

North southwards to Quang Binh Province (Nguyen et al.

2009; Luu et al. 2013).

Bonn zoological Bulletin 66 (1): 37—53

DISCUSSION

A total of 21 species of amphibians were recorded from

the Ha Lang District, Cao Bang Province. Rhacophoridae

and Ranidae were the two most species-rich families with

nine and five recorded species, respectively (Fig. 6). Three

species, Odorrana bacboensis, O. graminea, and Rha-

cophorus maximus, are recorded for the first time from

Cao Bang Province. Among the reported species, three

species, Odorrana mutschmanni, Gracixalus waza, and

Theloderma corticale, are currently known only from Viet-

nam. Remarkably, the karst forest of Ha Lang District al-

so harbors several threatened species, comprising one

species (Rhacophorus kio) listed as Vulnerable in the

IUCN Red List (2016) and two species (R. kio and Thelo-

derma corticale) listed as Endangered in the Red Data

Book of Vietnam (2007). Although the karst forest in Ha

Lang District harbors a considerable number of endemic

and rare species, its biodiversity is currently threatened due

to quarrying for cement and road construction, expand-

ing agriculture, and illegal timber logging (Pham et al.

2016c). Additional surveys are required to obtain further

data about the actual biodiversity of this karst forest. The

establishment of a new protected area should be serious-

ly considered in order to protect the remaining karst forests

and their unique biodiversity in Cao Bang Province.

Acknowledgements. We thank the directorate of the Forest Pro-

tection Department of Cao Bang for support of our field work

and issuing relevant permits. Many thanks D.T. Le (Hanoi), T.

Lehmann, M. Bernardes and M. van Schingen (Cologne) for their

assistance in the field. We thank E. Sterling (New York) and K.

Koy (Berkeley) for providing the map. For the support of joint

©ZFMK

52 Cuong The Pham et al.

research projects we cordially thank C.X. Le, T.H. Tran (IEBR,

Hanoi), and T. Pagel and C. Landsberg (Cologne Zoo). Field sur-

veys in Cao Bang Province were partially funded by the Nagao

Natural Environment Foundation (Japan), the Deutsche

Forschungsgemeinschaft (DFG) - International Collaboration

Grant (BO 1907/ 11-1), and the Cologne Zoo (Germany). Re-

search of T.Q. Nguyen in Germany and equipment were funded

by the Alexander von Humboldt-Foundation (VIE 1143441).

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Bonn zoological Bulletin 66 (1): 55—60

April 2017

Rediscovery and range extension

of the Guinean skink Trachylepis keroanensis (Chabanaud, 1921)

(Reptilia: Squamata: Scincidae)

Johannes Penner'”’, Joseph Doumbia’, N’Goran Germain Kouamé’, Laurent Chirio’,

Laura Sandberger-Loua’', Wolfgang Bohme® & Michael F. Barej'

' Museum ftir Naturkunde, Leibniz Institute Evolution and Biodiversity Science, Invalidenstr. 43, D-10115 Berlin, Germany

>" Corresponding author: University of Freiburg, Chair of Wildlife Ecology & Management, Tennenbacher Str. 4,

D-79106 Freiburg, Germany, johannes.penner@Qwildlife.uni-freiburg.de.de

> Envisud Guinée; Quartier: Kipé T2 commune de Ratoma; 030BP:558 Conakry, République de Guinée

* Jean Lorougnon Guédé University, UFR-Environment, Department of Biology & Animal Physiology,

Daloa, BP 150, Cote d’Ivoire

° PO. Box 87811, Riyadh 11652, Saudi Arabia

° Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute of Animal Biodiversity, Adenauerallee 160,

D-53113 Bonn, Germany

Abstract. We report the rediscovery of the skink Trachylepis keroanensis (Chabanaud, 1921) 90 years after its descrip-

tion. For the first time pictures of live specimens are shown and the known, now extended, distribution is presented. The

clear morphological differences (body shape, colouration and most notably ratio tail length to body length) towards Tra-

chylepis perrotetii (Duméril & Bibron, 1839), which justify the species status, are confirmed.

SHORT NOTE

On the African continent and neighbouring areas, the

genus Trachylepis is the most speciose one in the family

Scincidae. The genus currently holds at least 80 taxa (Uetz

et al. 2017) and occurs in all major biomes. However, tax-

onomy of many species is unresolved and likely to change

in the future.

In West Africa twelve species of the genus Trachylepis

are known, out of which eight can be found in Guinea

(Bohme et al. 2011; Trape et al. 2012). Most of them are

widespread and locally abundant. One exception is 7ra-

chylepis keroanensis (Chabanaud, 1921), which for a long-

time was considered a subspecies of Trachylepis perrotetii

(Duméril & Bibron, 1839).

Trachylepis perrotetii was first described in 1839 by

Dumeéril and Bibron as Euprepes perrotetii and 1s distrib-

uted all over West African savannahs, to Central Africa and

Sudan (Stoll 2008; Trape et al. 2012). The original descrip-

tion is based on a single specimen from Senegal (MNHN

2928). The genus name changed later to Mabuya Fitzinger,

1826; also erroneously spelled Mabuia (Cuvier 1829) or

Mabouya (Duméril & Bibron 1839). See Bauer (2003) for

a discussion on the correct genus name.

Nearly one century after the species description, the sub-

species Mabuya perrotetii keroanensis was described by

Chabanaud (1921; therein spelled as “Mabuia Perroteti

keroanensis). This author reported a number of herpeto-

Received: 10.11.2015

Accepted: 01.04.2016 / Updated: 31.03.2017

logical specimens collected on a survey in south-eastern

Guinea. In addition to Mabuya perrotetii, which he de-

scribed as very common in the area, Chabanaud recog-

nised the new taxon which he assumed to be closely re-

lated to the latter species. The description is based on two

specimens, “Kérouané” was given as the type locality and

the two syntypes are deposited in the Muséum d’ Histoire

Naturelle in Paris, France (MNHN 1921-323 & 1921-324).

Distinguishing characters from the nominate taxon were

a more slender body shape and a much longer tail in re-

lation to snout-vent length. Moreover, colouration differed.

T! keroanensis (Chabanaud, 1921) possessed a brown to

olive back and brown to black longitudinal lines (Cha-

banaud 1921). In addition, it exhibited white supralabial

scales which lead into a white lateral band ending at the

groin. Contrastingly, Trachylepis perrotetii (Duméril &

Bibron, 1839) was originally described as exhibiting a

“brown greyish” dorsal colouration with yellowish blotch-

es (Duméril & Bibron 1839; Fig. 1).

Nearly another century later, the genus name changed

from Mabuya to Trachylepis (Bauer 2003). Recently,

Bohme et al. (2011) provided an updated country wide

check list for Guinea. In accordance with Stoll (2008) they

noted that, based on the two syntypes, Trachylepis keroa-

nensis (Chabanaud, 1921) is indeed very different to Tra-

chylepis perrotetii (Dumeéril & Bibron, 1839) and resur-

rected the species, which was confirmed by Trape et al.

(2012). However, in the standard reference database the

Corresponding editor: Ph. Wagner

56 Johannes Penner et al.

& D; ZMB 82943: E & F).

taxon is still listed as a subspecies/synonym of 7! perrotetii

(Dumeéril & Bibron, 1839) (Uetz et al. 2017).

To our knowledge Trachylepis keroanensis (Chabanaud,

1921) has not been recorded since its description and was

not mentioned anywhere else in the literature. However,

the exact number of museum specimens remains unclear

because it cannot be ruled out that some specimens of 7.

keroanensis (Chabanaud, 1921) were misleadingly clas-

sified as T. perrotetii (Duméril & Bibron, 1839). In addi-

tion, a second slender skink of similar size and body shape

is known from Cote dIvoire and further eastwards (Trape

et al. 2012) — Trachylepis buettneri (Matschie, 1893). Tra-

Bonn zoological Bulletin 66 (1): 55-60

Pe he” 2

a? ta

~ , ~w! = j > "

on : med -

Bye BS =t Pati YS

Fig. 1. Photographs of live specimens Trachylepis perrotetii (ZMB 83362: A & B) and Trachylepis keroanensis (ZFMK 96261: C

chylepis buettneri was first collected in Bismarckburg (to-

day near Konkoa, prefecture Sotouboua), Togo, and de-

scribed by Matschie in 1893. Few differences exist (Tab.

1): mainly the black colouration on the back which con-

sists of irregular black spots in 7! buettneri (Matschie,

1893) (see photograph by Rodel in Trape et al. 2012) and

of distinct lines in 7’ keroanensis (Chabanaud, 1921) (Fig.

1). Trachylepis buettneri (Matschie, 1893) seems also to

be generally larger than 7. keroanensis (Chabanaud, 1921)

(Tab. 1). Head scales are very similar (Fig. 2).

From May to August 2011 three herpetological surveys

were initiated in eastern Guinea. On four occasions

©OZFMK

Rediscovery and range extension of the Guinean skink Trachylepis keroanensis S7

Fig. 2. Details of the dorsal head scales of the three taxa of slen-

der long-tailed skinks. From top to bottom: Trachylepis keroa-

nensis (ZMB 82943), Trachylepis buettneri (SMNS 08779) and

Trachylepis sudanensis (AMNH R10934). The scale bar repre-

sents 1mm.

(24.6.11, 28.6.11, 18.7.11, 22.12.11) and at four sites 7:

keroanensis (Chabanaud, 1921) was found. Three sites are

located around the town of Kérouané thus in close vicin-

ity of the type locality. The fourth one is north of Kissi-

dougou which is a larger town approximately 100km west

of Kérouané, Fig. 3. Three specimens were found during

the night, sleeping at 1m to 1.5m height in dense and high

grasses (Panicum sp.) at altitudes of 454m a.s.1., 664m

a.s.l. and 701m a.s.1. respectively. Interestingly, Trachylepis

buettneri (Matschie, 1893) was found under similar cir-

cumstances in Comoé National Park in Céte dIvoire

(Rodel et al. 1997). Specimens were caught by hand, pho-

Bonn zoological Bulletin 66 (1): 55-60

tographed, euthanised, DNA samples extracted and then

fixed in 4% Formaldehyde or 70% Alcohol. A fourth spec-

imen was collected by local villagers in Founouk-

ouroudou (545m a.s.1.) and was preserved as above. Spec-

imens are currently stored in the collections of the Natu-

ral History Museums in Bonn (ZFMK), Berlin (ZMB), in

the private collection of Laurent Chirio (Tab. 1) and in

Paris (MNHN, type specimens).

Our records were located close to rivers and fields and

place the species in the biome “Open broadleaved decid-

uous forest’, also called Guinean savannah. The precise

distribution and ecology remains unknown, and T: keroa-

nensis (Chabanaud, 1921) is speculated to be a rare species

with a larger distribution. Recently, this species was also

recorded from Mali (Kayes Province: Harvey et al., un-

published) and based on the distribution of the biomes we

speculate that its distribution might also extend into ad-

jacent areas in Cote d’Ivoire. Whether 7. keroanensis (Cha-

banaud, 1921) occurs in sympatry with 7 buettneri

(Matschie, 1893) or whether the two species exclude each

other is also unclear. Furthermore, the exact taxonomic re-

lationships between 7. perrotetii (Duméril & Bibron,

1839), 7. buettneri (Matschie, 1893), 7: keroanensis (Cha-

banaud, 1921) and 7. sudanensis (Schmidt, 1919) (a third

skink of similar body shape to 7. buettneri (Matschie,

1893) and T. keroanensis (Chabanaud, 1921), currently re-

garded as a synonym of 7! buettneri (Matschie, 1893); see

Uetz et al. 2017) require further investigations (BOhme et

al. 2011).

Trachylepis sudanensis was described from Faradje,

Garamba, South of Garamba River, Uele region and Yaku-

luku all in Sudan (Schmidt 1919) and according to the

original description lacks the distinct dorsal colouration

(present in 7? buettneri and T: keroanensis (Chabanaud,

1921)). In the following we summarise the current know-

ledge.

Our findings confirm distinctness on the species level

of T. keroanensis (Chabanaud, 1921) and 7. perrotetii

(Dumeril & Bibron, 1839). The main distinguishing char-

acters are body shape (slender in 7. keroanensis (Cha-

banaud, 1921) and stout in 7’ perrotetii (Duméril & Bibron,

1839), the ratio between tail and body length (between

2.63 and 3.4 in T. buettneri (Matschie, 1893), 7! keroa-

nensis (Chabanaud, 1921) and T. sudanensis (Schmidt,

1919) but between 1.57 and 1.68 in T. perrotetii (Duméril

& Bibron, 1839) as wells as the different colourations.

Colouration of live specimens is that only T. perrotetii

(Dumeéril & Bibron, 1839) has regular white spots on the

lateral side with a brown background on the upper half and

an orange to yellow lower background on the lower half

(Fig. 1). The other three species have a distinct white line

on the lateral side but different dorsal colourations: black

spots in 7? buettneri (Matschie, 1893), black lines in 7:

keroanensis (Chabanaud, 1921) and uniform brownish in

T! sudanensis (Schmidt, 1919).

©ZFMK

58 Johannes Penner et al.

Table 1. Selected measurements for the four species discussed. Literature records for T. perrotetii were chosen so that they represent proximate

populations. The column “source” gives the collection numbers for the new specimens of Trachylepis keroanensis or the literature source (1 = Trape

et al. 2012; 2 = Schmidt 1919). * Is a specimen in the private collection of Laurent Chirio. The following abbreviations (all measurements in mm)

are used: ToLe = total length, SVL = snout-vent length, TaL = tail length, TaL/SVL = ratio of tail to body, HL = head length, HW = head width,

SAM = scales around midbody, FLL = fore limb length, HL = hind limb length, SupC = number of supraciliar scales; R = right, L = left.

