Zoosyst. Evol. 96 (2) 2020, 397-410 | DO! 10.3897/zse.96.53660

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BERLIN

Molecular phylogenetic analysis of Punctoidea
(Gastropoda, Stylommatophora)

Rodrigo B. Salvador’, Fred J. Brook*, Lara D. Shepherd', Martyn Kennedy?

1 Museum of New Zealand Te Papa Tongarewa, 169 Tory Street, 6011, Wellington, New Zealand
2 Research Associate, Bernice Pauahi Bishop Museum, 1525 Bernice Street, 96817, Honolulu, Hawai‘i, USA
3 Department of Zoology, University of Otago, 340 Great King Street, 9016, Dunedin, New Zealand

http://zoobank.org/FC46F 2BD-E176-4B47-9835-13ACSA74B999

Corresponding author: Rodrigo B. Salvador (salvador.rodrigo.b@gmail.com)

Academic editor: Frank Kohler # Received 27 April 2020 # Accepted 27 May 2020 Published 23 June 2020

Abstract

A phylogenetic analysis using a combination of mitochondrial (COI, 16S) and nuclear markers (ITS2, 28S) indicated that Punc-
toidea, as previously interpreted, is polyphyletic. It comprises two main groups, containing northern hemisphere (Laurasian) and
predominantly southern hemisphere (Gondwanan) taxa respectively, treated here as separate superfamilies. Within Punctoidea sensu
stricto, Punctidae, Cystopeltidae and Endodontidae form separate monophyletic clades, but Charopidae, as currently interpreted, is
paraphyletic. Most of the charopid taxa that we sequenced, including Charopa coma (Gray, 1843) and other Charopinae, grouped ina
clade with Punctidae but some charopid taxa from Australia and South America grouped with Cystopeltidae. Cystopeltidae previous-
ly contained a single Australia-endemic genus, Cystopelta Tate, 1881, but our analysis suggests that it is considerably more diverse
taxonomically and has a much wider distribution. For taxonomic stability, we suggest that Charopidae be retained as a family-level
group for now, pending further study of the systematic relationships of its constituent taxa. A new superfamily, Discoidea, is erected
here for two Northern Hemisphere families, Discidae and Oreohelicidae, which were previously assigned to Punctoidea. The North
American species Radiodomus abietum, previously in Charopidae, is also here assigned to Discoidea. The phylogenetic relationships
of Helicodiscidae, previously assigned to Punctoidea, were not fully resolved in our analysis, but the family is apparently closely
related to Arionoidea Gray, 1840 and infraorder Limacoidel.

Key Words

Bayesian Inference, Discoidea, Helicodiscidae, land snails, maximum likelihood

Introduction

The Punctoidea Morse, 1864 is a group of stylommato-
phoran land snails that are typically of small to minute
size. As interpreted by Bouchet et al. (2017) it contains
eight families: Charopidae Hutton, 1884 (Australia, New
Zealand, New Caledonia, Malesia, Oceania, Central and
South America, St Helena, Southern Africa), Cystopelti-
dae Cockerell, 1891 (Australia), Discidae Thiele, 1931
(Holarctic), Endodontidae Pilsbry, 1895 (Oceania), He-
licodiscidae Pilsbry, 1927 (North and Central America,
Malesia, Australia), Oopeltidae Cockerell, 1891 (South-
ern Africa), Oreohelicidae Pilsbry, 1939 (North Amer-

ica), and Punctidae Morse, 1864 (nearly cosmopolitan,
except for Central and South America).

The classification of the group has been historically
unstable. Firstly, its family-level composition has dif-
fered markedly from author to author (e.g., Solem 1983;
Nordsieck 1986, 2014; Tillier 1989; Schileyko 2001,
2002, 2006, 2007; Bouchet and Rocroi 2005; Bouchet et
al. 2017). Secondly, many of the family-level taxa that
have been proposed have subsequently been treated as
synonyms. For instance, Bouchet et al. (2017) listed three
synonyms of Punctidae and ten of Charopidae. Those au-
thors erred in reassigning Oopeltidae to Punctoidea, with
anatomical and molecular phylogenetic studies (Sirgel

Copyright Rodrigo B. Salvador et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

398

2012; Teasdale 2017) indicating that this family is more
closely related to Arionoidea Gray, 1840.

Representatives of families Charopidae, Punctidae and
Discidae were included in ribosomal RNA phylogenetic
analyses by Wade et al. (2001, 2006). Those authors’ trees
showed, albeit with weak support, that Discidae are not
closely related to Punctidae and Charopidae. The system-
atic relationships of Punctidae and Charopidae were not re-
solved in those analyses, as noted by Bouchet et al. (2017).
In those works, Laoma Gray, 1850 (Punctidae) and Sute-
ria Pilsbry, 1892 (Charopidae) formed a poorly-supported
clade, with Otoconcha Hutton, 1883 (Charopidae: Otocon-
chinae Cockerell, 1893) as a sister group, thus rendering
Charopidae paraphyletic. Bouchet et al. (2017: 386) noted
that “if confirmed, it would indicate that the Charopidae in
the broadly defined sense of Solem (1983) would have to
be divided into separate families”. A phylogenetic study
of Panpulmonata by Teasdale (2017), using transcriptome
and exon capture, included two species of Charopidae,
from Australia and South Africa respectively, and one spe-
cies each of Cystopeltidae and Punctidae. This analysis
recovered a strongly-supported monophyletic Punctoidea,
closely related to Oopeltidae, Caryodidae Connolly, 1915
and Rhytidoidea Pilsbry, 1893. Within Punctoidea the cha-
ropid taxa Mulathena Smith & Kershaw, 1985 and Tra-
chycystis Pilsbry, 1893 grouped together, and Cystopelta
and the punctid taxon Paralaoma Iredale, 1913 formed a
separate, well-supported group.

The present study is a first attempt at determining a
global phylogeny of the Punctoidea, incorporating taxa
from all the constituent families listed by Bouchet et al.
(2017), except Oopeltidae, and using a combination of
mitochondrial and nuclear markers to infer a phylogeny
for this superfamily.

