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Journal of Natural History
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tnah20
Not just cryptic, but a barcode bush: PTP re-
analysis of global data for the bumblebee
subgenus Bombus s. str. supports additional
species (Apidae, genus Bombus)
Paul H. Williams
To cite this article: Paul H. Williams (2021) Not just cryptic, but a barcode bush: PTP re-
analysis of global data for the bumblebee subgenus Bombus�s.�str. supports additional
species (Apidae, genus Bombus), Journal of Natural History, 55:5-6, 271-282, DOI:
10.1080/00222933.2021.1900444
To link to this article: https://doi.org/10.1080/00222933.2021.1900444
Published online: 28 May 2021.
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Not just cryptic, but a barcode bush: PTP re-analysis of global
data for the bumblebee subgenus Bombus s. str. supports
additional species (Apidae, genus Bombus)
Paul H. Williams
Department of Life Sciences, Natural History Museum, London, UK
ABSTRACT
The subgenus Bombus s. str. (of the genus Bombus Latreille) includes
the bumblebee species of greatest commercial importance for
pollination world-wide as well as some of the bumblebee species
of greatest conservation concern. Species in this group have always
proved especially dicult to recognise because they are weakly
dierentiated, not only in morphology, but also in their evenly
branching ‘bushy’ patterns within the fast-evolving COI gene tree.
A previous analysis of this tree was unusual in nding no signicant
Yule-coalescent dierence between slow inter-species branching
and fast intra-species branching and consequently used an approx-
imation to recognise 17 species. The same global sample of 559
COI-barcodes is re-assessed here for evidence of species’ coales-
cents using Poisson-tree-process (PTP) models applied to the long-
est unique haplotypes. Results from this PTP re-analysis agree for 12
of the species but split ve of the previous ‘species’ with improved
support, so that either 22 or 23 species are recognised depending
on the choice of outgroup. PTP adds support for: B. czserkianus Vogt
stat. nov.; B. ganjsuensis Skorikov stat. rev.; B. mckayi Ashmead stat.
rev.; B. minshanicola Bischo stat. rev.; B. reinigi Tkalcu stat. rev.; and
possibly B. xanthopus Kriechbaumer stat. rev. These results provide
a more reliable and robust basis for faunal and conservation studies.
http://www.zoobank.org/urn:lsid:zoobank.org:pub:BCAED154-C643-4F3D-A766-C6AF343364B3
ARTICLE HISTORY
Received 1 February 2021
Accepted 2 March 2021
KEYWORDS
DNA barcode; bumble bee;
cryptic species; gene
coalescent; pollinator;
taxonomy
Introduction
A robust and reliable list of species is needed urgently for the bumblebees of the
subgenus Bombus s. str. (of the genus Bombus Latreille). These bumblebees include the
species most important for commercial pollination (Williams, Brown, et al. 2012), worth
billions of dollars annually (Dias et al. 1999; Winter et al. 2006; Goulson 2010; Ollerton
2020), and also include some of the species of highest conservation concern globally
(Colla and Packer 2008; Williams and Osborne 2009; Hateld et al. 2015a, 2015b,
Szymanski et al. 2016; Cameron and Sadd 2020). It is precisely because this group includes
species that are threatened by introductions of their commercially reared close relatives
CONTACT Paul H. Williams paw@nhm.ac.uk
JOURNAL OF NATURAL HISTORY
2021, VOL. 55, NOS. 5–6, 271–282
https://doi.org/10.1080/00222933.2021.1900444
© 2021 Informa UK Limited, trading as Taylor & Francis Group
Published online 28 May 2021
(e.g. Matsumura et al. 2004; Inoue et al. 2008) that identication diculties are of
particularly serious concern (Williams, An, et al. 2012).
Our grasp of biodiversity is ne-tuned through the process of revisionary taxonomy. If
we assume that species do actually exist in nature and that they can be discovered with
available techniques, then we expect taxonomic revisions to converge on broadly shared
interpretations of species (Williams et al. 2015). The introduction of molecular techniques
may have promised a faster approach to speed up taxonomy (Hebert et al. 2004; Brower
2010), but it has also brought pitfalls (Zamani et al. 2021). Molecular techniques have
certainly not resulted in instant, one-stop solutions for bumblebees, as exemplied by
recent studies of bumblebees of the subgenus Alpinobombus (Williams et al. 2015, 2019;
Thanoosing 2017; Martinet et al. 2018). However, there is an emerging consensus on the
advantages of an integrative approach that relies on corroboration among dierent
criteria for recognising species, including both morphological and molecular methods
(Padial et al. 2010; Schlick-Steiner et al. 2010). Long-term studies over the last decade on
multiple groups of bumblebees have shown that gradually improving sampling coverage
and improvements in molecular and integrative methods can result in convergence
through time on more consistent, stable estimates for the numbers of species (Williams
in press; Williams et al. 2020).