Species Source ToLe SVL TaL TaL/SVL HL HW SAM FLL HLL SupC

T. buettneri (1) 350 81 269 32 ND ND 26 ND ND 3-6

(rarely 28) (mostly 5)

T. keroanensis ZMB80183 238 56 182 3.25 11.7 7.8 29 22.1R 26.7R 4

22.0L 26.7L

T: keroanensis ZMB82943 228 60 168 2.80 10.6 7.3 28 20.2R 26.5R 6

20.3L 26.4L

T: keroanensis + ZFMK92661 178 49 129 2.63 9.0 6.0 28 SAR 20.0R 4

P20 19.8L

T. keroanensis 9114x* NA 61.5 NA NA 11 7.6 28 20.2R 25.2R 5/5

20.1L 25.2L

T. keroanensis (1) 230 ND ND ND ND ND 28 ND ND cs

254.3 58.5 199.1 3.40 1a 7.8 26 18.1 225

T: sudanensis (2) (221-304) (50-78) (192-226) (2.90-3.84) (11.7-15.0) (7.0-9.0) [n=8]or (16-21) (20-25) 5-7

[n=8] [n=10] [n=8] [n=10] [n=10] 28 [n=2] [n=10] [n=10]

28-30

T. perrotetii (1) 420 157 263 1.68 ND ND 30-32 ND ND 6

32-36

232.8 91.5 143.6 17. 20.4 135 32-34 26.0 33,3

T. perrotetii (2) (123-310) (47-123) (76-192) (1.56-1.62) (11.5—28.0) (7-19) [n=27] (14-33) (17-41) ND

[n=17] [n=27] [n=17] [n=27] [n=27] [n=27] [n=27]

Tissue samples of the morphologically similar 77 buet- REFERENCES

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synonym 7? sudanensis (Schmidt, 1919)) are not available,

so herein we cannot revise the respective taxa but point

out some morphological characters currently applied to

distinguish the 7rachylepis taxa of interest. The conser-

vation status of 7! keroanensis (Chabanaud, 1921) is un-

clear. Thus it is currently listed as “Data Deficient” on the

IUCN global Red list (UCN 2016) due to the absence of

ecological information. The species is very rarely encoun-

tered in the field, suggesting that it might be threatened

due to a possible low density and restricted distribution

range. However, it is completely unclear what natural

abundances are and how the species can cope with the cur-

rent anthropogenic alterations of its habitat.

Acknowledgements. We would like to thank all field guides and

involved parties who made the surveys possible. Our gratitude

also includes the Guinean authorities who issued the relevant per-

mits. Lauren Vonnahme (AMNH) kindly provided photographs

of Trachylepis sudanensis. Andreas Schmitz and one anonymous

reviewer provided helpful comments.

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tologique de I’ Afrique Occidentale. Bulletin du Comité d’E-

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imaux et d’ introduction a 1’ Anatomie Comparé. Nouvelle Edi-

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toire Naturelle Complete des Reptiles. Vol.5. Roret/Fain et

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©OZFMK

Rediscovery and range extension of the Guinean skink Trachylepis keroanensis 59

15° W 14° W 13° W 12° W

9°N

8° N-

7° N-

15° W 14° WwW 13° W 12° W

Globcover V2.2

[ 11 - Irrigated croplands

14 - Rainfed croplands

C_] 20 - Mosaic Croplands/Vegetation

C_] 30 - Mosaic Vegetation/Croplands

Se p ieee ie us

de =

Pie wt : 45 ty

Pay ry 9 J r rales

e 5 i a

11° W 10° W 9° W

12°N

10° N

- r 1

eS j Sp a f r =

sah 90 ia ; es

’ a? ai x

5 ts * Pa ey eS ty

: : - g : te

te 2 “ac r . aS = ae

3 v

re 7: F - & °

Bg SPS? -7°N

r “eve ae ‘ a"

b%: o ’ ‘ “

‘ ik

11° W 10° W

| 120 - Mosaic Grassland/Forest-Shrubland

| 130 - Closed to open shrubland

Cc] 140 - Closed to open grassland

Cc] 150 - Sparse vegetation

[| 40 - Closed to open broadleaved evergreen or semi-deciduous forest | 160 - Closed to open broadleaved forest regularly flooded (fresh-brackish water)

L | 50 - Closed broadleaved deciduous forest

C_] 60 - Open broadleaved deciduous forest

[| 70 - Closed needleleaved evergreen forest

{| 90 - Open needleleaved deciduous or evergreen forest

C_] 100 - Closed to open mixed broadleaved and needleleaved forest

L_] 110 - Mosaic Forest-Shrubland/Grassland

| 170 - Closed broadleaved forest permanently flooded (saline-brackish water)

| 180 - Closed to open vegetation regularly flooded

(| 190 - Artificial areas

| 200 - Bare areas

[Jj 210 - Water bodies

Fig. 3. Known occurrence records for the skink Trachylepis keroanensis in Guinea (its location on the African continent is depict-

ed in black in the bottom left corner). Underlying is the global land cover map of 2009 (after Arino et al. 2012). The star repre-

sents the city of Kérouané.

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60 Johannes Penner et al.

tetii (Reptilia; Squamata; Scincidae). Unpublished MSc The- Expedition, 1909-1915. Part I: turtles, crocodiles, lizards, and

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Bonn zoological Bulletin 66 (1); 55-60 ©ZFMK

Bonn zoological Bulletin 66 (1): 61-72

Community perception of natural history collections — an online survey

Jonas J. Astrin™ & Hannah C. Schubert!

' Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute of Animal Biodiversity,

Adenauerallee 160, D-53113 Bonn, Germany;

“E-mail: j.astrin@leibniz-zfmk.de

Abstract. Cultural, today mostly scientific, reflection on nature finds its physical manifestation in natural history collec-

tions (NHCs), which date back to the third century BCE at least. NHCs owe their existence to the formidable variation

of life and other natural (e.g., geological) phenomena. Documenting, ordering, understanding this variation, communi-

cating and showing it: these have been the classical functions of NHCs. What are the expectations for NHCs today, and

how is their performance judged? The present survey captured a snapshot of values and opinions regarding NHCs from

525 poll participants from predominantly North America and Europe, mostly based in academia (41%) and at NHCs (32%),

or students (10%). It was found that natural history collections are fascinating or highly interesting places for almost all

respondents (challenging the idea that the ‘dusty NHC’ is really such a wide-spread cliché). Basic research, collection

care, and educating the public were the three most often selected NHC core roles. The general importance of voucher-

ing is acknowledged by the poll participants, and treatment of type material is regarded as near-adequate. Molecular vouch-

ers, on the other hand, are considered to deserve more attention, reaching only medium levels of satisfaction, same as

NHC data accessibility, sample documentation, and taxonomic expertise at NHCs. Insufficient funding was the strongest

April 2017

concern of most survey participants.

Keywords. NHC; museums; herbaria; research collections; online poll; community opinion.

INTRODUCTION

Worldwide, an estimated 3,000,000,000 (3 billion) objects

(Soberén 1999, Wheeler et al. 2012) reside in about 8,000

natural history collections (NHCs): museums, herbaria,

botanical gardens, etc. (Kemp 2015). They form the ba-

sis of an object-oriented approach to the natural world

(Winker 2004) and offer the necessary baseline context for

obtaining new biodiversity knowledge (Cotterill 1995,

Lane 1996).

Together with their associated metadata, these unique

preserved objects, the “physical archive of our world”

(Pekarik 2003), inform taxonomy, systematics, evolution,

ecology, conservation, public and environmental health,

forestry, agriculture, and many other disciplines.

Classification of nature is a quintessentially human ac-

tivity (Foucault 1966, in Ellis 2008), and over the last cen-

turles, 1.e., since the introduction of Linnaean classifica-

tion (Linné 1758), a particularly strong focus of NHCs has

rested with creating stability in naming and with the sys-

tematization of life, producing a reliable and steadily up-

dated taxonomy (Tautz et al. 2003) within a pragmatic and

more or less harmonized system. This is an ongoing

process, and increasingly, molecular information is added

to morphological evidence, be it derived from ‘classical’

collections (Bi et al. 2013, Buerki & Baker 2016 and ref-

erences therein) or in the form of dedicated biobanks with-

in NHCs (Corthals & DeSalle 2005, Astrin et al. 2013).

Received: 27.01.2017

Accepted: 03.04.2017

But apart from a traditional focus on taxonomy (Wheel-

er 2009), systematics (Wen et al. 2015) and phylogeogra-

phy, over the last three decades, NHCs have consolidated

and are still expanding their role as a fundamental infra-

structural resource for ecological and environmental ques-

tions (Pyke & Ehrlich 2010) or conservation (Ponder et

al. 2001, Lyman 2011), including topics like climate

change, habitat conversion, pollutants and toxicants, bio-

diversity and ecosystem function loss, or introduction of

exotics (Dunnum & Cook 2012).

Analogously, public and environmental health benefit

are generated by NHCs, for example, by offering ways

of characterizing, mitigating, and predicting emerging in-

fectious diseases (DiEultis et al. 2016), as exemplified,

among others, by epidemiological investigations into Ly-

me disease (Persing et al. 1990), hantavirus (Sheldon &

Dittmann 1997), or West Nile virus (Fonseca et al. 2001).

Safety and food security issues (e.g., regarding agricul-

tural bioterrorism, Suarez & Tsutsui 2004) equally depend

on NHCs (SciColl International, in prep.).

The unifying feature in most of the manifold and in-

creasingly interdisciplinary approaches relying on NHC

specimens is their need for a retrospective view on nature.

Each of the specimens in NHCs is unique spatiotempo-

rally and opens a window on evolutionary processes

(Holmes et al. 2016) and past environmental conditions,

not uncommonly for species or populations already extinct

at time of investigation (e.g., Albano et al. 2014). Ideal-

Corresponding editor: R. S. Peters

62 Jonas J. Astrin & Hannah C. Schubert

ly, NHCs (individually or as a distributed network) offer

time series of objects that allow seamlessly monitoring and

investigating changing biodiversity and abiotic parameters

at different spatial scales and over years, decades, or some-

times even centuries (Shaffer et al. 1998). Specimens in

NHCs objectively document diversity and voucher our

planet’s historic conditions (Dunnum & Cook 2012).

A wealth of information results from these investigations

and resides in the specimens themselves, and the transfer

of such information is seen as one of the major responsi-

bilities of NHCs (O’Connell et al. 2004), relevant both in

the training of life and environmental science profession-

als (Cook et al. 2014, Wen et al. 2015) and in engaging

the public to become society’s biodiversity conscience (Kr-

ishtalka & Humphrey 2000).

To which extent are these published perceptions of

NHCs reflected in the community? The survey presented

here captured a snapshot of values and opinions regard-

ing natural history collections, both from NHC users and

NHC personnel. With its help we tried to roughly evalu-

ate the current view on roles, functions, performance and

appeal of NHCs.

MATERIALS & METHODS

The online poll was carried out over a period of two weeks

in November (2 to 17") 2015 using SurveyMonkey

(www.surveymonkey.com), a widely known platform for

online polling. The survey was conducted anonymously,

1.e., institutes’ and respondents’ names were not queried,

and IP data not logged. However participants were given

the opportunity to indicate their email address to receive

feedback on survey results.

The poll took about ten minutes to complete and encom-

passed 28 questions, most of which expected answers in

form of grades (1 to 5) or offered multiple-choice options

(usually with comment function). Only one question, Q

28, was purely open-ended (free text). No question was

defined as mandatory in order to complete the poll. Ques-

tions were grouped into two thematic blocks, the first one

containing seven inquiries on respondent background:

country, discipline, type of work place, frequency of con-

tact with NHCs, own experience with sample depositions,

type of samples, and type of characters / materials. The

remaining 21 questions focused on NHC roles and func-

tions (8 questions general in nature or weighing relevance

of specific NHC functions, 13 questions evaluating cur-

rent NHC performance, condition, or appeal): most im-

portant roles of NHCs, importance of voucher deposition,

need for NHC growth, international legislation, current ac-

cessibility, importance of offering NHC data digitally,

quality of maintenance, suitability for present-day science,

attention given to molecular samples, relevance of molec-

ular samples, attention given to type material, quality of

Bonn zoological Bulletin 66 (1): 61—72

sample documentation, need for libraries at NHCs, scien-

tific relevance of NHC journals, importance of engaging

the general public, adequacy of taxonomic expertise, over-

all satisfaction, appropriateness of funding, performance

of NHCs in one’s country, general appeal of NHCs, per-

ceived deficits. The full questions alongside answer op-

tions are listed in Appendix I.

Requests to participate in the online poll were distrib-

uted via email lists, fora and social media, asking for opin-

ions on natural history collections, their roles and servic-

es. We addressed the following mailing lists or fora:

EvolDir, Taxacom, NHCOLL-L, ISBER, GfBS, CETAF,

Bonn biology student listserv, and the following groups

within social networks: LinkedIn: Biodiversity Profession-

als, Ecology & Evolutionary Biology, ESBB, GGBN, ED-

IT groups and Facebook: Natural History Collection

group. We estimate that our announcement to participate

in the survey may have reached 60,000 recipients altogeth-

er.

Survey results were exported from SurveyMonkey and

processed with Microsoft Excel. We counted responses

within categories and calculated arithmetic means (in the

grading-mode questions, Q 9—Q 27) as well as proportions

(other questions). A contingency table was generated to

show average answer values dependent on respondent

background.

RESULTS & DISCUSSION

We received 525 responses altogether (see Appendix II),

more than 90% of these submitted within the first week

of the poll. The calculated response rate lay at 0.9%, but

can be assumed to be higher, as recipients are often sub-

scribed to two or more of the lists/groups. Of all 525 par-

ticipants, more than 90% gave an answer to all questions

(without counting the purely free-text based question, Q

28).

Respondents’ backgrounds (Q 1-7):

Respondents are scattered over 39 countries. 44% of them

are based in North America, 43% in Europe, 7% in Ocea-

nia, 3% in South America, 1% in Africa and 1% in Asia.

The individual country contributing by far most respons-

es is the US (39% of all responses). Preponderance of re-

sponses from the United States is partly a consequence of

distribution channels used in announcing the poll, but part-

ly also reflects the dominance in the NHC scene, as ca.

1/4 of the registered collections and 1/3 of all NHC sam-

ples worldwide are located in the US (Ellwood et al. 2015,

Kemp 2015).

All queried disciplines are well represented, with evo-

lution, taxonomy, phylogeny and ecology most prominent,

population biology least prominent. From those respon-

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Survey on natural history collections 63

dents indicating their professional environment, 215 are

based in academia, while 166 work at a NHC and 55 are

university students. 328 participants indicated daily or ca.

monthly contact with natural history collections, 160 a few

times a year or more infrequently. Only 21 participants

have never had contact with NHCs. 87% of interviewed

taxonomists have frequent (daily or monthly) contact with

NHCs, as have conservation professionals and phyloge-

neticists in almost 70% of the cases. Representatives of

the other explicitly queried disciplines (ecology, evolution,

population biology, molecular biology) had frequent NHC

contact in 50-57% of the cases. 74% of the respondents

have at least once deposited samples at a NHC (as have

more than a third of the participating students). More than

2/3 of the interviewees (368) work with animal samples,

followed by plants (128), then fossils (78). Respondents

also marked geological (42), fungal (37), micro-organis-

mal (35), and human (22) samples. 75% of the participants

(answering this question) indicated that they work with

morphology, 60% work with molecular samples (e.g., with

DNA), 44% with images, 38% only with metadata, 8%

with acoustic data. The contingency table in Appendix IV

shows average answer values for questions 8—27 depend-

ent on respondent background.