Material and methods

Over 50 museums and universities worldwide were con-
tacted in search of specimens, but only seven of those
were able to provide preserved material that was suitable
for molecular analysis (a few institutions had suitable
specimens but declined to loan them). We tried to obtain
representatives of as many genera, subfamilies and fam-
ilies of putative Punctoidea as possible, with preference
given to type species of genera (and type genera of fami-
ly/subfamily), and specimens from or near type localities.
The difficulty of obtaining specimens suitable for mo-
lecular analysis was not entirely unexpected. From our
experience, tissues of punctoid snails, especially minute
ones, are commonly in poor condition in museum collec-
tions. There are two main reasons for this: (1) snails sort-
ed from soil/leaf litter samples can be dead and partly de-
composed prior to preservation. (2) Live specimens that
are killed by being put directly into ethanol retract into
their shell, sometimes with copious production of mucus,
and this can prevent ethanol penetrating all tissues (some
decomposition then occurs in those tissues).

zse.pensoft.net

Salvador, R.B. et al.: Molecular phylogeny of punctoid snails

Overall, we obtained specimens of 50 species from
seven of the eight punctoid families recognized by Bou-
chet et al. (2017) (Table 1). We did not include any rep-
resentatives of Oopeltidae, which is more closely related
to Arionoidea (see above). Our analysis included puta-
tive punctoid species assigned to families Charopidae (27
species), Cystopeltidae (1 species), Discidae (15 species),
Endodontidae (1 species), Helicodiscidae (2 species),
Oreohelicidae (4 species) and Punctidae (6 species). It
included taxa from South Africa (1 species), Australia (4
species), New Zealand (17 species), Oceania (2 species),
Central and South America (7 species), North America
(23 species) and Europe (4 species). For three species, we
included two specimens each from different geographic
regions (1.e., USA vs Canada, NE vs SE Brazil). Data
for three additional punctoid species were gathered from
NCBI GenBank (Table 1); we used only sequence data
stemming from published works with reliable identifica-
tions, voucher specimens, locality data, and sequence data
for our markers of interest. All the specimens sequenced
herein had their identification determined by comparison
with type material or illustrations of type material where
feasible, or from taxonomic literature and reference ma-
terial in museum collections (details listed in Suppl. ma-
terial 1: Part I).

We used as outgroups two species of Hygrophila, one
of Succineidae, and one of Rhytididae, rooting the phy-
logeny using Hygrophila; Rhytididae was used to test
the monophyly of Punctoidea in the first instance (see
below). Sequence data of these species were taken from
GenBank (Table 1), with the exception of the succineid,
which was sequenced by us.

The specimens that we analyzed had either a small sec-
tion of the foot clipped or (in the case of extremely min-
ute specimens) were completely used for DNA extraction
(standard protocol, QIAGEN DNEasy Blood & Tissue
Kit; or 5% Chelex 100 solution, see Spencer et al., 2006).
Roughly one third of our extractions failed due to poor
specimen preservation, as explained above. We targeted
four markers: (1) the barcoding fragment of the mitochon-
drial COI gene (primers LCO and HCO; Folmer et al.,
1994), with circa 650 bp; (2) the mitochondrial 16S rRNA
gene (primers 16SarL and 16SbrH; Simon et al., 1994),
with circa 450 bp; (3) and (4) a continuous fragment of
nuclear DNA encompassing the 3’ end of the 5.8S rRNA
gene, the ITS2 region, and the 5’ end of the 28S rRNA
gene, with a total of around 1,300 bp, that was amplified in
two fragments. The primers used were LSU-1 and LSU-3
for the first fragment and LSU-2 and LSU-5 for the second
fragment (Wade and Mordan 2000; Wade et al. 2006).

PCR amplification for COI and 16S involved an ini-
tial denaturation at 96 °C (2 min); followed by 35 cycles
of denaturation at 94 °C (30 s), annealing at 48 °C (1
min) and extension at 72 °C (2 min); finishing with a
final extension at 72 °C (5 min). The PCR protocol for
ITS2+28S was performed with an initial denaturation at
95 °C (3 min); then 40 cycles of denaturation at 95 °C
(30 s), annealing at either 50 °C (ITS2 section) or 45 °C

Zoosyst. Evol. 96 (2) 2020, 397-410

(28S section) (1 min) and (4) extension at 72 °C (2 min);
followed by a final extension at 72 °C (4 min). Small var-
iations of these protocols (e.g., annealing temperature,
length of cycle steps) were used for some samples that
initially failed to amplify.

PCR products were quantified via agarose gel electro-
phoresis, cleaned with ExoSAP-IT™ (Affymetrix Inc.),
and Sanger sequenced. Sequences were assembled in
Geneious Prime (v. 2019.0.3, Biomatters Ltd.), quali-
ty-checked, and uploaded to GenBank (Table 1). Align-
ment of sequences was also done in Geneious Prime with
the MUSCLE plugin (Edgar 2004) using default settings
(i.e., optimized for accuracy). The resulting alignment of
each marker was manually proofed for errors and then
run through Gblocks (Talavera and Castresana 2007),
with the least restrictive settings available, in order to
eliminate poorly aligned and divergent positions that
might interfere with the analyses.

The sequences of each marker (COI, 16S, and ITS+28S)
were then concatenated for a single phylogenetic analysis.
Before concatenation, however, each marker was analyz-
ed separately to search for conflicts between the resulting
trees; no meaningful conflict was found. Phylogenetic
analyses were performed with MrBayes 3.2.6 (Ronquist
et al., 2012) for Bayesian Inference (henceforth BI) and
the PhyML 3.0 online portal (Guindon et al. 2010) with
maximum likelihood (henceforth ML).

For BI two concurrent analyses were run, each with
four Markov chains of 20 million generations with the first
20% of samples discarded as ‘burn-in’, the default priors,
nst = 6, rates = invgamma, temperature parameter = 0.1,
sampling every 1,000 generations and the substitution
model parameters unlinked across the three loci. MCMC
convergence was assessed by examining the standard
deviation of split frequencies and effective sample sizes
(ESS) values in Mr Bayes and examining likelihood plots
in Tracer v.1.7.1 (Rambaut et al. 2018). For ML, we used
smart model selection (Lefort et al. 2017) with Akaike
Information Criterion (AIC), subtree pruning-regrafting
branch swapping and 2,000 bootstrap replicates.

A subset of our Punctoidea ingroup (17 species) was
used alongside 23 other stylommatophoran snails (and 2
Hygrophila as outgroup) to further investigate the poly-
phyletism of Punctoidea and the position of its compo-
nent branches within the whole group. The methodology
is similar to the above and is discussed in detail in the
Suppl. material 1: Part II, including a list of all species
and their GenBank accession numbers (Suppl. material
1: Table S1).