The bumblebee subgenus Bombus s. str. has a long history of studies showing how
challenging its taxonomy has been, because the morphological variation is very subtle
and many of the species are morphologically cryptic (reviewed in Williams, Brown, et al.
2012). In the same 2012 study, an international consortium collaborated to compile
a global set of standard (‘barcode’) sequences for the fast-evolving cytochrome
c oxidase subunit 1 (COI) gene, to assess the species present in the sense of evolutio-
narily independent lineages (De Queiroz 2007). Species were recognised by identifying
species’ gene coalescents from tting single-threshold General Mixed Yule-Coalescent
(GMYC) models (Monaghan et al. 2005). Unusually for GMYC analyses, in this case the
solution obtained was not statistically signicant (p = 0.15: Williams, Brown, et al. 2012
their table 2), so the results were not as robust as was hoped and a best available
approximation had to be chosen. This diculty in tting the GMYC models is considered
to have arisen from the weak dierentiation of the species in terms of unusually similar
lineage-branching patterns within and between species (a barcode ‘bush’).
Consequently, although there is a long-standing consensus that many separate species
do exist in the subgenus Bombus s. str. (e.g. Skorikov 1923; Krüger 1951, 1954, 1956,
1958), many of these species are not only morphologically cryptic (Rasmont 1984) but
also poorly dierentiated by changes in the branching pattern of the tree even for a fast-
evolving gene.
More recently, the Poisson-tree-process (PTP) method has been claimed to perform
better than GMYC for identifying species’ gene coalescents (Zhang et al. 2013). When
applied to samples with adequate coverage of all global species and from throughout
their global ranges, PTP has been shown to give similar results to GMYC for bumblebees
(Williams et al. 2015, 2016) that can be robust and stable once samples are adequate
(Williams et al. 2020). Here I t PTP models to re-assess the species of the subgenus
Bombus s. str.
272 P. H. WILLIAMS
Materials and methods
When possible, methods for revisionary taxonomy should include (Williams et al. 2020): (1)
an explicit statement of the species concept and the appropriate practical criteria that need
to be met for recognising species; (2) representation of all species and taxa world-wide from
the entire monophyletic group under revision; (3) representation of variation from across
the entire geographical ranges of all of those species, covering all constituent taxa and
representing all apparent gradients and clines; (4) iterative tree estimation as sampling
develops until convergence on robust trees is attained; and (5) examination and compar-
ison of type specimens for all of the named taxa in order to apply names. For revising
Bombus s. str.: (1) this study accepts species in theory as evolutionarily independent lineages
that may be detected in practice by PTP for species coalescents in fast-evolving genes (op .
cit.); (2, 3, 5) a sucient sample is provided by the data compiled for the earlier study by
Williams, Brown, et al. (2012) (data from the online BOLD database at boldsystems.org,
Ratnasingham and Hebert 2007, project BBBO). All specimens were identied to taxa by
comparison with the primary types and original descriptions (enumerated in the Appendix
by PW in Williams, Brown, et al. 2012), including proxy type specimens and sequences used
to assign names. For requirement (4), iterative estimates have been run on these data
every year since 2012, while adding distinctive new sequences each time to test for possible
new species (as discussed in Williams in press), but no further species have been detected
among the new sequences since the 2012 analysis. Therefore to keep this analysis compar-
able to the 2012 study, only the original data are re-analysed.
For estimating the metric phylogenetic tree needed for PTP, unique haplotype
ltering has been recommended to avoid uneven over-sampling (Williams in press;
Williams et al. 2020). Of the 559 COI-barcode sequences obtained by Williams,
Brown, et al. (2012) from a broad range of sites across species’ global distribution ranges
(mapped in Figure 1), the 121 longest unique haplotypes were used. Phylogenetic
Figure 1. Global distribution of sample sites for the samples of the subgenus Bombus s. str. with the
559 COI-barcode sequences from Williams, Brown, et al. (2012) that are re-analysed here (these
bumblebees are not indigenous to sub-Saharan Africa, the Arabian peninsula, lowland India,
Southeast Asia, Australia, or Central and South America). Spots are coloured to show the site elevation
(scale in metres a.s.l. at left). Cartesian orthonormal projection, north at the top of the map.