Ranking of NHC roles (Q &):

Asked to identify the one to three most important role/s

of natural history collections within society and science,

75% of the respondents (counting also abstentions) high-

lighted basic research, 61% collection care and loans, 58%

educating the public, 30% collecting new samples, 27%

training students, 22% performing applied research, 5%

coordinating citizen science, and 5% shaping science pol-

icy (see Appendix IV). Most of the 5% free-text answers

focused on the NHC role to safeguard collections and to

document biodiversity, a function we missed to add more

explicitly to the simple label ‘collection care’.

The most prominent role survey participants saw in

NHCs was that of the active research institute, with a fo-

cus on mostly basic, but also on applied research. Although

the poll does not offer a direct means to infer this, we hy-

pothesize that a strong focus of this perceived necessity

for research lies with the collections themselves (cf. Dos-

mann 2006). We base this assumption on the fact that al-

most 2/3 of the participants selecting basic research

checked also the second most frequent answer, curation

of collections. Furthermore, there exists a natural connec-

tion: with the physical collections, i.e., the “museum’s

‘soul’ and raison d’étre” (Alberch 1993), NHC research

has a unique resource directly at hand.

A look into respondent backgrounds revealed that opin-

ion on most relevant NHC roles does not seem to differ

strongly between North America and Europe, although

some differences exist (e.g., participants from North

Bonn zoological Bulletin 66 (1): 61-72

America put somewhat more emphasis on applied research

and training students, participants from Europe on edu-

cating the public and on citizen science). As other conti-

nents were less well represented, results for these must be

taken with a (sometimes rather big) grain of salt, but av-

erage ranking hinted at higher valuation of science poli-

cy by respondents based in Oceania (37 participants), of

academic training in South America (17 participants), or

of educating the public in Africa and Asia (6 participants

each). Other background traits had influence on respons-

es as well, to eclectically name some examples (see Ap-

pendix IV for full data): on average, sample depositors and

NHC staff give more importance to collection care, but

less to educating the public. Students, ecologists and mi-

crobiologists see above-average relevance in teaching at

NHCs (an activity that overall only about 1/4 of partici-

pants ranked among the 3 most relevant). Of all sample

types, participants working with molecular samples see the

highest necessity for gathering more samples, as do, in

terms of discipline, phylogeneticists and taxonomists.

Perceived relevance of selected NHC activities and per-

formance (QO 9-28):

Questions 9-27 were answered by using a grading

scheme from | to 5, where consequently 3 would count

as a neutral answer. A “5” denotes “high relevance’,

“good”, or “much”. Table | lists the mean values for all

responses. These are also presented 1n the text below, high-

lighting, as before, conspicuous correlations with respon-

dents’ backgrounds. Not all correlations can be discussed

here, and the selection is necessarily biased by our own

(author) background. We therefore encourage readers to

access the primary data in Appendix III, or for easier ref-

erence the contingency table in Appendix IV.

QO 9: Asked about the scientific relevance of depositing

vouchers, the mean of all answers lay at 4.6. Of the inter-

viewed persons, 77% graded with 5, and 1% with 1. There

existed an obvious correlation between perceived rele-

vance and frequency of contact with NHCs. The high im-

portance assigned to the vouchering concept and also, as

shown below, the overall positive evaluation of type dep-

osition practice at NHCs can be seen as evidence for the

long-established role of vouchers at NHCs as the materi-

al guarantors against which research is calibrated (Ellis

2008). Vouchers and associated databases form the nexus

that links individual studies with past or future inferences

(Astrin et al. 2013), and the costs for non-reproducibili-

ty in life science have been estimated to be very high

(Freedman et al. 2015).

How much growth is still necessary for NHCs to com-

prehensively answer most biological / environmental ques-

tions? This question, O 10, was answered with 4.2 on av-

erage. While NHCs are confronted with often serious

budgetary bottlenecks (see below), the necessity for col-

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64 Jonas J. Astrin & Hannah C. Schubert

Table 1.

Mean responses on NHC functions and performance (Q 9-27), sorted by response value. There existed 5 answer op-

tions: 1, 2,3, 4, and 5. A 5 usually denotes high relevance, good, much, etc. In Q 11 on current legislation, 1 indicates a major per-

ceived problem, 5 none.

survey question

mean response

Q 27: Do you perceive NHCs as interesting places? 4.8

Q 9: Depositing vouchers — how relevant is this for science? 4.6

Q 17: How relevant are molecular samples in life sciences? 4.6

Q 13: How important is it for NHCs to offer their collection data digitally? 4.6

Q 22: How important is it for NHCs to engage the general public through, 4.5

e.g., exhibitions, events, or social media?

Q 10: How much growth is still necessary for NHCs to comprehensively answer 4.2

most biological/environmental questions?

Q 20: Are libraries necessary at NHCs? 4.1

Q 18: Is type material given the necessary importance at NHCs? 4.0

Q 24: Overall, how happy are you with the services offered by the community of NHCs? A

Q 21: How relevant (scientifically) are journals edited at / issued by NHCs? A

Q 14: Are the collections you know maintained properly? 3.7

Q 26: Overall, do your country’s collections offer better or worse services 3.6

than those in most other countries?

Q 15: Do you feel NHCs are up to present-day science? 3.5

Q 12: Are collections sufficiently accessible for science? 3.4

Q 19: On average, is sample documentation (sample metadata) in NHCs sufficient? 3.2

Q 16: Are NHCs paying enough attention to molecular samples (e.g., DNA used in publications)? 3.1

Q 23: Is there sufficient taxonomic expertise at NHCs? 3:0

Q 11: Is the current international legislation Dats

(esp. Nagoya Protocol) an obstacle for collection-based work?

Q 25: Are NHCs funded appropriately? ee

lections to continuously keep growing is recognized by the

largest part of the community (as captured by this survey).

It is obvious that NHCs cannot stop collecting activities

if they are to inform seamlessly on changes in biodiver-

sity and in environmental conditions (e.g., Brooke 2000,

Winker et al. 2010, Habel et al. 2013, Rocha et al. 2014).

However, it has been critically pointed out that haphaz-

ard growth often prevails (Alberch 1993, Ponder et al.

2001, Pyke & Ehrlich 2010). Targeted collecting strate-

gies should consider, as Pyke & Ehrlich (2010) note, the

increasing relevance of ecological and environmental is-

sues in addition to taxonomic and geographic considera-

tions. For instance, continuous time series for common

species hold considerable long-term value (Winker 2004,

Kiister et al. 2015), as do bulk samples from ecological

studies (Schilthuizen et al. 2015) and monitoring projects.

QO 11: Only 15 of all respondents have the opinion that

current international agreements (esp. Nagoya Protocol)

is no obstacle (= answer “5”’) for collection-based work.

With 2.7 as the arithmetic mean (2.5 by NHC-based re-

spondents), most answers revealed that current legislation

was seen as a rather strong obstacle. Although obviously

Bonn zoological Bulletin 66 (1): 61-72

not the only regulatory framework governing NHC sam-

ple acquisition and/or transfer (e.g., Fowler et al. 2007,

Vernooy et al. 2010), the Nagoya Protocol of the Conven-

tion on Biological Diversity (UNEP 2011) that came in-

to effect in 2014 to cut back on biopiracy is placing a

heavy bureaucratic burden (Comizzoli & Holt 2016) al-

so on non-commercial research (Schindel et al. 2015) and

necessitates major revision of workflows and capacity-

building at NHCs (Davis et al. 2015).

QO 12: Current access to collections was evaluated with

an average of 3.4, 1.e., only somewhat better than neutral.

Participants from South America (2.9) and especially

Africa (2.3) chose lower values on average (but note the

limited number of responses from these continents). How-

ever, when asked about the general importance of digital

access to collections (Q /3), more than 90% responded

with a 4 or 5 (mean: 4.6). Participants working with ge-

ological (4.2) or human (4.3) samples rated somewhat low-

er, whereas a raised answer value was apparent in respons-

es from Africa (5.0), Asia (4.8) and South America (4.7)

as well as from students (4.8). It seems obvious that those

parts of the world farthest away from the bulk of NHC

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Survey on natural history collections 65

specimens (in North America and Europe) and with of-

ten weaker economies are in especially acute need for dig-

ital access to collections.

Since earlier calls to better integrate biodiversity infor-

mation sources (Soberon 1999, Krishtalka & Humphrey

2000), the situation has changed thanks to new biodiver-

sity data architecture (e.g., Edwards 2004, Graham et al.

2004, Ratnasingham & Hebert 2007, Constable et al. 2010,

Wheeler et al. 2012, Droege et al. 2014, Kelbert et al.

2015, Belbin & Williams 2016, Schindel et al. 2016). Al-

so, efforts are under way to systematically digitize collec-

tions (e.g., Baird 2010, Nelson et al. 2012, Heerlien et al.

2015), some taking advantage of public participation (EIl-

wood et al. 2105), automation (Blagoderov et al. 2012,

Hudson et al. 2015), or DNA barcoding campaigns

(Hebert et al. 2003a,b). However, the poll mirrors the still

considerable need for digitizing and linking NHC data.

This becomes also apparent from answers to Q 28, where

almost a third of respondents submitting free-text com-

ments had explicit issues with current amount of data dig-

itization or access to collections, and where some concern

(5% of free-text answers) was expressed that NHCs were

not networking sufficiently.

Adequacy of collection maintenance was the focus of

QO 14. The overall average answer lay at 3.7. Respondents

with zoological (3.7) and botanical or paleontological (3.6)

background mostly defined this value. Micro-organismal

or human background correlated with lower answer val-

ues (3.4). In terms of data type / material, responses from

participants involved with acoustics were higher than oth-

ers (4.0). Many free-text comments in Q 28 indicated that

additional funds would be necessary to appropriately cu-

rate collections.

Do you feel NHCs are up to present-day science? The

average opinion on Q /5 condensed into a value of 3.5.

Frequent NHC contact on average resulted in a slightly

higher value (3.7). This difference may be explained with

the notion that museums are sometimes perceived as dusty

places stifling intellectual excitement (Brooke 2000).

However, the feedback received in this survey on appeal

of NHCs indicated that natural history collections are com-

monly seen as “fascinating” places (Q 27, see below). This

finding challenges the idea that the ‘dusty NHC’ is real-

ly such a wide-spread cliché. Accordingly, NHC image

cannot be used in explaining why collections are perceived

to only moderately well meet the requirements of present-

day science. The scope and focus of research carried out

at NHCs is likely not the reason either, as no indication

could be found among free-text answers (Q 28). On the

other hand, insufficient funding is seen as a considerable

obstacle (Q 25, 28), which supports the idea that missing

or out-dated infrastructure as well as scarce or poorly

trained staff pose problems for cutting-edge research at

NHCs.

Bonn zoological Bulletin 66 (1): 61-72

In answering this question, Q 15, molecular biology

backgrounds were associated with somewhat lower val-

ues. This connects to the following question. The impor-

tance that is currently being paid to molecular samples

(e.g., DNA used in publications) at NHCs (Q /6) was

ranked with an average 3.1, one of the lowest marks in this

survey. In contrast to this result stands the fact that 90%

of the respondents consider the general relevance of mo-

lecular samples in life sciences (Q 17) to be high or very

high. This is mirrored in the overall mean value for the

answer: 4.6. Where explicitly expressed as free-text (Q 28),

issues were usually that more (sub)samples should be pre-

served specifically for molecular analysis, and/or that a

more liberal regime to sample also ‘morphological’ spec-

imens should be applied. The high expectations towards

molecular samples, taken together with the mediocre NHC

performance perceived by the community in this context,

suggest that NHCs are lagging behind and partially miss-

ing out on an important chance. As NHCs can curate spec-

imen vouchers cross-referenced with molecular vouchers

(DNA, tissue, etc.), they are an ideal place for establish-

ing molecular collections or biobanks (Astrin et al. 2013)

and can play a central role in the integrative study of bio-

diversity (Whitfield & Cameron 1994, Cristescu 2014).

Efforts are under way to virtually unite as many molecu-

lar collections as possible under the single access point of-

fered by the Global Genome Biodiversity Network, GGBN

(Droege et al. 2014), and an increasing number of NHCs

are joining this endeavor. However, the fact that NHC staff

responded with a slightly lower value to this question (Q

17) indicates that NHCs have not yet fully understood the

potential of molecular samples. Biodiversity biobanking

is still in its infancy and needs to be ramped up, which

becomes obvious when compared to human biobanking

(Astrin & Betsou 2016).

The question (Q /8) whether type material was given

the necessary importance at NHCs scored a mean answer

of 4.0, the highest value among all those answers that eval-

uated NHC performance (see above), although consider-

able variation existed among countries.

For instance, the overall result was worse for QO 19,

which found satisfaction with sample documentation

(sample metadata) at NHCs to be rather low: 3.2. Partic-

ipants based in Australia checked considerably higher val-

ues on average (3.8; possibly influenced by data presen-

tation and accessibility in the Atlas of Living Australia, see

Belbin & Williams 2016). 15 respondents explicitly

wished to see more updates on specimen records (e.g., re-

/identification of specimens) or added metadata in free-

text question Q 28. As NHC samples are put to an ever-

growing range of uses, one should keep in mind the high

demand for detailed metadata, also by “new clients” in en-

vironmental, ecological, societal, and management-relat-

ed areas (Winker 2004). Although sometimes an arduous

task for both sides involved, it is an inevitable necessity

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66 Jonas J. Astrin & Hannah C. Schubert

that collection managers and curators try to obtain from

the depositors as much information on samples as anyhow

possible. Data from subsequent analysis, like bibliograph-

ic information on resulting publications, database acces-

sion numbers, etc., should equally be fed back to the col-

lection — a widespread but not always implemented pre-

requisite for loans. Adding high-resolution specimen im-

ages greatly enriches NHC datasets and can be performed

semi-automatically (e.g., Balke et al. 2013). Metadata can

be detected, mined, and semantically enhanced in automat-

ed fashion from already existing datasets (e.g., van den

Bosch et al. 2009, Guralnick et al. 2016).