Results

Taxonomic coverage

Our analysis was based on sequence data from taxa in
seven of the eight families that Bouchet et al. (2017) as-
signed to Punctoidea, but coverage was not equal for all

399

families (Table 1). Charopidae, Discidae, Oreohelicidae
and Punctidae were each represented by multiple samples.
Just under half the sampled species belong to Charopidae,
with three of the presently recognized subfamilies being
represented, namely Charopinae, Otoconchinae and Ro-
tadiscinae Baker, 1927. Cystopeltidae and Endodontidae
were represented by just one species each. Helicodiscidae
was represented by GenBank data only (DNA extraction
from additional helicodiscid specimens that we procured
was unsuccessful). In any event we achieved relatively
broad coverage for our ingroup, which included 53 spe-
cies and 56 terminal branches (as there are three species
each represented by two individuals).

Sequence data

After selection through Gblocks, our resulting concate-
nated alignment was 2196 bp long, with 1176 variable
characters of which 935 were parsimony informative.
Gblocks maintained 683 bp in the COI fragment, 387 bp
in the 16S, and 1126 bp in the IT2+28S. We were unable
to obtain high-quality 16S sequence data for four spe-
cies (Table 1).

Phylogenetic analyses

The BI and the ML analyses returned nearly identical
trees, so we present here the Bayesian phylogeny only
(Fig. 1, but also including the ML support values). The
ML tree had some minor differences regarding the place-
ment of the charopid taxa Al/odiscus Pilsbry, 1892, Chal-
cocystis Watson, 1934, Otoconcha, and Chilean Radio-
discus sp., but all with very little support. For clarity, we
refer below only to BI posterior probability (PP) values,
while the ML support values can be seen in Fig. 1.

The resulting tree shows that Punctoidea is not
monophyletic (Fig. 1), a possibility that had already
been alluded to by some previous authors (e.g., Wade et
al. 2001, 2006; Holyoak et al. 2011; Nordsieck 2014).
Rather, it is widely polyphyletic (see also the more com-
prehensive polyphyletism test in the Suppl. material 1:
Part II), consisting of three distinct and well-supported
groups within suborder Helicina: (1) a group containing
Discidae and Oreohelicidae (1.0 PP), which we refer to a
new superfamily Discoidea, based on the earliest avail-
able family-group name; (2) the Helicodiscidae, which
forms a separate strongly supported group (1.0 PP) of
uncertain affinity within suborder Helicina, in Stylom-
matophora; and (3) the Punctoidea sensu stricto, con-
taining Endodontidae, Cystopeltidae, Punctidae and par-
aphyletic Charopidae (1.0 PP). Because the fossil record
of Punctoidea sensu stricto is poorly understood (see
Discussion below), and some of the internal branches
of our phylogeny were not strongly supported, we have
not attempted to estimate divergence times based on the
molecular data.

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Molecular phylogeny of punctoid snails

Salvador, R.B. et al.:

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Zoosyst. Evol. 96 (2) 2020, 397-410

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zse.pensoft.net

402

Discussion
Systematics: Discoidea

This superfamily is strongly supported (Fig. 1) and is
overall very well resolved, with all internal branches
equally well supported. It contains two distinct groups,
the families Oreohelicidae and Discidae. Our analysis of
wider relationships within Stylommatophora (see Suppl.
material 1: Part IT) placed Discoidea close to infraorder
Helicoidei, with strong support in the BI tree but weak
support in the ML tree.

Oreohelicidae: This family, which 1s endemic to
North America, is a strongly supported (1.0 PP) mono-
phyletic group that is separate from Discidae and basal
within Discoidea.

Discidae: This is a well-supported (1.0 PP) mono-
phyletic group, which includes Anguispira Morse, 1864
and Discus Fitzinger, 1833. Our analysis indicates that
the former genus is monophyletic, but the latter, as cur-
rently interpreted, is paraphyletic. This is not unexpected
as Discus has been used a wastebasket taxon for North
American and European discoid species, both Recent and
fossil. However, what was surprising is that whereas two
European species of Discus formed a separate basal clade
(1.0 PP), a third European species, which was identified
as D. ruderatus (Hartmann, 1821), the type species of the
genus, grouped with North American species (1.0 PP).
Further work is required to resolve the genus-level taxon-
omy of the species presently assigned to Discus, as well
as the phylogenetic relationships of putative discid taxa
from the Canary Islands (Holyoak et al. 2011; Cameron
et al. 2013).

Our analysis indicated that samples identified as An-
guispira alternata (Say, 1817) from the USA and Cana-
da were very similar genetically and probably conspecific
with one another. In contrast, the samples identified as A.
kochi (Pfeiffer, 1846) from the USA and Canada differed
markedly from one another, indicating that this taxon,
which has a complex synonymy (MolluscaBase 2020), and
is currently recognized as having a strongly disjunct distri-
bution in North America, is probably a species complex.

Discoidea incertae sedis: The monotypic North Amer-
ican genus Radiodomus H.B. Baker, 1930 has previously
been classified in subfamily Rotadiscinae of Charopidae,
although Pilsbry (1948b) noted that the type species, Ra-
diodomus abietum Baker, 1930, differed anatomically
from other rotadiscines. Our phylogenetic analysis indi-
cates that Radiodomus belongs instead in Discoidea, but
further work is required to determine if it should be treat-
ed as the basal taxon in Discidae, or assigned to a sepa-
rate, new family-level group within Discoidea.

Systematics: Helicodiscidae

This family is native to Central and North America (Zilch
1959). A species of helicodiscid that has been described

zse.pensoft.net

Salvador, R.B. et al.: Molecular phylogeny of punctoid snails

from southeastern Brazil (Simone 2006) 1s actually an ad-
ventive North American species (Silva et al. 2020). Stenopy-
lis coarctata (MoOllendorff, 1894), which is apparently native
to Malesia and northern Australia, has also been assigned to
Helicodiscidae (e.g., Solem 1984; Stanisic et al. 2010), but
this family-level classification requires reevaluation.