QO 20: The general necessity for maintaining libraries at

NHCs was perceived as rather high with a mean answer

of 4.1. Taxonomists (4.4) and conservation professionals

(4.3) on average saw a higher need for these, as did NHC

staff (4.4). Libraries are a fundamental research infrastruc-

ture, and represent yet another collection type at NHCs

in addition to those focusing on natural history samples

and metadata on these. Facilities are steadily evolving in-

to hybrid libraries (Rusbridge 1998) that offer digital in-

formation alongside printed documents. The quasi-legal

status of taxonomic literature (Minelli 2003) demands the

archival of publications associated with nomenclatural

acts, irrespective of age — an atypical situation in common

libraries. Taxonomic literature and many other biodiver-

sity documents are being digitized at NHCs libraries and

other institutions (e.g., Pilsk et al. 2016), often in target-

ed projects and employing high throughput workflows

(BHL 2017). The role of NHC libraries as information bro-

kers will become even more relevant once library discov-

ery systems and catalogs are deeply integrated with col-

lection databases and with aggregator portals on sample

data. There remains little doubt that such a fusion of phys-

ical evidence plus metadata (on particular instances of nat-

ural history) with the accumulated knowledge on various

‘units’ of nature (e.g., taxa, as in EOL: Wilson et al. 2003)

will be the future of biodiversity informatics and NHC-

based LIS (library and information science) approaches.

Uniting and unifying these ‘universes’ allows powerful da-

ta mining if the necessary ontologies are in place (e.g.,

Tochtermann et al. 1997, Vogt 2009, Seltman et al. 2012,

Walls et al. 2014, Thessen et al. 2015).

QO 21: The poll participants evaluate scientific relevance

of NHC-edited or NHC-issued journals with 3.7. Elevat-

ed relevance was tied to background traits paleontology

(4.0), taxonomy (3.9), NHC staff (3.9), decreased mean

levels to bioacoustics (3.4), micro-organisms (3.4) or mo-

lecular and population biology (3.5).

QO 22: The importance to engage the general public

through, e.g., exhibitions, events, or social media was re-

garded as very high (4.5), with little variation depending

on background (but mark the average 4.8 in participants

from UK or Africa). Exhibiting objects to the general pub-

lic has a long-standing tradition at NHCs (Maerker 2005).

Bonn zoological Bulletin 66 (1): 61—72

Apart from exhibitions that illustrate life science or envi-

ronmental topics, the research performed at NHCs is of-

ten put into focus. At some museums, the actual workflows

of collection staff or researchers can be observed (like dig-

itization, Heerlien et al. 2015). Based on the many free-

text comments (in Q 28) on profile and education, NHCs

should use all outreach means available to more convinc-

ingly explain and justify to general public and policy mak-

ers the outstanding importance of active and growing re-

search collections for life and environmental sciences (e.g.,

Krishtalka & Humphrey 2000). Raising public awareness

for the relevance of collections and collection-based re-

search can foster participation in citizen science projects

(e.g., Hill et al. 2012, Geiger et al. 2016) and vice versa.

Regarding adequacy of taxonomic expertise (OQ 23) at

NHCs, the survey revealed a heterogeneous notion with-

in the community. The overall answer was neutral (3.0),

and no individual answer reached more than 28% of the

votes. Respondents from the Southern hemisphere re-

sponded with lower values than colleagues from North-

ern countries. Students (3.4) and respondents who had nev-

er deposited material (3.6) saw less of a taxonomic deficit

at NHCs then NHC staff (2.8) or taxonomists (2.8). In this

question on adequacy of taxonomic expertise as well as

in the following one (Q 24), we see a logical correlation

with funding issues (see Q 25).

QO 24: The overall satisfaction with NHC services

reached 3.7, was elevated with conservation profession-

als and with students (both 3.9), and reduced in respon-

dents working with human (3.3), fungal (3.4), or micro-

organismal (3.4) samples.

Prompted on funding (Q 25), 54% of the interviewed

persons indicated they considered funding of natural his-

tory collects as “insufficient” (lowest answer value “1’’).

Only 0.6% considered it “appropriate” and 3% near-ap-

propriate. Overall, the arithmetic mean of all answers lay

at 1.7, marking the lowest of all answer values in the sur-

vey. This finding coincides with the often raised argument

that investments in NHCs are insufficient (e.g., Cotterill

1995, Lane 1996, Dalton 2003, Suarez & Tsutsui 2004,

Dosmann 2006, Andreone et al. 2014, Kemp 2015, Stok-

stad 2015), endangering specimens and positions, and

compromising research, infrastructure, outreach and

NHC services (including loans, sample digitization, vis-

itor programs, etc.). More than 130 free-text answers in

Q 28 elucidate this problem (see Appendix V). Notwith-

standing, the cost-efficiency and the economic benefit of-

fered by NHCs are evident (Suarez & Tsutsui 2004, Over-

mann 2015) and will become even more pronounced

thanks to the constantly growing number of disciplines and

approaches using NHCs (Winker 2004, Pyke & Ehrlich

2010, Kemp 2015, McLean et al. 2016).

As Q 26 sets NHC performance in the participants’

country into relation with NHCs abroad, the overall mean

(3.6) holds limited information value. We therefore extract-

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Survey on natural history collections 67

Table 2. Mean values for Q 26 (“Overall, do your country’s collections offer better or worse services than those in most other countries?”),

grouped geographically. Left side of table: list of all countries represented with 10 or more answers. Right side: grouped by continent (Asia and

Africa represented by only 6 answers each).

country mean answer number of answers continent mean answer number of answers

USA 4.0 204 North America 4.0 224

UK ao 35 Oceania 37 36

Germany 339 47 Europe 333 216

Australia Sat 26 Africa 20 6

New Zealand cae! 10 South America 2.4 17

Austria 3.4 17 Asia 2.0 6

Canada Sal 16

Belgium Sal 13

France 3.0 27

Brazil 2.3 12

Portugal 1.8 13

ed all countries with 10 or more responses and list the da-

ta for these along with information on continents in Table

2. Respondent happiness with NHC services varied great-

ly depending on geography, from an average “4” in North

America to “2” in Asia. Although countries from especial-

ly Africa, Asia and South America are only marginally rep-

resented in this survey, geographical bias seems to be a

fact: respondents from the mentioned continents have

more issues on average with overall NHC services in their

country. In the light of the current biodiversity crisis (Wil-

son 1985), one should keep in mind that it is on these con-

tinents where most of our planet’s biological diversity is

localized and where NHCs are especially challenged (e.g.,

Arbelaez-Cortés et al. 2015).

Q 27: Asked whether they see NHCs as interesting

places (see also above), 82% of the survey participants in-

dicated they found them “fascinating” by assigning a “5”

(as opposed to “boring”, answer “1”, which did not receive

any hits at all). The arithmetic mean for all answers is 4.8,

the highest value reached in this survey.

The last question, Q 28, collected free-text opinions on

the most fundamental critique points: “If anything, what

should improve most at NHCs?” We received 240 respons-

es, which we cannot discuss individually (although many

provide food for thought, and we recommend browsing).

Therefore we tagged responses according to categories.

Appendix V shows our — often multiple — tag assignments

and explains details on category tags. More than half of

the answers (134) target the general lack of appropriate

funding, apparent in staffing (and staff education), cura-

tion / collection care, infrastructure, etc. 72 respondents

had explicit issues with current amount of databasing, da-

Bonn zoological Bulletin 66 (1): 61—72

ta digitization, or digital/physical access to collections. 42

answers suggested sharpening or modifying NHC profiles,

or commented on NHC strategies and on reception by pol-

icy-makers, while measures towards engaging the gener-

al public or training students and professionals were tar-

geted in 34 comments. 22 respondents stressed the neces-

sity to collect molecular samples more rigorously, or ex-

pressed their unhappiness about limited options to subsam-

ple morphological specimens for molecular studies. 16 an-

swers pointed out the general need to add new samples to

the collections. 15 participants explicitly wished to see

more updates on specimen records (e.g., re-/identification

of specimens) or added metadata. 12 comments suggest-

ed NHC need a stronger focus on networking efforts, e1-

ther among themselves or with other institutions. Issues

with either the way research 1s carried out at NHCs, with

ABS and legal regulations, or with loans were represent-

ed in less than 10 responses each.

CONCLUSION

The survey results show a very positive connotation as-

sociated with natural history collections. ‘Traditional’

NHC roles and values are not questioned (emphasis on col-

lections, research, engaging the public, etc.), and at the

same time the importance of comparatively ‘new’ concepts

in the NHC task spectrum becomes apparent (data digi-

tization, molecular samples). Nevertheless, current per-

formance of NHCs_ in_- various areas (Q

12,14-16,18,19,21,23,24) was graded with an average 3.5

points (out of 5), i.e., only somewhat better than neutral

©OZFMK

68 Jonas J. Astrin & Hannah C. Schubert

(3.0). We interpret this mostly as a consequence of the rel-

ative scarcity in NHC funding, for which both this survey

and the literature give evidence. Notwithstanding, inde-

pendently from funding, there seem to be topics on which

NHC staff need to pick up (see Appendix III). There are

two sides to this coin, and the user community seems in-

sufficiently informed on some efforts already undertak-

en at NHCs. Transferring this information is the task of

NHCs, and we wonder if self-critical voices within the mu-

seum community have been sufficiently heard (Alberch

1993, Krishtalka & Humphrey 2000).

Our hope, bolstered by the survey results, is to see an

unabated — or better even — a strengthened focus on col-

lections, by performing and facilitating state-of-the-art bio-

diversity and environmental research on them, by active-

ly and confidently advocating and communicating their

immense and growing value, by using them for public and

academic education, by safeguarding the legacy of exist-

ing collections and coordinating a targeted collection

growth that meets the demands of the various traditional,

emergent, and prospective NHC user communities — for

the benefit of society and biodiversity. We further hope

for this process to take place across the whole globe, with

no geographic areas left behind.

Acknowledgements. David Schindel and two anonymous re-

viewers kindly provided many helpful comments on the manu-

script. We would like to sincerely thank all those people who took

this survey and shared their view on natural history collections.

Furthermore, we want to express our apologies for not being able

to target all domains and aspects of NHCs equally (e.g., being

ourselves based in biology, we lack the focus on geology and

other areas which have their place in many NHCs as well). We

are also aware that in formulating our poll (questions as much

as answer options), we had to simplify a complex world so that

participants would still be able to complete the survey in a rea-

sonable amount of time.

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©OZFMK

Survey on natural history collections 71

APPENDIX I

List of questions and possible answers from the online poll

(NB: “...” indicates an open-ended answer, all other an-

swers were predefined as ‘multiple choice’).

About yourself...:

1) Which country are you based in? (hint: use your key-

board for the first letters)

— [dropdown menu with country list as supplied by

SurveyMonkey]

2) Which discipline are you associated with (can choose

various)

— ecology / conservation biology / taxonomy / phylo-

genetics / evolution / population biology / molecular bi-

ology / other (please specify)...

3) Where do you work

— academia / freelance biologist / education / natural

history collection / non-academic research organization

/ science policy / zoological/botanic garden / I am a stu-

dent / I earn my money elsewhere, but biology is my

passion / other (please specify)...

4) How often do you have contact with natural history

collections

— daily / roughly monthly / once or a few times a year

/ seldom / never

5) Have you ever deposited material at a collection?

— yes / no

6) Samples you routinely work with (can choose vari-

ous)

— fungal / animal / plant / micro-organismal / viral /

human / fossil / geological / mixed environmental / oth-

er (please specify)...

7) Characters or materials you work with (can choose

various)

— morphology / molecules (e.g., DNA) / acoustics /

images / pure metadata (e.g., from collection databases)

/ other (please specify)...

Questions concerning natural history collections

(NHCs):

8) Which is the most important role of natural history

collections (NHCs)? (check up to 3 — if 4 or more are

checked, this question will not be evaluated)

— educating the public / training students / collection

care and loans / collecting new samples / performing

basic research / performing applied research / shaping

science policy / coordinating citizen science / other

(please specify)...

9) Depositing vouchers — how relevant is this for sci-

ence?

— not relevant 1 2 3 4 5 relevant

Bonn zoological Bulletin 66 (1): 61—72

10) How much growth is still necessary for NHCs to

comprehensively answer most biological/environmental

questions?

— no growth needed | 2 3 45 much growth needed

11) Is the current international legislation (esp. Nagoya

Protocol) an obstacle for collection-based work?

— major obstacle 1 2 3 45 no obstacle

12) Are collections sufficiently accessible for science?

— not accessible 1 2 3 4 5 accessible

13) How important is it for NHCs to offer their collec-

tion data digitally?

— not important | 2 3 4 5 important

14) Are the collections you know maintained properly?

— inadequately | 2 3 45 well-maintained

15) Do you feel NHCs are up to present-day science?

— antiquated 1 2 3 4 5 up to date

16) Are NHCs paying enough attention to molecular

samples (e.g., DNA used in publications)?

— not enough | 2 3 45 sufficient

17) How relevant are molecular samples in life sci-

ences?

— not relevant 1 2 3 4 5 relevant

18) Is type material given the necessary importance at

NHCs?

— not enough | 2 3 4 5 appropriate

19) On average, is sample documentation (sample meta-

data) in NHCs sufficient?

— insufficient 1 2 3 45 sufficient

20) Are libraries necessary at NHCs?

— not necessary | 2 3 4 5 necessary

21) How relevant (scientifically) are journals edited at /

issued by NHCs?

— not relevant 1 2 3 4 5 relevant

22) How important is it for NHCs to engage the general

public through, e.g., exhibitions, events, or social me-

dia?

— not important | 2 3 4 5 important

23) Is there sufficient taxonomic expertise at NHCs?

— insufficient | 2 3 45 sufficient

24) Overall, how happy are you with the services of-

fered by the community of NHCs?

— unsatisfied 1 2 3 45 happy

25) Are NHCs funded appropriately?

— insufficiently 1 2 3 4 5 appropriately

26) Overall, do your country’s collections offer better or

worse services than those in most other countries?

— below average | 2 3 4 5 above average

27) Do you perceive NHCs as interesting places?

— boring | 2 3 45 fascinating

28) If anything, what should improve most at NHCs?

If you would like to be updated on results from this sur-

vey, type in your (plain) email address here:

=> eee

©ZFMK

7D Jonas J. Astrin & Hannah C. Schubert

APPENDIX II

List of networks and list-serves used to announce the on-

line poll. Membership figures for the respective groups

were obtained at time of the survey.

Mailing lists and fora

EvolDir mailing list (and platform) (http://life.mcmas-

ter.ca/evoldir.html), ca. 10,000 recipients.