In our phylogeny Helicodiscidae is represented by two
North American species of Helicodiscus Morse, 1864
that form a strongly supported (1.0 PP) clade. Although
previously included in Punctoidea, our analysis suggests
that Helicodiscidae does not belong in either Discoidea or
the redefined Punctoidea. Its phylogenetic relationships
with other taxa have not been precisely determined (see
Suppl. material 1), but both our ML and BI trees position
it (albeit with low support) close to Arionoidea and the
‘limacoid clade’ (now infraorder Limacoidei; Bouchet et
al., 2017). As such the family is treated here as incertae
sedis within suborder Helicina (in Stylommatophora),
pending further work. Oopeltidae has also been previous-
ly classified in Punctoidea (Bouchet et al. 2017), although
shown to be more closely related to Arionoidea (Sirgel
2012); whether or not Oopeltidae is closely related to
Helicodiscidae requires investigation.

Systematics: Punctoidea

The Punctoidea, as redefined here, is a strongly sup-
ported clade (1.0 PP) clade containing representatives
of Endodontidae, Cystopeltidae, Punctidae and Charop-
idae (Fig. 1). We could not reliably determine its posi-
tion within Stylommatophora: our ML tree placed it as
the basal group within suborder Helicina, while our BI
placed it in a more derived position within Helicina (see
Suppl. material 1: Part II).

Endodontidae: In our analysis, this family is repre-
sented by one species only, in the Polynesian genus Lib-
era Garrett, 1881, but its split from the other punctoids is
clear and strongly supported (1.0 PP). As such, Endodon-
tidae is basal in the redefined Punctoidea, and is the sister
taxon of the clade formed by the other punctoid families,
as redefined below.

Cystopeltidae: Previously this family was interpret-
ed as containing a single genus of semi-slugs, Cystopelta
Tate, 1881, endemic to southeastern Australia, but our
analysis indicated strong support (1.0 PP) for a mono-
phyletic family-level group comprising two strongly sup-
ported clades (both 1.0 PP): one containing Cystopelta
bicolor Petterd & Hedley, 1909, and two Tasmanian land
snail taxa that were previously assigned to Charopidae,
Diemenoropa kingstonensis (Legrand, 1871) and Sce-
lidoropa officeri (Legrand, 1871); and the other con-
taining South American land snail species in the genera
Lilloiconcha Weyrauch, 1965 and Zilchogyra Weyrauch,
1965, which were previously assigned to Charopidae as
well. These two clades possibly warrant separate sub-
family-group status, but further work is required to test
this. Our results indicate that the genus- and species-level

Zoosyst. Evol. 96 (2) 2020, 397-410

Acroloxus lacustris "—

1.0/100

1.0/86
1.0/100
1.0/90 7.0/93
0.97/51

1.0/92
1.0/89)

1.0/80
1.0/71

0.94/86

1.0/100

1.0/100}

1.0/83
0.94/50

0.84/25

1.0/74

1.0/80 1.0/99

1.0/98

1.0/83}

1.0/83 1.0/88

0.60) 0.93/44!

1.0/97'

403

Planorbis planorbis

Succinea manaosensis
Oreohelix idahoensis
Oreohelix vortex
Oreohelix strigosa depressa
Oreohelix subrudis
Radiodomus abietum
Discus perspectivus
Discus rotundatus
Discus shimeki

0.99/98

1.0/100

Oreohelicidae

Discus ruderatus

T0788) Discus whitneyi Discoidea
1.0/100& Discus catskillensis
Discus patulus Discidae
1.0/100 Discus nigrimontanus
Anguispira nimapuna
Anguispira kochi [USA]
Anguispira kochi [Canada]
0.99/84, Anguispira strongyloides
Anguispira jessica
Anguispira alternata [Canada]
1.0/100L Anguispira alternata [USA]
Rhytida greenwoodi
Helicodiscus parallelus ] Helicodiscidae
Helicodiscus barri
Libera fratercula 7] Endodontidae
Diemenoropa kingstonensis
Scelidoropa officeri
0.99/43 Cystopelta bicolor t
Lilloiconcha cf. gordurasensis [Alagoas] Cystopeltidae
Lilloiconcha superba
0.95/61 Lilloiconcha gordurasensis [Sao Paulo]
1.0/1008 Zilchogyra sp.
Chalcocystis aenea
Suteria ide
Radiodiscus sp. [Brazil]
Radioconus amoenus
Stenacapha hamiltoni
Mocella eta
Otoconcha dimidiata
Charopa coma
Sinployea atiensis Punctoidea
Fectola infecta
Alsolemia longstaffae Charopinae | “Charopidae”

1.0/86

0.2

Mitodon wairarapa
Ranfurlya constanceae
Flammulina zebra
Radiodiscus sp. [Chile]
Allodiscus dimorphus
Phenacohelix pilula
Phacussa helmsi
Therasia thaisa
Neophenacohelix giveni
Phrixgnathus celia
Laoma leimonias
Paralaoma servilis
Punctum californicum
Punctum randolphi
Punctum pygmaeum

1.0/100

ie Punctidae

1.0/100

Figure 1. Bayesian tree for the “Punctoidea”, rooted by the Hygrophila. Numbers shown on nodes are BI posterior

probabilities (0 to 1) followed by ML bootstrap values (0 to

classification of Lilloiconcha and Zilchogyra is in need of
revision, as already alluded to by previous authors (e.g.,
Salvador et al. 2018b; Salvador 2019).

The charopid taxa that grouped in Cystopeltidae in our
analysis have very similar shell morphology to some cha-
ropid taxa in the Punctidae + Charopidae clade (below).
For the South American cystopeltid branch at least, a
smooth protoconch might be a diagnostic character (Schi-
leyko 2001). However, for many charopid genus groups it
may not be possible to assign taxa to either family on the
basis of shell characters alone. Further work is required
to determine the family-level placement of the numerous
extant taxa that are currently assigned to Charopidae but
which were not included in our analysis, as well as to de-
termine reliable family-level diagnostic characters.

The phylogenetic relationships of Cystopeltidae in
our analysis appear to differ from the findings of Teas-
dale (2017), which indicated that Cystopelta purpurea
Davies, 1912 (Cystopeltidae), and a putative represent-
ative of Punctidae that was identified as Paralaoma sp.
(misspelled as Paraloama in the original), were sister
Species, separate from a group of two charopid species
from Australia and South Africa, respectively. The rea-
sons for this difference are unclear. It may be an artefact
of the small number of punctoid samples and restricted
geographic range in Teasdale’s (2017) analysis compared
with our study.