Taxacom list (http://mailman.nhm.ku.edu/cgi-bin/mail-

man/listinfo/taxacom): more than 2000 members.

Natural History Collections listserver, NHCOLL-L

(http://mailman. yale.edu/mailman/

listinfo/nhcoll-l), 1775 members.

International Society for Biological and Environmental

Repositories, ISBER, forum (http://www.isber.org/):

over 1000 members.

Mailing list of the German Society for Biological Sys-

tematics, GfBS (http://www.gfbs-home.de/?L=1): 285

subscriptions.

Consortium of European Taxonomic Facilities, CETAF

(http://cetaf.org/) mailing list and website: 170 members

(mailing list).

Listserv for biology students in Bonn, Germany (bio-

stuff@listserv.uni-bonn.de): number of subscriptions

could not be determined

Social Networks

LinkedIn (https://www.linkedin.com/) groups:

Biodiversity Professionals: 25,000 group members.

Ecology & Evolutionary Biology: 17,000 members.

European, Middle Eastern & African Society for Biop-

reservation& Biobanking, ESBB: 3000 members.

Global Genome Biodiversity Network, GGBN: 80

members.

European Distributed Institute of Taxonomy, EDIT: 220

members .

Facebook (https://www.facebook.com/) Natural History

Collection group: almost 1000 members.

Bonn zoological Bulletin 66 (1): 61—72

APPENDIX III

(electronic supplement, available at www.bonnzoologicalbulletin.de)

Table containing the full data collected in the survey (email

addresses excluded).

APPENDIX IV

(electronic supplement, available at www.bonnzoologicalbulletin.de)

Mean values for questions 8—27 and overview of average

answer values for the respective background traits.

APPENDIX V

(electronic supplement, available at www.bonnzoologicalbulletin.de)

Categorization of open-ended answers to question 28. The

file consists of two spreadsheets: 1) the original answers

along with category tags assigned by us, and 2) a sum-

mary as well as explanatory note on how we defined tags.

©ZFMK

Bonn zoological Bulletin 66 (1): 73-84

The advertisement calls of Epipedobates anthonyi (Noble, 1921) and

Epipedobates tricolor (Boulenger, 1899)

(Anura: Dendrobatidae: Colostethinae): intra- and interspecific comparisons

Alice Marie Scherges" & Dennis Rodder'

Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute of Animal Biodiversity,

Adenauerallee 160, D-53113 Bonn, Germany;

E-mails: s6alsche@uni-bonn.de,; d.roedder@leibniz-zfmk.de

Abstract. Vocal communication is the main mechanism to exchange information in many groups of animals, and anu-

rans represent an impressive example of complex acoustic communication signals. To date several calls with different

and partly multiple meanings have been described for numerous species. Anuran calls were found to be species-specif-

ic, providing important taxonomic information, and advertisement calls are frequently used for systematic assignments.

The closely related poison dart frogs Epipedobates anthonyi and Epipedobates tricolor inhabit geographically disjunct

areas in Ecuador and Peru. Both species are very similar in appearance and have frequently been confused. Consequent-

ly, multiple scientific publications on E. tricolor refer to E. anthonyi instead and vice versa. This is also true for the de-

scriptions of the species’ advertisement call. So far, no comparative study on the species’ calls exists. Here we analyze

and compare the calls of E. anthonyi and E. tricolor. We found the advertisement calls of both species to be very simi-

lar in general but to differ in pulse frequency and interval. Thus, they can be distinguished with certainty. However, a

comparison of characteristic call patterns between populations revealed intraspecific variation to be higher than interspe-

cific variation, which questions the usefulness of calls for species delimitation of frogs for the genus Epipedobates.

April 2017

Keywords. Acoustic communication, anurans, bioacoustics, species delimitation, systematics.

INTRODUCTION

Communicational signals are primarily visual or acoustic,

but can also be chemical, seismic, electrical or tactile

(Pough 2001, Janik & McGregor 2014) and often combi-

nations of different signals are used (Starnberger et al.

2014). Anurans primarily use acoustic communication

which plays a significant role in reproduction and social

interactions (Salthe & Mecham 1974). Capranica & Rose

(1983) showed that the anuran auditory system is highly

adapted to the species-specific dominant frequencies and

furthermore functions as a filter, improving the detection

of important signals and suppressing distracting back-

ground noises. According to Wells (2007) the whole fe-

male sensory system is tuned to detect certain spectral and

temporal characteristics of calls, including frequency and

pulse rate, whereas the ranges of the characters depend on

the species. This ability also allows discrimination between

conspecific and heterospecific calls (Fritsch et al. 1988)

through recognition of specific spectral and temporal com-

ponents (Salthe & Mecham 1974; Wells 1977; Ryan 1985;

Cocroft & Ryan 1995).

Many different types of calls and their respective func-

tions have been described (e.g., Borgert 1960; Littlejohn

1977; Hddl 1996; Wells & Schwartz 2006; Wells 2007).

In most anuran species only males produce advertisement

Received: 17.10.2016

Accepted: 04.04.2017

calls during the breeding season to attract females

(Bogert 1960; Wells & Schwartz 2006; Wells 2007). How-

ever, acoustic signals may also have territorial (Bogert

1960) or competitive (Wells & Schwartz 2006; Wells

2007) meaning, are used for localization (Whitney &

Krebs 1975; Wells 1977, 1978), or as defense, distress, or

alarm signal (Bogert 1960; Wells 2007). Previous research

reveals that quantitative and qualitative parameters of anu-

ran advertisement calls are species-specific and thus fa-

cilitate systematic assignments, particularly in poison-ar-

row frogs (Wells 1977; Zimmermann & Zimmermann

1988; De la Riva et al. 1996; Lotters et al. 1999; Napoli

& Cruz 2005; Padial et al. 2008, Mayer et al. 2014).

In this study, we characterize and analyse the advertise-

ment calls of two species of poison dart frogs, E. antho-

nyi and E. tricolor. The distributional range of the poison

dart frog Epipedobates anthonyi stretches from southwest-

ern Ecuador to neighboring areas of northwestern Peru

(Zimmermann 1983a; Lotters et al. 2007). Within this

range twelve different populations with distinct morpho-

types exist; however, precise information on the distribu-

tion of most is lacking (Ostrowski & Mahn 2016). The

species’ natural habitat covers hot and humid forests,

gallery forests and rivers which are surrounded by dry and

sparsely vegetated landscapes at an elevation of up to

1,800 m (Lotters et al. 2007). Different call types have

Corresponding editor: W. Bohme

74 Alice Marie Scherges & Dennis Rédder

Fig. 1. Photographs of selected specimens of all study populations of Epipedobates anthonyi and Epipedobates tricolor. A: E.

anthonyi |: “Buena Esperanza”. B: E. anthonyi II: “Rio Jubones”. C: E. anthonyi Il: “Rio Saladillo”. D: E. anthonyi IV: “Tierra

alta”. E: E. tricolor V: “Moraspungo”. F: E. tricolor VI: “Rio Soloma”’.

been reported for this species and besides the known func-

tions (courtship behavior, territorial behavior, competition,

localization, defense signal, distress signal and alarm sig-

nal) (e.g., Bogert 1960; Whitney & Krebs 1975; Wells &

Bonn zoological Bulletin 66 (1): 73-84

Schwartz 2006; Wells 2007), their advertisement call al-

so triggers specific short-term behavior (females show

positive phonotactic responses on the day of oviposition,

which are head lifting, persevering, orienting by head and

©ZFMK

Intra- and interspecific comparisons of advertisement calls of poison dart frogs TS

body movements, zigzag hopping towards sound source,

tactile contact to sound source) and reproductive state

(Zimmermann & Rahmann 1987). According to Hermans

et al. (2002), specific calls might also serve to identify al-

ready occupied territories. The closely related species

Epipedobates tricolor inhabits premontane forests and

sparsely vegetated margins of streams in western and cen-

tral Ecuador (Graham et al. 2004; Lotters 2007).

Silverstone (1976) recorded a population of E. antho-

nyi from the province Azuay in Ecuador which he erro-

neously identified as E. tricolor. Graham et al. (2004) cor-

rected the mistake several decades later by detecting sig-

nificant genetic differences that correspond to the geo-

graphically disjunct distribution ranges, separated by a cor-

ridor of 200 km. Based on the assignment by Silverstone

(1976), multiple scientific papers confused the two

species including the description of the advertisement call

of E. tricolor which actually corresponds to E. anthonyi

in Zimmermann & Rahmann (1987). For their studies

Zimmermann & Rahmann (1987) used specimens from

their own breeding stock (Zimmermann 1983b), which

were identified following Zimmermann (1983a).

According to Zimmermann & Rahmann (1987), the ad-

vertisement call, emitted by males during the breeding sea-

son, is a long trilling high-pitched sound with a duration

of 2.15—5.2 s, on average 40-115 pulses per trill, and a

dominant frequency ranging between 4,160—4,530 Hz. The

pulses last about 15-25 ms, whereas the pulse intervals

show a length of 30-50 ms. However, the advertisement

call of E. tricolor remained yet undescribed. Here, we pro-

vide a description of the advertisement call of E. tricolor

and compare spectral and temporal call characteristics be-

tween the closely related species to assess whether

acoustic-based species discrimination is feasible. In ad-

dition, we quantify intraspecific variation in call patterns

by comparing calls of geographically isolated populations.

MATERIAL AND METHODS

To compare acoustic signals of Epipedobates anthonyi and

Epipedobates tricolor, advertisement calls of four popu-

lations of E. anthonyi, and two populations of E. tricolor

were recorded. Study populations for E. anthonyi comprise

I: “Buena Esperanza” (n=5), II: “Rio Jubones” (n= 6) and

Il: “Rio Saladillo” from geographically ranges in the

vicinity of El, Oro, Ecuador (Ostrowksi & Mahn 2016)

and IV: “Tierra alta” (n=4) which occurs along the bor-

der of Loja and Peru (Ostrowski & Mahn 2016). Study

populations for E. tricolor comprise V: Moraspungo (n=6)

from Cotopaxi and VI: “Rio Soloma” (n=4) from Bolivar

(Ostrowski & Mahn 2016) (Fig. 1). All frogs were obtained

from the pet trade and represent F, from wild caught spec-

imens (Understory Enterprises Inc. [www.understoryen-

terprises.com] via Peruvian frog import [www.peruvian-

Bonn zoological Bulletin 66 (1): 73-84

frogimport.com now available at https://nasuta.nl/] and

Raymond Kuijf, both the Netherlands). Populations were

held in separate enclosures measuring 70 x 60 x 50 cm

(H x W x D) or 40 x 40 x 50 cm. Enclosures were dense-

ly vegetated with tropical plants such as Pilea sp., bromeli-

ads like Neoregelia schultesiana, some mosses and ten-

drils and equipped with a small pond of water. The bot-

tom was covered with wet sponges and walls were lined

with cork for all populations except “Tierra alta”, for

which Hygrolon was used instead. The air temperature was

kept between 22 and 24 °C with constant conditions dur-

ing all recordings; rain was simulated by irrigation sys-

tems running three times per day for 30 minutes, and wa-

ter was sprayed three times per day for 30 seconds, to

maintain humidity.

The calls were recorded using a PMD620MKII Profes-

sional Handheld Recorder (Marantz Professional, Kawasa-

ki, Japan) by placing the recorder on the sound-permeable

ceiling of the enclosures and maintaining a distance of sev-

eral meters from the enclosures to keep disturbance to a

minimum. To ensure suitable recordings, the irrigation sys-

tem was deactivated during recordings. All calls were

recorded, cut and processed using Audacity V. 2.0.5.

(Audacity Development Team, available at

http://audacity.sourceforge.net). For visualization we used

the seewave package for Cran R 3.3.2 (Sueur et al. 2008).

Spectrograms were utilized to measure call length, pulse

length, pulse intervals and number of pulses; frequencies

of each call were plotted using a Hanning Window with

a size of 512 and a linear representation to determine dom-

inant frequencies, fundamental frequencies and _har-

monies. Images from oscillograms and spectrograms were

produced for each population. In addition, minima, max-

ima and mean values of important call features (call du-

ration, number of pulses, pulse duration, pulse frequen-

cy, pulse interval length and important frequencies) were

computed. Furthermore, one-way ANOVA tests were per-

formed to test for significant differences between impor-

tant call parameters of E. anthonyi and E. tricolor; post

hoc Tukey HSD tests were used to assess intra- and inter-

specific differences.

RESULTS

A total of 401 calls were recorded and analyzed. For E.

anthonyi 282 calls were recorded (89 for I: “Buena Es-

peranza’”’, 39 for II: “Rio Jubones”, 64 for III: “Rio Sal-

adillo”, 90 for IV: “Tierra alta’), 119 calls were recorded

for E. tricolor (68 for V: “Moraspungo”’, 51 for VI: “Rio

Soloma’’).

The advertisement calls of both species were found to

be high-pitched and trilling with numerous pronounced

pulses per call, six harmonies located above and a funda-

mental frequency located below the dominant frequency

©ZFMK

Frequency (kHz)

Amplitude

Frequency (kHz)

Amplitude

Frequency (kHz)

Amplitude

Frequency (kHz)

Amplitude

Alice Marie Scherges & Dennis R6dder

A, Bueno Esperanza Speen es pee

co 1 i) i} t

en Ga oA oe ie er rp ee Pepe hese peepee th pore pe ere Pee ee heehee rea , 9

| ,t ' wee Weare petted © BEL CR hag heute OR ihsnae Riee to Gabba Ceo bed aia d i ieeb webs Boe hk ee Boat honad ie get hs Pep ail

Peewee A POPPER GCE UE PARE OD tee Ph Ode PEPE Ode PRP TRO ee

| Li AsAAAAAAMAAA AAS AAALIAAAMAMALAARAMASMOAASAnMAMaaAaAaA bth be

: Bk. (s) ° :

8 Rio Jubones

[eam seen dGpedy Seaton! j a "I ye an 2 i, xe , me rs Gouna - rere repr’ PP PP: eats Het Sutteaay io sen Realeclgataecig ives goblet gesamness Gpeg ian atta ae

: Time (s) ; ;

Cc Rio Saladillo

A RD tote BB Ts eee teen Cee ce

| ahh ‘

! repel Gh ars 7

* «

‘ big ta! ee “ane

8 Uae ia

Time (s)

EAN NS NIG

Ae RR HY RR gn

aed aa .

0 1 2 3 4

Time fs)

Fig. 2. Spectrograms and oscillograms of the recorded advertisement calls of Epipedobates anthonyi A: “Bueno Esperanza”, B:

“Rio Jubones’’,, C: “Rio Saladillo” and D: “Tierra Alta”.