Punctidae + Charopidae clade: Our analysis indi-
cates strong support (1.0 PP) for a clade incorporating

100%). Scale bar is substitutions per site.

taxa that were previously assigned to Punctidae and Cha-
ropidae (excluding those that grouped with Cystopeltidae,
see above). The phylogenetic relationships determined
here suggest that whereas Punctidae, as previously inter-
preted, is monophyletic, Charopidae sensu Solem (1983:
47) and later authors is paraphyletic. At present there is
insufficient information to determine whether Charopidae
Hutton, 1884 would be best treated as a junior synonym
of Punctidae Morse, 1864, or split into a series of sepa-
rate monophyletic family units. In the meantime, for tax-
onomic stability, we suggest that Charopidae should be
retained as a separate, paraphyletic family-level group,
pending further work to determine the phylogenetic rela-
tionships of its constituent taxa (below).

The family-group name Punctidae is used here for a
well-supported clade (1.0 PP), within which there is a
strongly supported (1.0 PP) basal group containing the
endemic New Zealand taxa Laoma Gray, 1850 and Phrix-
gnathus Hutton, 1882, corresponding to Laominae Suter,
1913, and a weakly supported group (0.56 PP) containing
Paralaoma, which is native to Australasia but has a wide
adventive distribution, and type genus Punctum Morse,
1864. As presently interpreted the latter genus has a pre-
dominantly Holarctic distribution in North America, Ja-
pan and extratropical Eurasia, but with records also from
Central America, Hawai’i and tropical Africa (Pilsbry
1948b; Cowie et al. 1995; Wronski and Hausdorf 2010;
de Winter 2017; Horsak and Meng 2018). Punctidae
probably also includes other New Zealand punctid taxa

zse.pensoft.net

404

listed by Spencer et al. (2009) and Australian punctid taxa
listed by previous authors (e.g., Smith 1992: Schileyko
2002; Stanisic et al. 2010, 2018).

The family-group name Charopidae is provisionally
retained here for charopid taxa other than those reas-
signed to Cystopeltidae (above). It includes taxa pre-
viously assigned to Charopinae Hutton, 1884 (in part),
Phenacohelicidae Suter, 1892, Otoconchinae Cockerell,
1893, Flammulinidae Crosse, 1895, Patulastridae Steen-
burg, 1925, Rotadiscinae, Trachycystidae Schileyko,
1986, Ranfurlyinae Schileyko, 2001, and Therasiinae
Schileyko, 2001. This diverse group of taxa has a very
wide distribution that includes South America, South Af-
rica, Australia, New Zealand and Oceania. The relation-
ships within this group are as yet poorly resolved (see
below), but our analysis indicates that it contains at least
one strongly-supported group (1.0 PP), corresponding to
Charopinae sensu stricto, which includes the type genus
Charopa Albers & Martens, 1860, some other New Zea-
land taxa, and Sinployea Solem, 1983 from Oceania. Two
of the constituent taxa, Flammulina E. von Martens, 1873
and Ranfurlya Suter, 1903, are the type genera of Flam-
mulinidae and Ranfurlyinae, respectively, confirming that
the latter two taxa are synonyms of Charopinae. Con-
versely, our analysis indicates that Charopinae does not
include some genus-groups such Mocella Iredale, 1915,
Stenacapha Smith & Kershaw, 1985 and Suteria Pilsbry,
1892, that were assigned to it by previous workers (e.g.,
Schileyko 2001).

Many of the charopid taxa in our analysis could not
be reliably assigned to subfamily groups. The basal-most
charopid taxon in our phylogeny is the African genus
Chalcocystis Watson, 1934. It has been referred to the
subfamily Trachycystinae (e.g., Schileyko 2001), but
other authors have treated this subfamily as a synonym
of Charopinae (e.g., Bouchet et al. 2017). This branch 1s
strongly separated from the remaining punctoids, which
suggests that Trachycystinae may have some biological
reality if restricted to African taxa. Analysis of a larger
sample of African taxa, including the type genus of the
subfamily, 1s required to reliably determine the systemat-
ic relationships of this group.

The genus of semi-slugs Otoconcha forms a separate
lineage in our analysis, albeit with poor support (0.55
PP). Otoconcha and Maoriconcha Dell, 1952 have been
assigned to the endemic New Zealand subfamily Oto-
conchinae (e.g., Schileyko 2001), but further work 1s
required to determine the phylogenetic relationships of
these genera and the taxonomic status of Otoconchinae.

The New Zealand charopid taxon Suteria Pilsbry,
1892 also forms a separate lineage with poor support (0.6
PP) in our analysis. It was previously included in Cha-
ropinae (e.g., Schileyko 2001). Four other New Zealand
“charopid” taxa, Neophenacohelix Cumber, 1961, Phena-
cohelix Suter, 1892, Phacussa Hutton, 1883 and Thera-
sia Hutton, 1883, formed a poorly supported group (0.65
PP). The two first-named and two last-named taxa were
previously assigned to Phenacohelicinae and Therasiinae,

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Salvador, R.B. et al.: Molecular phylogeny of punctoid snails

respectively. In our Bayesian tree, the New Zealand taxon
Allodiscus Pilsbry, 1892, previously assigned to Phenaco-
helicinae (e.g., Schileyko 2001), grouped with these four
taxa albeit with poor support (0.69 PP); in the ML tree,
however, it was the sister taxon to Punctidae, again with
poor support (50).

Stenacapha Smith & Kershaw, 1985 from Australia
and Mocella Iredale, 1915 from New Zealand, both for-
merly included in Charopinae, formed a separate group in
our analysis, albeit with moderate support only (0.93 PP).

Three of the South American taxa that were includ-
ed in our analysis belong in two separate groups within
the Punctidae + Charopidae clade. Radioconus amoenus
(Thiele, 1927) and the Brazilian Radiodiscus sp. form a
strongly supported group (1.0 PP), but the Chilean Radi-
odiscus sp. belongs to a separate lineage. Radiodiscus, as
previously interpreted, is evidently polyphyletic; this is
not unexpected, as the genus has historically functioned
as a wastebasket taxon for South American charopids.
Whether one or both these groups should have subfam-
ily status, and whether or not either of them corresponds
to Rotadiscinae, has not been determined. In any event,
it 1s clear that New Zealand taxa that were assigned to
Rotadiscinae by Climo (1989) and subsequent workers,
including the genera A/solemia Climo, 1981 and Mitodon
Climo, 1989, belong instead in Charopinae (Fig. 1).