Intra- and interspecific comparisons of advertisement calls of poison dart frogs Te:

M U rospu ngo Amplitude (dB)

oO 4; i} i) 7

H / a

—

Pf OCONEE ED OCOEO DEER D ED ODER 6

& 10

s MPRAE RR AREA / ita

' ‘ Gre CRPP ER THT ETP GREET TEER ECD EP EFO RP ERELEEOETRE CREED TG EI ET EPUB DERE E g

‘ ; a eRe) ; hy Ye

ee { ba ee Tg

iyir eM Er FE TAR og %

4 ; j wall i? er i . ¢ ‘. . ee ay i

o 0 bait) u i Pines! oy bits LJ ds ’ rs f t e]

a RUNNIN VN UN PTTLVNVATENENTACVTVLTTINTTI

: QUAL ALA

0 1 2 3 4

Time (s)

B Rio Soloma

it t THERESE ECL EL P pep ey pek pnt

NOR eae aa aera eee ARP a i Ree Pee weer ei WIP Cer errr ta Fae ee) POPU PP OCORM ERR POLOCNP a EL IISLES

y ‘ Y i

= OO EAN COUPLE ERE ECEL UCR ER ERE CEREI EDR ECR EGER CECA REEL Cbd GUM bE Hy a

= ‘ * ’ ‘ . s hae Bt ae

rc} i ye ( (ity, | (pul roth ere Tee \ y

2 40 mbiryenedes kets why hy Ch T wee Ee dees DEO he bebe bead Cs 2 ee ee ae ere ree corre

o : ‘ ‘en :

=] ¥ : OY

2 N OT Rv CONE A (eee See ee ee ee SO ee ee

im \ v ' : ' i D a BO

At Re ew eee dovig 9, A af bap I

5 4 eee yl beesssesteede . oF rh ae 3 Mis ae “ee

Ruts oe es

i active ; A igal t; wen ‘ Sat Te “f

a a sande Be KA SN

N Ae Ve mo whe eee SF 3 " Nhe cay

@ 0 ; ee

se]

‘a

2 3 4

Time (s)

Fig. 3. Spectrograms and oscillograms of the recorded advertisement calls of Epipedobates tricolor. A: “Moraspungo” and B:

“Rio Soloma’’.

(Figs. 2, 3). In general, dominant frequencies, fundamen-

tal frequencies and harmonies remained constant from the

beginning to the end, but for some calls slightly lower ini-

tial frequencies could be recognized followed by stabiliza-

tion at higher values. Comparable to this, pulse frequen-

cy and arrangement of pulses remained relatively regular

maintaining similar distances to the neighboring pulses

during the whole call, but sometimes this was modulat-

ed. We observed that pulse frequency for some calls was

lower at the beginning and increased after an initial phase.

Also, in the final stage an increasing pulse frequency can

be recognized for some signals. However, the pulse pat-

tern is occasionally irregular with two or more pulses

forming groups which are clearly separated from other

groups of pulses, so that calls consist of several notes with

a variable number of pulses per note. Although this was

observed in both species, it remains an exception. Due to

automatic recording, calls could not be related to specif-

ic individuals within the monitored tank.

Bonn zoological Bulletin 66 (1): 73-84

The trilling signals of E. anthonyi had an average du-

ration of 2.55 s + 1.267 (range: 0.5—5.6 s; n=282), were

composed of 48.521 pulses + 20.971 (range: 9-99 puls-

es; n=282) per call and had a pulse frequency of 20.293

pulses/s + 5.02 (range: 8.776—40 pulses/s; n=282; com-

puted for each call by dividing the number of pulses

through call length). Every pulse possessed a length of

17.091 ms + 4.041 (range: 9-33 ms; n=282), and the av-

erage length of intervals between pulses was 32.645 ms

+ 7.618 (range: 14-71 ms; n=282). The dominant frequen-

cy with an average value of 4.24 kHz + 0.672 (range:

3.126—5.67 kHz; n=282) was located above the fundamen-

tal frequency of 2.196 kHz + 4.737 (range: 1.456—-4.584

kHz; n=229) and below up to six harmonies occupying a

frequency band of 4.497—18.135 kHz. The first harmony

averaged on 5.918 kHz + 0.483 (range: 4.441—7.151 kHz;

n=262), whereas the last harmony was observable at a fre-

quency of 16.997 kHz + 1.138 (range: 12.330—-18.135

kHz; n=150). Detailed harmony frequency values are de-

picted in Table 1.

©ZFMK

78 Alice Marie Scherges & Dennis Rédder

Table 1. Properties of the advertisement calls of Epipedobates anthonyi and Epipedobates tricolor in comparison. Values are giv-

en as mean + standard deviation (SD).

Feature

Call duration (s)

Total number of pulses per note

Pulse frequency (pulses/s)

Pulse duration (ms)

Pulse interval length (ms)

Dominant frequency (kHz)

Fundamental frequency (kHz)

1. Harmony (kHz)

2. Harmony (kHz)

3. Harmony (kHz)

4. Harmony (kHz)

5. Harmony (kHz)

6. Harmony (kHz)

Bonn zoological Bulletin 66 (1): 73-84

Epipedobates anthonyi

2.550 + 1.267

(0.5-5.6) n=282

48.521 + 20.971

(9-99) n=282

20.293 + 5.02

(8.776-40) n=282

17.091 + 4.041

(9-33) n=282

32.645 + 7.618

(14-71) n=282

4.240 + 0.672

(3.126-5.67) n=282

2.196 + 4.737

(1.456-4.584) n=299

5.918+ 0.483

(4.441-7.151) n=262

8.154+0.619

(6.364-9.659 kHz, n=250)

10.210 + 0.773

(8.131-12.469) n=211

12.171 £ 0.958

(9.174-13.493) n=213

14.400 + 1.066

(11.157-15.819) n=191

16.997 + 1.138

(12.330-18.135) n=150

Epipedobates tricolor

2.016 + 0.337

(0.6-2.6) n=119

60.765 + 11.682

(14-79) n=119

30.051 + 3.039

(17.5-33.75) n=119

13.958 + 2.584

(9-22) n=119

19.748 + 2.324

(13-26) n=119

4.081 + 0.218

(3.551-5.431) n=119

2.229 + 0.153

(2.036-2.814) n=96)

5.941 + 0.336

(4.719-6.567) n=119

8.267 + 0.345

(7.103-9.985) n=114

10.365 + 0.348

(9.36-10.879) n=116

12.240 + 0.480

(10.664-13.06) n=114

14.428 + 0.350

(13.579-15.131) n=105

16.289 + 0.302

(15.45-16.869) n=97

©OZFMK

Intra- and interspecific comparisons of advertisement calls of poison dart frogs

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Bonn zoological Bulletin 66 (1): 73-84

Intra- and interspecific comparisons of advertisement calls of poison dart frogs 81

For the advertisement calls of E. tricolor an average

length of 2.016 s + 0.337 (range: 0.6—2.6 s; n=119) was

measured. The total number of pulses per note was 60.765

+ 11.682 (range: 14-79; n=119) with 30.051 + 3.039 puls-

es per second (range: 17.5—33.75 pulses/s; n=119). The

single pulses on average persisted for 13.958 ms + 2.584

(range: 9-22 ms; n=119) and maintained intervals for

19.748 ms + 2.324 (range: 13-26 ms; n=119). A domi-

nant frequency of 4.081 kHz + 0.218 (range: 3.551—5.431

kHz; n=119) and a fundamental frequency of 2.229 kHz

+ 0.153 (range: 2.036—2.814 kHz; n=96) was measured.

As for E. anthonyi, six harmonies above the dominant fre-

quency could be recognized, occupying a frequency band

of 4.719-16.869 kHz. For detailed results see Table 1. In

general, observed variations were lower in E. tricolor than

in E. anthonyi.

One-way ANOVA tests were used to assess statistical-

ly significant differences between all local variants of E.

anthonyi and E. tricolor for the most relevant call prop-

erties: call length (Fis.305=77.16; p<0.001), fundamental fre-

quency (Fo3i9=6.031; p<0.001), dominant frequency

(Fis.395=62.97; p<0.001), pulse interval (Fo395=117.8;

p<0.001), number of pulses per call (Fis395=46.8: p<0.001),

pulse frequency (Fo305=200.4; p<0.001), pulse length

(Fo395=33.84; p<0.001).

Intraspecific differences between the populations of E.

anthonyi and E. tricolor were detected in terms of stan-

dard deviation. While there are very slight differences be-

tween E. tricolor V: “Moraspungo” and VI: “Rio Soloma”

in matters of some harmony frequencies (first, second,

fourth and fifth harmonies), huge discrepancies were

found between the populations of E. anthonyi. Compar-

ing the means and standard variations of call properties

our results suggested that the variation between the E. an-

thonyi populations was much greater than the differences

between E. anthonyi and E. tricolor. With a call duration

of 3.750 s + 1.343, the call of I: “Buena Esperanza” was

significantly longer than for the remaining populations,

which all lasted on average 2 s. Furthermore the total num-

ber of pulses per call is highest for I as well (63.970 s +

24.349). For HI: “Rio Saladillo” and IV: “Tierra alta” we

found a slightly elevated pulse frequency (22.875 s + 6.284

and 20.500 s + 1.94) in comparison with I (17.198 s +

3.153) and I (16.153 s + 2.980). The dominant frequen-

cy of I (3.649 kHz + 0.264) is lower than the dominant

frequencies of the other populations, all averaging above

4.000 kHz. Additionally, smaller values for the fifth and

the sixth harmonies were recognized for I, and III also

showed a decreased average for the sixth harmony. Be-

sides, we noticed slight variations between all populations

of E. anthonyi in terms of the first and the fourth harmo-

ny. In general, population I was most variable showing the

highest variability across all populations in terms of stan-

dard deviation. Intraspecific differences are depicted in

Table 2.

Bonn zoological Bulletin 66 (1): 73-84

Post hoc Tukey HSD tests revealed E. tricolor to show

statistically significant differences across both populations

in the number of pulses per call (p<0.05), however, dom-

inant and fundamental frequency, pulse frequency, pulse

length, pulse interval and call duration do not vary sig-

nificantly. In E. anthonyi there were statistically signifi-

cant differences between population I and III for all pa-

rameters that were included (p<0.01 each). In contrast,

population II and IV only statistically significantly differ

in number of pulses per call, pulse frequency, pulse length

and pulse interval length (p<0.01 each), whereas call du-

ration (p=0.957), dominant frequency (p=0.999), and fun-

damental frequency (p=0.999) do not show statistically

significant differences. Pairwise comparison of all remain-

ing combinations of the populations proved at least five

statistically significant different parameters of included

call properties. For detailed results see Table 3.

DISCUSSION

Properties of the advertisement call of Epipedobates an-

thonyi in the present study were generally in accordance

with the physical characteristics determined by Zimmer-

mann & Rahmann (1987), but call patterns were revealed

to be more variable. Although the note length (2.15—5.2

s) described by Zimmermann & Rahmann (1987) match-

es the computed average call duration observed in this

study, we found that the lower limit reported by Zimmer-

mann & Rahmann (1987) was higher than observed here.

This was also true for the total number of pulses per note,

which Zimmermann & Rahmann (1987) reported to range

from 40 to 115 but was found to range from 9 to 99 in the

present study. Due to very few short calls (0.5 s) the av-

erage call length was slightly shorter than in previous stud-

ies (Zimmermann & Rahmann 1987). However, this might

be attributed to elevated levels of disturbance in our ani-

mal keeping facility which may have sometimes led to pre-

mature interruption of calls. Zimmermann & Rahmann

(1987) reported pulse durations between 14—25 ms with

intervals of 30-50 ms. Although pulse duration and inter-

vals determined in this study were both longer, results are

comparable in general. While Zimmermann & Rahmann

(1987) studied a single population we studied four, which

could account for the variation in recorded pulse duration

and intervals. Zimmermann & Rahmann (1987) found

most calls recorded to exhibit only a single harmony with

a frequency around 8 kHz while up to six distinct har-

monies were present in the majority of the calls in the pres-

ent study. The few cases in which no or only few har-

monies could be identified were caused by background

noises or low calls. Pulse frequency and fundamental fre-

quency were not reported previously.

A comparison of the call characteristics of both species

yielded small variations. While patterns in EF. anthonyi

©ZFMK

82 Alice Marie Scherges & Dennis R6dder

were very similar in terms of standard deviation in note

duration, number of pulses per note, pulse duration and

all important frequency values, standard deviations of E.

tricolor revealed similarity of calls to be restricted to calls

to note duration, number of pulses per call, and all impor-

tant frequencies except for the last harmony. As we stud-

ied four populations of E. anthonyi but only two popula-

tions of E. tricolor, these discrepancies between the stan-

dard deviations might be attributed to sample size.

Despite the similarities, calls of both species clearly dif-

fer in pulse interval length (EZ. anthonyi: 32.645 + 7.618

vs. E. tricolor: 19.748 + 2.324) which results in a signif-

icantly higher pulse frequency for EF. tricolor than for E.

anthonyi (E. anthonyi: 20.293 + 5.02 vs. E. tricolor:

30.051 + 3.039), which might be attributed to local vari-

ation across their different distribution areas. Pulse fre-

quency is known to be important for anuran communica-

tion (Cocroft & Ryan 1995) and according to Cocroft &

Ryan (1995) this feature seems to be the most divergent

character in advertisement calls for the genera Bufo and

Pseudacris and is different in almost every species includ-

ed in their study. Given the clear differences in pulse fre-

quency and interval length, which were found between E.

anthonyi and E. tricolor, these characteristics might prove

to be relevant characters for species delimitation in

Epipedobates (significantly different in intraspecific

comparisons: 100%, intraspecific comparisons: 71.5 %).

Only few descriptions of the advertisement calls of oth-

er species of Epipedobates exist, except for a comprehen-

sive description for EF. boulengeri (Barbour, 1909) (Lot-

ters et al. 2003) who found distinct advertisement calls for

two populations from Colombia and Ecuador, respective-

ly. While the population from Colombia was found to ex-

hibit calls consisting of 1—3 notes, calls of the population

from Ecuador comprised 6—12 significantly shorter notes

with lower numbers of pulses per note. Calls consisting

of several notes were occasionally recognizable for E. an-

thonyi and E. tricolor as well, but these calls remained ex-

ceptions. The dominant frequencies of 5.192 kHz (Colom-

bia) and 5.198 kHz (Ecuador) for E. boulengeri are high-

er than in the present study, thus, their calls differ in at

least two characteristics from E. anthonyi and E. tricol-

or. Further studies are required to facilitate a more thor-

ough comparison. Because of their different advertisement

calls, populations of E. boulengeri were thought to rep-

resent a species complex and Grant et al. (2006) proved

E. boulengeri not to be monophyletic based on genetic da-

ta.

We found huge intraspecific disparities across popula-

tions of E. anthonyi with population I being the most di-

vergent. However, as the geographic distribution of the

species is poorly known, a character comparison relating

to geographical distribution and environmental influences

was not possible. Given that previous research on various

taxa revealed varying signals between and within geo-

Bonn zoological Bulletin 66 (1): 73-84

graphically separated units and conspecific populations

and that variations increase with geographical distances

(Nei 1972; Wo6hrmann & Jain 1990; Hutchinson & Tem-

pleton 1999), it appears likely that population I exhibits

the largest geographic distance to all other populations of

E. anthonyi. Intraspecific differences of E. tricolor were

very low and their distribution is poorly known as well.

Considering huge call similarities, both populations

might show small geographical distance.

Previous research demonstrated that depending on the

species, some call characteristics are more important for

species discrimination than others, and sometimes only a

single feature varies between two closely related species,

for example, in some species of Bufo (Cocroft & Ryan

1995). Thus, calls can be very helpful for taxonomic mat-

ters, facilitating acoustic signal- based species discrimi-

nation (Wells 1977; Zimmermann & Zimmermann 1988;

De la Riva et al. 1995, 1996; Lotters et al. 1999; Padial

et al. 2008). However, calls are not always suitable to dis-

criminate between closely related taxa in amphibians (L6t-

ters et al. 2001; Napoli & Cruz 2005; Tsuji-Nishikidoet

et al. 2012). Tsuji-Nishikidoet et al. (2012) showed that

differentiation is not possible based on acoustic signals for

the closely related species Allobates nidicola and A. mas-

niger (Aromobatidae), which occupy similar areas along

the Madeira River. Additionally, previous research on frogs

of the genus Allobates (Aromobatidae) supports that dis-

crepancies in call characters are often associated with ge-

netic differences in amphibians but do not necessarily in-

dicate different species (Sim6es et al. 2008). Advertise-

ment call characteristics were shown to be very different

even within a species depending on temperature, distribu-

tion area and other aspects (Bernal et al. 2005; Marquez

& Eekhout 2006; Lotters et al. 2009); this is now also

shown across different populations of E. anthonyi. Huge

differences in call features within E. anthonyi question the

suitability of advertisement call characteristics for species

identification. Between E. anthonyi and E. tricolor signif-

icant intraspecific variation could be detected for pulse fre-

quency and interval length which can be used for species

identification. Thus acoustic based classification should

be regarded with suspicion for at least those two species

of Epipedobates.

The present research raises new questions, which require

further investigation. First, to show if pulse frequency and

interval length, which differ between E. anthonyi and E.

tricolor, are sufficient for females to discriminate between

Species supporting the genetic assignment by Graham et

al. (2004), further behavioral studies should be conduct-

ed: According to the biological species concept, a species

is a group of potentially interbreeding individuals in time,

thus, mating experiments between E. anthonyi and E. tri-

color could be investigated to detect if pulse frequency and

pulse interval length are relevant for female attraction and

species recognition. Besides, acoustic comparison of more

©OZFMK

Intra- and interspecific comparisons of advertisement calls of poison dart frogs 83

populations would help to determine whether observed

variation in pulse frequency and interval length was attrib-

uted to the restricted sample size or whether these char-

acteristics still differ from each other in a more extensive

study and thus proving them to be relevant for species

identification. Further advertisement call comparison of

more species of the genus Epipedobates would help to de-

tect if pulse frequency and pulse interval length are im-

portant for species discrimination for Epipedobates in gen-

eral. In addition, investigations in more populations of both

Species are required to determine whether intraspecific dif-

ferences are also observable across other populations. Eco-

logical mating experiments could help to indicate if fe-

males are attracted by calling males from other popula-

tions despite intraspecific differences and thus support the

populations belonging to the same species.

Sometimes species are difficult to distinguish solely

based on their genetic or morphological characters (Ru-

binoff et al. 2006). Thus the anuran advertisement call

characteristics can be useful to help classify taxa. To de-

termine which and how many acoustic characters need to

be used to identify diverging species, more extensive call

comparison studies have to be conducted. Due to intraspe-

cific variations for E. anthonyi and E. tricolor, acoustic

based discrimination should be interpreted with care. Al-

though we did not find out whether the observed differ-

ent call characteristics facilitate species discrimination be-

tween E. anthonyi and E. tricolor or if identification based

on advertisement calls is possible, this study is a starting

point for further investigations in acoustic based-identi-

fication of Epipedobates.

Acknowledgements. The manuscript benefited from critical in-

put of two anonymous reviews and Ralph Peters (ZFMK). We

are grateful to Flora Ihlow, Timo Hartmann and David Hoérnes

for logistic support, as well as to the Alexander-Koenig-

Gesellschaft (AKG) for financial support.

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©OZFMK

Bonn zoological Bulletin 66 (1): 85—93

April 2017

Feeding ecology of the invasive gecko species

Hemidactylus mabouia (Moreau de Jonnes, 1818) (Sauria: Gekkonidae)

in Sao Sebastiao (Brazil)

Yvonne Driike' & Dennis Rodder™

' Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute of Animal Biodiversity,

Adenauerallee 160, D-53113 Bonn, Germany;

“E-mail: d.roedder@leibniz-zfmk.de

Abstract. In this study the feeding ecology of Hemidactylus mabouia (Moreau de Jonneés, 1818) is analyzed based on

stomach contents of specimens collected in 1964, 1965 and 1967 in Sao Sebastiao, southeastern Brazil. Hemidactylus

mabouia is an alien invasive species, especially in South America, with known negative impacts on its environment. Our

results suggest that the studied population shows a generalist feeding habit. We detected a nearly complete niche overlap

in the diet of males, females and juveniles. The most important prey items of all specimens were lepidopterans followed

by dipterans. In general, most prey items were winged insects, which may be characteristic for populations living close

to human civilization. Different to other studies on the diet of H. mabouia juveniles showed the highest trophic diversi-

ty.

Key words. Gekkonidae, naturalized and invasive species, diet composition.

INTRODUCTION

On a global scale, alien invasive species are an increas-

ing problem with unpredictable negative effects like niche

displacement, competitive exclusion, mutualism, extinc-

tion, and biodiversity loss (Mooney & Cleland 2001;

Clavero 2005). Members of the genus Hemidactylus, al-

so called “house geckos”, live synanthropically (Vanzoli-

ni 1980; Howard 2001), which is also true for Hemidacty-

lus mabouia (Moreau de Jonnés, 1818) (Spawls 2002).

Among other countries (Fuenmayor 2005) H. mabouia oc-

curs native in Central and East Africa and Madagascar and

is an alien invasive species in many states of South Amer-

ica, for example, Brazil (Vanzolini 1978).

The gecko is characterized by small body-size (maxi-

mum 70 mm) (Hodge et al. 2003). It is able to change its

body color, varying from greyish white to dark brown, and

the body is drawn with v-shaped bands (Murphy 1997).

All over its dorsum it has small and granular scales and

weakly keeled tubercles arranged in transverse rows

(Hodge et al. 2003). It is primarily a nocturnal predator

(Dixon & Soini 1986) hiding during the day (Avila-Pires

1995) and reaching its activity maximum in the evening

(Branch 1988). One reason for the synanthropy of the

gecko may be the availability of a magnitude of potential

prey items attracted by light at night, for example, flies,

bugs and moths (Howard 2001; Pianka & Vitt 2003).

Due to increasing worldwide trade H. mabouia was able

to colonize new territories (Carranza & Arnold 2006). Po-

tential reasons why H. mabouia is such a successful in-

Received: 19.10.2016

Accepted: 03.04.2017

vader are that it is a good colonizer (Hughes et al. 2015)

and is often described as a generalist predator (Zamprog-

no 1998) making it easy for the gecko to survive in new

areas. Multiple introduction pathways have been proposed,

including natural rafting (Kluge 1969) and transport via

slave trade ships (Goeldi 1902; Breuil 2002). As addition-

al invasion pathway it has been observed that the species

is able to stick its eggs to fishing ships (Breuil 2009).

Once introduced H. mabouia is known to negatively af-

fect populations of other geckos (Short & Petren 2012),

as was also suggested for other geckos of the genus Hemi-

dactylus (e.g., Cole et al. 2005). Therefore it is important

to study the ecology of these species in order to assess the

effects of the invasive species on its new environment. In

the present study stomach contents of H. mabouia speci-

mens from Sao Sebastido (state SAo Paulo, Brazil) were

analyzed and the trophic niche was assessed including on-

togenetic and sexual differences in diet composition. The

results are compared to the trophic niche of a native gecko

species (Gymnodactylus darwinii [Gray, 1845]) which oc-

curs sympatrically in Sao Paulo state and Pernambuco

state (e.g., Oliveira et al. 2016). We assessed if there is a

possible competition for food between the two species. Al-

though G. darwinii occupies diverse kinds of environments

such as rainforests and forest edges, it occupies also hu-

man populated regions (Teixeira 2002; Almeida-Gomes et

al. 2008). Since both species overlap in habitat preferences

as well as in the time of foraging (Pellegrino 2005), a com-

petition for prey items is likely.

Corresponding editor: W. Bohme

86 Yvonne Dritike & Dennis R6odder

MATERIAL AND METHODS

Specimens were collected in Sao Sebastiao (Brazil) at dif-

ferent locations and times and preserved in 70% Ethanol.

Six samples were collected in October 1964 (ID

3238-3243), 15 samples are part of a batch collected in

October 1964 and March 1965 (ID 3255-3269, no exact

month is traceable for single specimens) and an addition-

al 48 specimens were collected in August 1967 (ID 3190-

3237). All specimens originated from the collection of P.

Muller (Trier, Germany, original IDs given) (Miller 1968,

1969), which was recently transferred to the Zoologisches

Forschungsmuseum Alexander Koenig, Bonn (ZFMK).

All specimens were confirmed to be identified as Hemi-

dactylus mabouia. Main identification characters are:

lamellae on the fourth toe do not reach the base of digit

and the numbers of scansors beneath the first toe (Mur-

phy 1997). Furthermore, H. mabouia has enlarged and

weekly keeled tubercles on the back, which are arranged

in 12 to 18 transverse rows (Spawls 2002). Male speci-

mens could be identified by the presence of preanal pores

(Avila-Pires 1995).

Snout-vent length, mouth-width and head-width of all

specimens were measured with a digital caliper (accura-

cy +0.01mm). According to Dixon & Soini (1986) adult

male specimens may reach a snout-vent length between

58 and 67 mm and females a length between 61 and 70

mm. We made three specific categories: male, female or

juvenile (< 55 mm). Additionally, females and males were

jointly analyzed as the category of adults. In total, there

were 69 tested specimens: 11 males, 23 females (equals

34 specimens in the category of adults) and 35 juveniles.

All specimens were dissected with a scalpel by cutting

from anterior to posterior of the ventral side. The dermis

was fixed laterally with pins, and often parts of the intes-

tinal tract had to be taken sideward to reach the stomach

which was then removed with two cuts. The first cut was

placed at the approximate place of the duodenum and the

second directly beneath the esophagus. Thereafter the

stomach was taken out and kept in 70% ethanol. Stom-

achs were opened with a scalpel by a longitudinal cut and

all content was removed with a dissection needle and trans-

ferred to a Petri dish for identifications. All stomachs and

prey items are kept in the collection of the ZFMK.

All prey items were identified under a stereomicroscope

(Olympus) to the level of order following Miller (2011).

In the order Hymenoptera, Formicidae (ants) were iden-

tified to family level. Some prey items were too fragment-

ed to be identified and were categorized as “others”. They

have been considered in context with volumetric calcula-

tions only. Length and width of single prey items was

measured with a digital caliper (accuracy +0.01mm) and

the volume of each item was estimated with the formula

for the volume of an oblate ellipsoid (Magnusson 2003),

Bonn zoological Bulletin 66 (1): 85—93

because the form of an oblate ellipsoid is close to the shape

of most insects:

_ 12

/=—*T* =

1 Ww

where / is the length and w the width of the food item.

Number, volume and frequency of the different prey cat-

egories were used to calculate the importance value of prey

(IV; Gadsen 1997) of each prey category following:

Where V;; = Volume of food item i in predator j, S Vij

= total volume of all stomachs, N;;= number of elements

of food item 7 in predator 7, SN;; = total number of prey

items in sample, F;; = number of stomachs of predator 7

in which food item i was found, SN; = total number of

stomachs (Gadsen 1997).

To determine whether specimens show a generalist or

a specialist feeding habit, different indices were used: the

Simpson’s Index considers diversity richness and evenness

and is therefore well suited to assess if a species is rather

a generalist or a specialist predator. Results close to zero

indicate nearly no diversity of prey items and if the index

equals one this would imply infinite diversity (Simpson

1949). The Simpson’s Index of Diversity was calculated

with the following formula:

where n; = number of food item 7 in all stomachs and n

= total number of prey items. The Shannon-Index (Shan-

non 1948) indicates the trophic diversity in prey consump-

tion of the different groups:

H, = —Y p; *1n (p;) and corresponding H,,,, = In(N)

where p; = number of all prey items of category i divid-

ed by number of all prey items of all categories and N =

number of all prey items of all categories. The higher

the value of the index the more diverse is the diet. By

dividing H, by H,,,, the evenness Ey was calculated:

Hmax

E, =

E}, ranges between 0 and 1. Results close to one indi-

cate an even distribution of prey items and close to zero

©OZFMK

Feeding ecology of Hemidactylus mabouia in Sao Sebastiao (Brazil) 87

indicate an irregular distribution. To test for niche-over-

lap between the different groups (males, females and ju-

veniles) Pianka’s Index was used (Pianka 1973):

rer Pay * Pix

0, = a

: V ve Pajz * D Prinz

Where P;; and Pj; are the frequencies of consumed prey

item i in the different categories j and k. The result of the

Pianka’s index can vary between 0 and 1.A value of zero

represents no similarities in prey consumption between the

tested groups and values closer to one indicate a high niche

overlap.