Several family-level taxa that have previously been
treated as synonyms of Charopidae, or subfamily-groups
within Charopidae, were not included in the analysis.
These include (in chronological order): Amphidoxinae
Thiele, 1931 (Chile); Dipnelicidae Iredale, 1937 (Aus-
tralia); Hedleyoconchidae Iredale, 1942 (Australia);
Pseudocharopidae Iredale, 1944 (Lord Howe Island);
Semperdoninae Solem, 1976 (Micronesia); Trukcharopi-
nae Solem, 1983 (Micronesia); and Flammoconchinae
Schileyko, 2001 (New Zealand). Thysanotinae God-
win-Austen, 1907 (southern Asia and Pacific islands) has
been included in Charopidae by some authors (e.g., Bou-
chet et al., 2017), but ongoing studies suggest that it does
not belong in Punctoidea (Fred Naggs, pers. comm.).

The poor resolution in our analysis of some phyloge-
netic relationships within Charopidae may have been be-
cause of insufficient sequence information or inadequate
sampling of taxa. The latter is more likely, given that the
sequence data were sufficient to resolve phyletic relation-
ships with strong support within the other families that
were examined. Although the analysis included samples
of 24 genus-level charopid taxa (Table 1), this represents
only a very small proportion of the overall diversity of
this paraphyletic group. For instance, the Australian fau-
na includes 104 named genus groups of charopids (Stani-
sic et al. 2010, 2018), of which we sampled three taxa (c
3%) only. In the New Zealand fauna, there are 45 named
charopid genera (Spencer et al. 2009), 14 of which (31%)
were included in our analysis. The fauna of Oceania in-
cludes 20 named charopid genera (Solem 1983), of which
we sampled one taxon (5%) only. For large, diverse and
reasonably old groups, it 1s deemed that adding taxa usu-

Zoosyst. Evol. 96 (2) 2020, 397-410

ally outweighs adding sequence data (Pollock et al. 2002;
Zwickl and Hillis 2002; Heath et al. 2008; Nabhan and
Sarkar 2011). Obtaining a better resolution of the sub-
family-level groups within the clade of Punctidae + Cha-
ropidae will require a broader coverage of species, both
taxonomically and geographically.

Paleobiogeography: Discoidea

This superfamily has a Laurasian distribution. Based on
our present phylogeny of extant species, Oreohelicidae
and Radiodomus are North American, and the most basal
Discidae are European, while a group of more derived
discids includes both European and North American taxa.
The phylogenetic relationships of purported Discidae
from the Canary Islands are as yet undetermined.

Records of land snails from the Carboniferous of North
America that were attributed to Discidae and other sty-
lommatophoran groups by Solem and Yochelson (1979)
are now considered to be non-stylommatophoran eupul-
monates (e.g., Bandel 1991, 1997; Mordan and Wade
2008). The oldest known fossil taxa assigned to Discoidea
are from the Late Cretaceous of Alberta, Canada. They in-
clude Discus sandersonae (Russell, 1929), in family Dis-
cidae (Pilsbry, 1939), and Oreohelix obtusata (Whiteaves,
1885), Radiocentrum anguliferum (Whiteaves, 1885) and
R. thurstoni (Russell, 1926), all in family Oreohelicidae,
(Henderson 1935; Tozer 1956; Roth 1986). Other fossil
species of Radiocentrum Pilsbry, 1905 are known from
the Paleocene of Alberta, Eocene of Wyoming, and Oli-
gocene of Colorado, whereas the Quaternary distribution
of this genus group is restricted to southwestern USA and
northwestern Mexico (Roth 1986; Hochberg et al. 1987).
Fossil species of Oreohelix Pilsbry, 1904 are known from
Late Cretaceous, Paleocene and Eocene faunas from Al-
berta to Utah (Roth 1986). Oreohelix is the most diverse
genus group in the extant North American land snail fau-
na, with 79 species recorded from western Canada, USA
and Mexico (Pilsbry 1948a, 1948b; Nekola 2014).

In North America relatively few fossil species of
Discus sensu lato are known from the Cenozoic, with
records from the Late Paleocene/Early Eocene of Utah,
Eocene of Wyoming and Montana, and Miocene of Or-
egon (Pilsbry 1939; La Roque 1960; Pierce and Conste-
nius 2014). In Europe the oldest known fossil taxon in
Discidae is Discus perelegans (Deshayes, 1863) from the
Late Paleocene/Early Eocene of the Paris Basin, France
(Wenz 1923). Discus sensu lato evidently underwent an
extensive radiation in the mid Paleogene of Europe, with
several species represented in fossil faunas of Eocene age
from southern England and the Paris Basin (Preece 1982;
Pacaud and Le Renard 1995). The Neogene land snail
fauna of Europe also contains numerous fossil species
that have been assigned to this paraphyletic genus group
(e.g., Harzhauser et al. 2014; Holtke et al. 2016, 2018).

Anguispira has a fossil and extant distribution re-
stricted to North America. The oldest known fossil is

405

Anguispira cf. alternata (Say, 1816) from the Eocene of
Montana, USA (Pierce and Constenius 2014), indicating
that the split between this genus and Discus sensu lato
took place in the Eocene or earlier. The Discidae presum-
ably diverged from the Oreohelicidae and Radiodomus
lineages in the Late Cretaceous or earlier.

Paleobiogeography: Helicodiscidae

Fossils of helicodiscid taxa are known from the Ear-
ly Miocene of Europe (genus Lucilla Lowe, 1852;
Nordsieck 2014; Salvador 2014) and the Late Miocene
of North America (Liggert 1997; Gladstone et al. 2019),
indicating a former wider Laurasian distribution.

Paleobiogeography: Punctoidea

This superfamily is distributed almost worldwide, but
given that the greatest diversity of extant taxa is in the
Southern Hemisphere, with one genus only in the North-
ern Hemisphere, it is likely of Gondwanan origin. Inter-
pretation of the biogeographic history of the Punctoidea
is hindered by a relatively sparse fossil record, and the
difficulty in reliably assigning fossil material, which in
many cases is poorly preserved, to family-level groups
on the basis of shell morphology alone. Our finding that
some extant taxa that were previously assigned to Cha-
ropidae actually belong in Cystopeltidae has further com-
plicated matters, because, as noted above, shell characters
of charopid genus groups do not appear to be a reliable
indicator of family-level phylogenetic relationships. De-
spite these limitations, some useful biogeographic infor-
mation can be gleaned from the fossil record.

The oldest known fossil taxon that could possibly be
assigned to Punctoidea is Radiodiscus santacrucensis
Morton, 1999, from the Lower Cretaceous of Argentina
(Morton 1999; Rodriguez et al. 2012), although the ge-
nus-level placement of this species is probably incorrect
and requires re-evaluation (Salvador et al. 201 8a), and the
family-level placement is unclear. All other known fossils
of Punctoidea are from the Cenozoic.

The oldest fossil species that can be reliably assigned to
Endodontidae is Cookeconcha subpacificus (Ladd, 1958)
from the Lower Miocene of Bikini Atoll, Marshall Islands
(Ladd 1958). It is most closely related to Pleistocene and
Recent congeners from Midway Atoll and Hawai'i, re-
spectively (Solem 1976, 1977, 1983). The monotypic fos-
sil taxon Hebeispira hebeiensis Youluo, 1978, of “Early
Tertiary” age from the Bohai coastal plain in North China,
was assigned to Endodontidae. However, examination of
images of type material (Youluo 1978: pl. 30, figs 12-14)
indicates that it belongs in neither Endodontidae nor Punc-
toidea and is likely a freshwater Planorbidae. Likewise,
records of undetermined Endodontidae from the Early/
Middle Miocene of Germany by Moser et al. (2009) have
been refuted (Nordsieck 2014; Salvador and Rasser 2014).

zse.pensoft.net

406

The Endodontidae are otherwise known from Oceania
only, on volcanic and uplifted islands between Tuvalu, Pit-
cairn Islands and Hawai'i, with an outlying genus-group
in Palau, Micronesia (e.g., Solem 1976, 1983; Abdou and
Bouchet 2000; Brook 2010; Sartori et al. 2014). No en-
dodontids are known from the Holocene faunas of Mar-
shall Islands and Midway Atoll. Taxa that were present
there during mid to late Cenozoic time when these islands
were high-standing presumably became extinct when the
islands subsided and became atolls (Solem 1976). Sim-
ilar histories of endodontid species colonizing oceanic
islands by over-water dispersion, undergoing radiations
at species and sometimes also genus level, and becoming
extinct when host islands subsided to, and below, sea lev-
el, probably played out across much of Oceania during
the Neogene and Quaternary, and probably also earlier in
the Paleogene (see below).

Thirteen species of Cenozoic fossil land snails from
South America have been included in Punctoidea with
varying degrees of confidence (Miquel and Bellosi 2007;
Rodriquez et al. 2012; Miquel and Rodriguez 2015; Sal-
vador et al. 2018a). This includes species in extant genus
groups that we have assigned to Cystopeltidae (1.¢e., Lil-
loiconcha, Zilchogyra) and the Punctidae + Charopidae
clade (1.e., Punctum, Radiodiscus), along with other ex-
tant and extinct genus groups whose family-level place-
ment has not been determined. The earliest fossil records
of Lilloiconcha, Radiodiscus and Zilchogyra are from the
Eocene of Argentina (Miquel and Bellosi 2007; Rodri-
quez et al. 2012), and the earliest (and only) record of
Punctum from South America is from the Early/Middle
Miocene of Argentina (Miquel and Rodriguez 2015; Sal-
vador et al. 2018a). Even with uncertainties, this indicates
that Cystopeltidae, Punctidae and “Charopidae’ existed in
South America as separate lineages by Eocene time.

In New Zealand, where extant Punctoidea are extreme-
ly diverse at both genus and species level, the pre-Qua-
ternary fossil record is unfortunately very limited. The
oldest known fossils are seven species of Early Miocene
age from Otago (Marshall and Worthy 2017). All but one
of these species have been assigned to extant genera, with
one in Punctidae (i.e., Paralaoma), two in genera that
our analysis indicated belong in Charopinae (1.e., Cha-
ropa, Fectola Iredale, 1915), and one other charopid ge-
nus (Neophenacohelix). The extinct genus Atactolaoma
Marshall & Worthy, 2017 probably belongs in Punctidae,
but the family and subfamily status of the charopid taxa
Cavellia Iredale, 1915 and Dendropa Marshall & Worthy,
2017 has not been determined. As yet, we do not know
if Cystopeltidae are and/or were ever present in the New
Zealand region.

In Oceania, the only known pre-Quaternary fossil
charopid is Vatusila eniwetokensis (Ladd, 1958) from
the Late Miocene of Eniwetok Atoll, Marshall Islands
(Solem 1976, 1983). This genus, which is genetically
closely related to Sinployea (M. Kennedy, unpublished
data) and probably belongs in Charopinae, has a Holo-
cene distribution extending from Tuvalu south to Tonga

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Salvador, R.B. et al.: Molecular phylogeny of punctoid snails

and Niue (Solem, 1983). As with Endodontidae on Mar-
shall Islands and Midway Atoll (see above), the distri-
bution of Vatusila within Oceania evidently changed
markedly during the Neogene, in response to patterns of
over-water dispersion and the emergence and foundering
of oceanic islands.

In Europe and North America, the Punctoidea is repre-
sented by one genus only, as noted above. The oldest pu-
tative fossil Punctum in Europe is P. oligocaenicum Zin-
ndorf, 1901 of Late Oligocene age from Germany (Wenz,
1923). However, Harzhauser et al. (2014) noted that this
species may not belong to Punctum, and the family place-
ment therefore also requires re-evaluation. Fossil species
that undoubtedly belong in Punctum are well represented
in Neogene strata in continental Europe (Harzhauser et al.
2014; Holtke et al. 2016). In North America the oldest pu-
tative fossil Punctum, and the only pre-Quaternary record
of this genus, is P. alveus Pierce, 1992, from the Late Ol-
igocene/ Early Miocene of Montana, USA (Pierce 1992).