For results of prey size, snout-vent length, head-width

and mouth-width a Mann-Whitney U-test was performed

to test if differences between categories were significant

(Whitney 1947). To account for allometric growth the ra-

tio of head-width to snout-vent length and the ratio of

mouth-width to snout-vent length were used to test for sig-

nificant differences. All data was analyzed using Microsoft

Excel 2010.

RESULTS

Of the 69 specimens five (7.25%) had empty stomachs.

Hence, for feeding ecology analyses 64 samples remained,

of which nine were males, 22 females and 33 juveniles.

In total, 474 single prey items could be identified belong-

ing to 14 different categories (Table 1). 13 categories rep-

resented Arthropods: one in the category of Crustacea

(Isopoda), ten of the class Insecta (Blattodea, Coleoptera,

Diptera, Hemiptera, Hymenoptera, Formicidae and oth-

ers), Lepidoptera, Lepidoptera larvae and Orthoptera and

two categories of the class Arachnida (Araneae and Ixo-

dida). In some stomachs we found small stones represent-

ing an additional category. In total there were eight items

of Crustaceans, 440 of Insecta and 21 items of Arachni-

da (see Table 1). On average the specimens had 7.4 prey

items in their stomachs. In all samples the most common

prey items found were Diptera (N = 243) followed by Lep-

idoptera (N = 102) and Hemiptera (N = 37), though the

Importance Value (IV) is slightly higher for Lepidoptera

(IV = 1.25) than for Diptera (IV = 1.23). This is mainly

caused by the volumetric distribution of prey items (Table

1).

Volumetrically Lepidoptera represent the largest part of

all stomach contents with a volume of 53.84% followed

by Orthoptera (V = 11.25%) and Coleoptera (V = 8.62%).

Although Diptera occurred in the highest number they on-

ly represented 7.5% of the volume of all stomach contents

(Table 1). Frequently detected categories were Diptera (F

= 68.75%) and Lepidoptera (F = 51.56%), followed by

Bonn zoological Bulletin 66 (1): 85—93

Hemiptera and Coleoptera (both F = 23.44%) and

Araneae and Orthoptera (both: F = 15.63%) (Table 1).

Prey composition in males

In total there were 11 males available of which two had

empty stomachs. Additionally two males were infected

with nematodes within their stomachs. In terms of prey

consumption nine of the twelve different prey categories

were found in males, which is the smallest quantity found.

On average in male specimens we detected 7.56 items per

stomach. Numerically Lepidoptera were the most common

item N = 22 (36.07%) followed by Diptera N = 13

(21.31%) and Isopoda N = 7 (11.48%). In terms of vol-

ume Lepidoptera (V = 35.73%) were most important,

whereas Diptera only represented 1.75% of the total vol-

ume. Lepidoptera are the most significant food item with

an Importance Index of 1.24, followed by Diptera (IV =

0.88) and Coleoptera (IV = 0.86). In terms of frequency,

Diptera were found in six of nine stomachs (66.67%),

Coleoptera and Lepidoptera in five of them (55.56%). One

stone was found in a stomach and male specimens are the

only category where Isopoda were found (Table 2).

Prey composition in females

One of the female samples had an empty stomach, hence

22 remained for analyses. On average they had fewer items

per stomach than males (6.18 vs. 7.56). In terms of num-

bers, Lepidoptera N = 45 (37.19%) and Diptera N = 37

(30.58%) are dominant. Also, Lepidoptera were represent

the highest volume with 59.93% of all stomach content

of female samples, followed by Orthoptera (V = 14.44%).

Diptera were found in 16 (72.73%) of the total 22 stom-

achs, Lepidoptera in 13 (59.09%), which were also most

important (IV = 1.52) followed by Diptera (IV = 1.09)

(Table 2).

Prey composition in juveniles

Of the 35 juveniles examined two had empty stomachs

while the remaining 33 specimens had on average 9.58

food items per stomach, which was the highest number of

all three classes. In terms of quantity Diptera were most

prevalent with 193 (66.1%) items, followed by Lepidoptera

with 35 (11.99%) and Hemiptera with 23 (7.88%). Though

Diptera represented 66.1% of all found items, Lepidoptera

was again the most important category with 59% of all

stomach contents in terms of overall volume, followed by

Diptera with 10.43%. Diptera occurred in 22 (66.67%)

stomachs, Lepidoptera in 15 (45.45%) and Hemiptera in

10 (30.3%). Diptera was the most significant food item

©ZFMK

88 Yvonne Driike & Dennis R6odder

Table 1. Overall results of analyzed stomach contents (n = 64). N represents the number of prey items found of this category, %

(N) gives the corresponding percentage in relation to all found items. V (in mm’) is the total volume contributed by the item, %

(V) is the percentage of the volume compared to the total volume. F'is the frequency and shows in how many stomachs the item

is found and % (F) shows the corresponding percentage. /V is the Importance Value which results out of N, V and F.

Prey catagory N N% Vv V% F F% IV

Crustacea

Isopoda 8 1.54 3294.05 3.14 2 Ss, 0.08

Insecta

Blattodea 1 0.19 296 0.28 1 1.56 0.02

Coleoptera 27 5.19 9050.90 8.62 15 23.44 ea.

Diptera 243 46.73 7878.38 7.50 te 68.75 1.23

Hemiptera 37 Fle 2948.16 2.81 IS 23.44 0:33

Hymenoptera

Formicidae 6 | Sele) 242 0.23 5 7.81 0.09

others q 1.35 482.95 0.46 + 6:25 0.08

Lepidoptera 102 19.62 $6527.28 53.84 33 Sn56 1.25,

Lepidoptera larvae 1 Os19 583 0.56 1 1.56 0.02

Orthoptera 16 3.08 11810.98 1625 10 15.63 0.30

Others = = 9596.05 9.14 = > =

Arachnida

Araneae 19 3.65 1690.87 1.61 10 15.63 0.21

Ixodida 2 0.38 0.32 0.00 2 Sr18 0.04

Stones 0.96 591.16 0.56 5 7.81 0.09

total 474 104991.94

with an Importance Value (IV) of 1.38, followed by Lep-

idoptera (IV = 1.16) and Hemiptera (IV = 0.45) (Table 2).

Diversity indices and niche overlap

Simpson’s Index of Diversity (D) indicates little differ-

ences between males, females and juveniles. Males

showed the highest trophic diversity (D = 0.79), followed

by females (D = 0.73). When grouping all male and fe-

male adults together, the Simpson’s Index (D) is 0.76. Prey

diversity is smallest in juveniles (D = 0.52). If all sam-

ples are grouped together D equals 0.66, which suggests

that the animals are likely generalist predators (Table 3).

Results of Simpson’s — and Shannon Index suggest sim-

ilar patterns (Table 3). In only one case regarding the ju-

venile group did Shannon’s Index (H,) differ from Simp-

son’s Index. Juveniles show highest diversity with H, =

2.25 and E,= 0.91, indicating that the diet composition

of juveniles is quite evenly distributed. Males (H, = 1.79;

E7,= 0.81) and females (H, = 1.64; Ey, = 0.71) have a low-

er trophic diversity and also a more uneven distribution

Bonn zoological Bulletin 66 (1): 85—93

of prey consumption. Assessing the category of adults the

Shannon-Index was H, = 1.75 and Evenness E, was 0.73

(Table 3).

Pairwise comparison between males, females and juve-

niles using Pianka’s Index (O,;) for niche overlap suggests

highly overlapping trophic niches. The most remarkable

difference detected was between males and juveniles (O;;

= 0.85). The remaining comparisons indicated almost a

complete niche overlap (Table 4).

Body measurements and prey size

The difference in snout-vent length, mouth-width and

head-width of males and females was not significant. Sig-

nificant differences only occurred between females and

juveniles. Prey length is on average highest in males (4.66

mm + 3.28 mm), whereas female prey width is highest on

average (1.68 mm + 1.12 mm). The longest prey item was

detected in a female (SVL 16.74 mm) and although juve-

niles commonly consume smaller prey a very long prey

item with 15.76 mm was also found (Table 5). Prey length

©OZFMK

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©OZFMK

Bonn zoological Bulletin 66 (1): 85-93

90 Yvonne Dritike & Dennis R6odder

Table 3. Results of Simpson’s Index (D) and Shannon-Index (H,) and related Evenness (Ep) for the different categories.

Group Simpson’s Index Shannon Index

D Hy Ep

all 0.66 1.56 0.61

adults 0.75 1.75 0.73

males 0.79 179 0.81

females 0.73 1.64 0.71

juveniles 0.52 225 0.91

Table 4. Results of pairwise comparison of the different cate-

gories with Pianka’s Index Oj,

comparison Ojx

males — females 0.94

males — juveniles 0.85

females — juveniles 0.94

juveniles — adults 0.93

and width were only significantly different between males

and juveniles.

The average prey volume was 3062.27 mm?’ in males,

1939.17 mm’ in females and 1054.1 mm? in juveniles

(Table 5). The lowest stomach content volume was found

in a juvenile with 14.49 mm’ and the highest volume in a

male with 9417.33 mm? (Table 5). The only significant dif-

ference in the volume of consumed prey occurred between

males and juveniles.

DISCUSSION

Hemidactylus mabouia is known to have a generalist feed-

ing habit (Vitt 1995; Zamprogno & Teixeira 1998), which

is also the conclusion of this diet analysis. The animals

mainly feed on arthropods, mostly insects. Rocha & An-

jos (2007) studied a population of H. mabouia in an in-

selberg area in south-eastern Brazil where they found a

higher proportion of Araneae, representing numerically

22.4% of the total diet. In a study performed by Alves

(2013) Araneae are the most important food item for H.

mabouia in terms of frequency (20.51%). According to our

data Araneae only make up 4.01% of the total number of

prey items and occurred with a frequency of only 15.63%

in all samples (Table 1). Another prey category that is very

Bonn zoological Bulletin 66 (1): 85—93

different between the studied populations is Diptera. In the

study of Rocha & Anjos (2007), Diptera only contributed

1.9% of the total number of prey items, whereas in this

study they represent more than a half (51.27%) of all con-

sumed prey items (Table 1). In this study, the number of

individual prey items and total volume of Lepidoptera

made up high proportions contrary to the study of Rocha

& Anjos (2007) where Lepidoptera were detected in just

6.2% in terms of number and 2.8% in terms of volume.

Whereas in this study Lepidoptera make 21.52% of food

items and are also the most prevalent and important food

item constituting a volume of 53.84% of all consumed

prey (Table 1). Furthermore, in the inselberg area studied

by Rocha & Anjos (2007) the authors found a higher num-

ber of different prey categories such as gastropods or

diplopods, than we did. These results may be related to

the fact that the population examined in the present study

was collected in an urban area according to the prey com-

position. Here a higher proportion of winged insects oc-

curs compared to natural habitats, as they are attracted by

artificial light (e.g., as suggested by Bonfiglio et al. 2006).

In addition, in concordance with Bonfiglio et al. (2006),

these results may be explained by differences in analyzed

microhabitats providing different proportions of prey cat-

egories.

Intraspecific comparisons

There were no noteworthy differences in the diet of males

and females. In juveniles, stomachs numerically contained

66.1% Diptera, only 21.31% in males and 30.58% in fe-

males (Table 2). Only in juveniles, Diptera have the high-

est importance (IV = 1.38) (Table 2), whereas highest im-

portance was detected for Lepidoptera in males and fe-

males. One reason for the dominance of Diptera in the di-

et of juveniles could be that juveniles in general have a

smaller mouth-width which excludes large prey items.

Whereas males and females show no significant difference

in prey size, the consumed prey of juveniles is significant-

©OZFMK

Feeding ecology of Hemidactylus mabouia in Sao Sebastiao (Brazil) 91

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Bonn zoological Bulletin 66 (1): 85-93

ly smaller (Table 5).There was no significant difference

found for body size, head-width or mouth-width between

males and females, in agreement with the results from pre-

vious studies (Anjos & Rocha 2008; Iturriaga 2013).

According to Pianka’s index, the diet composition of

males, females and juveniles is similar and has high niche

overlaps. There is nearly a complete niche overlap between

males/females and females/juveniles (Oix = 0.94) (Table

4). Males and juveniles differ the most (O;; = 0.85), but

still have a large overlap in diet composition which cor-

responds with the results presented by Rocha & Anjos

(2007).

Shannon and Simpson Diversity Indices results are con-

tradictory: According to Simpson’s Index males and fe-

males have a higher trophic diversity than the juvenile cat-

egory (Table 3), whereas Shannon Index indicates juve-

niles have the highest diversity and males and females have

a lower diversity in diet composition (Table 3). Compar-

ing adults, males, females and all samples together Shan-

non and Simpson’s indices suggest similar patterns, be-

sides for juveniles where the Shannon Index is higher than

one would expect regarding Simpson’s Index (Table 3).

Simpson’s Index gives more weight to dominant prey cat-

egories and can be interpreted as an abundance index, un-

like the Shannon Index (Hill 1973). The extremely high

number of Diptera (N = 193) (Table 4) in juvenile prey

composition could therefore influence Simpson’s index.

Interestingly, in the stomach of one juvenile we found 83

Dipterans which makes nearly half (43%) of all Diptera

detected in this group. This could negatively influence the

result of Simpson’s Index for the juvenile group. We con-

clude that the Shannon Index is more reliable for this study

suggesting that juveniles have the highest trophic diver-

sity.

Interspecific comparisons

Comparing the feeding ecology of H. mabouia with the

diet of Gymnodactylus darwinii studied by Almeida-

Gomes (2013), a competition for food seems quite unlike-

ly. Whereas H. mabouia is able to prey on a broad spec-

trum of arthropods, in this study 13 different prey cate-

gories were found (stones excluded), there are only five

different categories found (plant material excluded) in the

study by Almeida-Gomes (2013) for G. darwinii. If both

diets are compared with Pianka’s Index, it results in a

niche-overlap of O;, = 0.19, indicating nearly no overlap

between the two gecko species.

CONCLUSIONS

In conclusion, a moderate ontogenetic shift in prey con-

sumption was detected. There are no differences between

©OZFMK

92 Yvonne Dritike & Dennis R6dder

the two sexes. Though diet composition slightly differs

compared to other studies, H. mabouia is a generalist and

opportunistic predator with a high trophic plasticity. Re-

gional differences in diet composition are likely related

to differences in prey availability in different microhabi-

tats or different seasons. Further studies should focus on

arthropod availability at the different locations and sea-

sons in order to examine the reasons for the differences

in prey consumption.

Acknowledgements. Ursula Bott supported us with her proof

reading skills and Trevor Burt improved the writing style. We

are grateful to an anonymous reviewer for constructive comments

which helped us to improve an earlier version.

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