Australia, like New Zealand, has a diverse extant punc-
toid fauna, but whereas the New Zealand fauna is domi-
nated at the species level by Punctidae, the Australian fau-
na is dominated by charopid taxa. Our analysis showed
that the Tasmanian charopid fauna includes represent-
atives of Cystopeltidae and the Punctidae + Charopidae
clade, but the family-group affinities of the vast majority
of Australian taxa have not yet been determined, and the
paleobiogeographic history of the Australian punctoid
fauna is not known. Similarly, the family-group affinities
and paleobiogeographic histories of charopid taxa from
Africa, New Caledonia and Saint Helena, are not known.

In summary, some extant punctoid genera are inter-
preted as having stratigraphic ranges extending back to
the lower Neogene or middle Paleogene, and fossil as-
semblages from South America, New Zealand and Oce-
ania also include extinct punctoid genera (e.g., Solem
1977; Miquel and Rodriguez 2015; Marshall and Wor-
thy 2017). The fossil record in South America indicates
that Cystopeltidae, Punctidae in the restricted sense and
‘Charopidae’ existed as separate family-level groups by
Eocene time, and thus must have diverged sometime pri-
or to that. The oldest known fossils of Endodontidae are
Early Miocene in age, but the basal position of this family
in Punctoidea suggests that it diverged from the lineages
giving rise to Cystopeltidae and the Punctidae + Charop-
idae clade in the Paleocene or earlier. The oldest known
fossils assigned to Punctidae and Charopinae are of Late
Oligocene and Early Miocene age, respectively (Wenz
1923; Pierce 1992: Marshall and Worthy 2017), indi-
cating that these groups had diverged by the Late Paleo-
gene. By Early Miocene time the Punctidae had attained
avery wide distribution, with at least two genus groups in
New Zealand (Atactolaoma, Paralaoma), and species of
Punctum in South America, North America and Europe,
but the latter genus (and Punctidae in general) evidently
subsequently became extinct in South America. The basal
group of Punctidae in our phylogenetic analysis contains
the New Zealand genera Laoma and Phrixgnathus. These

Zoosyst. Evol. 96 (2) 2020, 397-410

two genera are not known from any pre-Quaternary fossil
assemblages in New Zealand or elsewhere, but must have
diverged from the group of Paralaoma and Punctum in
the Oligocene or earlier. The shells of Laoma and Phrix-
gnathus typically have a color pattern of radial stripes and
zigzags, whereas shells of Paralaoma and Punctum are
generally smaller and uniformly brown in color. Whether
the Laoma-Phrixgnathus \ineage originated in the New
Zealand region in the Paleogene, or dispersed there from
elsewhere later in the Cenozoic, is not known.

From a morphological and evolutionary perspective it
is interesting to note that, although the vast majority of
punctoid taxa have coiled external shells that animals can
fully retract into, shell reduction leading to limacization
has occurred independently in the endemic Australian
genus Cystopelta (Cystopeltidae), and in separate line-
ages within the Punctidae + Charopidae clade, including
the endemic New Zealand genera Ranfurlya (Charopi-
nae) and Otoconcha (Otoconchinae). The phylogenetic
relationships of Flammoconcha Dell, 1952, another en-
demic New Zealand genus of punctoid semi-slugs, have
not yet been determined. There are, however, no known
cases of limacization within Endodontidae, which might
have been precluded by aspects of their pallial anatomy
(Solem, 1976).

Conclusion

Based on our results, we propose the following revised
taxonomic classification.

Superfamily Discoidea Thiele, 1931 (1866)
Family Discidae Thiele, 1931 (1866)
Family Oreohelicidae Pilsbry, 1939
Superfamily Punctoidea Morse, 1864
Family Endodontidae Pilsbry, 1895

Family Cystopeltidae Cockerell, 1891
Family Punctidae Morse, 1864

Family Charopidae Hutton, 1884

Helicina incertae sedis

Family Helicodiscidae Pilsbry, 1927

The North American genus Radiodomus Baker, 1930
is transferred from Charopidae and treated here as in-
certae sedis within Discoidea. In Punctoidea, family
Cystopeltidae has been expanded to include not only the
type genus Cystopelta, but also some other Australian
and South American genera. Whether or not any cha-
ropid genus groups from Africa, New Zealand, New
Caledonia and Oceania also belong in Cystopeltidae has
not yet been determined. Charopidae is provisionally re-
tained as a family-level name for a paraphyletic group
of taxa, pending further study of phylogenetic relation-
ships within Punctoidea. The relationships of Helicodis-
cidae within Helicina remain uncertain, but it is an in-
dependent branch that is separate from both Punctoidea
and Discoidea.

407

Acknowledgements

We are extremely grateful to the following people for
providing specimens or tissue samples: Lily Berniker
and Mark Siddall (AMNH, USA), Michal Manas (Czech
Republic), Jochen Gerber (FMNH, USA), Gary Barker
(Landcare, New Zealand), Kristine Greke (Latvia), Owen
Griffiths (Madagascar), Rafael Araujo (MNCN, Spain),
Simone Lira and Luiz R.L. Simone (MZSP, Brazil),
Robert G. Forsyth (NBM, Canada), Jennifer Gallichan
(NMW, UK), Henry Choong, Heidi Gartner and Hugh
McIntosh (RBCM, Canada), Simon Grove and Kirrily
Moore (TMAG, Australia). We are especially thankful to
R.G. Forsyth, J. Gerber, and M. Manas for the identifica-
tion of some specimens (see Suppl. material 1: Part I). We
are likewise grateful to Chris M. Wade for the initial help
with primers and PCR protocols; to Carlos Lehnebach
and Barbara M. Tomotani for help with lab work; to Nick
S. Gladstone for sharing the sequence data of H. barri; to
Fred Naggs for sharing unpublished information regard-
ing the affiliation of Thysanotinae; to Timothy Pearce and
Barry Roth for sharing information on fossil Oreohelici-
dae; to John Stanisic, Frank Kohler and an anonymous
reviewer for the helpful comments. RBS acknowledges
the bequest of the Bruce Fraser Hazelwood Fund and the
MNZ for this project. MK received support from the Uni-
versity of Otago and Allan Wilson Centre.

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Supplementary material |

Species identification and
stylommatophoran phylogeny

Authors: Rodrigo B. Salvador, Fred J. Brook, Lara D.
Shepherd, Martyn Kennedy

Data type: species data

Explanation note: The supplement contains: (1) further
information regarding species identification; and (2) a
large-scale molecular phylogeny of Stylommatophora,
made to test the polyphyly of Punctoidea.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://doi.org/10.3897/zse.96.53660.suppl1