JOURNAL OF NATURAL HISTORY 273
relationships were estimated with MrBayes (Ronquist and Huelsenbeck 2003) using:
a general time-reversible evolutionary model with a gamma frequency distribution of
changes among sites; four Markov-chain Monte-Carlo chains; the temperature set to 0.2,
for 10 million generations; and a burn-in excluding the rst 5% of the trees. The analysis
was repeated four times in order to assess the stability of the results, twice with
B. (Alpinobombus) alpinus (Linnaeus) and twice with B. (Pyrobombus) vagans Smith as
the outgroup to root the tree. These species represent the two most closely related
subgenera. The PTP method was applied using the online bPTP server (species.h-its.org)
with the default bPTP options.
The candidate species identied here from coalescents in a fast-evolving gene are
compared with morphological characters examined previously with a binocular micro-
scope from the sequenced samples (Williams, Brown, et al. 2012). Names were applied to
the resulting nal species by reviewing the type material and using the proxy-type
procedure of Williams, Brown, et al. (2012 and its Appendix).
Results
Assessment of species’ coalescents in COI barcodes for the subgenus Bombus s. str. with
PTP shows highest local support for either 22 or 23 species depending on the choice of
outgroup (e.g. Figure 2 with B. vagans as the outgroup: 23 species with 95% condence
limits 22‒55 species). These results support splitting four or ve former ‘species’ into
coalescent candidate species.
Among the newly supported candidate species, the most weakly supported is
B. xanthopus, which is supported by PTP only when B. vagans is the outgroup (not
when B. alpinus is the outgroup) and then only with a low PTP support value (p = 0.66).
When B.alpinus is the outgroup, the highest local PTP support is for the group terrestris +
xanthopus, but is much lower (p = 0.38). Note that this support value is not a statistical test
of dierence, but shows the highest probability in this part of the tree that all of the
included groups are parts of a single species. When B. vagans is the outgroup, there is no
subset or superset of the taxon xanthopus that is supported more strongly as a separate
species (e.g. combining the taxa xanthopus and terrestris is supported much less strongly,
p = 0.14). Bombus xanthopus diers conspicuously from its sister group, B. terrestris, in that
the colour pattern of the hair (pubescence) lacks a yellow band on the anterior part of the
thoracic dorsum.
The ve other cases of candidate species newly supported by PTP in Figure 2 are
all further corroborated by consistent morphological character-state dierences: (1)
for B. czerskianus by the more extensively yellow hair and shorter hair compared with
B. sporadicus (Tkalců 1967); (2) for B. reinigi by what is described as the slightly more
chagrined outer surface of the hind tibia (Tkalců 1974) and for B. longipennis by the
more densely plumose hair on the head and scutellum and more extensively black
hair compared with B. minshanicola; (3) for B. mckayi by the yellow metasomal band,
yellow scutellar band, and longer hair compared with B. occidentalis (Williams, Brown,
et al. 2012; Williams et al. 2014; Sheeld et al. 2016); and (4) for B. ganjsuensis by the
narrow pale bands, often (not always) pink or orange hair of terga 4‒5, and the hind
basitarsus on its central outer surface with shorter hairs than on the posterior margin
274 P. H. WILLIAMS
Figure 2. Estimate of phylogeny from MrBayes for the fast-evolving COI gene of Bombus s. str. as
a metric tree from the longest examples of sampled unique COI-barcode haplotypes (from data for
559 sequences from Williams, Brown, et al. 2012), combined with the Bayesian Poisson-tree-process
(PTP) solution with spots showing the nodes with the highest support for coalescents of candidate
species by maximum likelihood (outgroup B. vagans not shown). Values above the nodes are MrBayes
Bayesian posterior probabilities, showing branch support for groups; values below the nodes are PTP
maximum-likelihood support values that all daughter haplotypes are parts of a single species. The
scale bar is calibrated in substitutions per nucleotide site. Each sample sequence is labelled with: the
sequence length in number of nucleotides; a taxon name, often referring to a particular colour pattern;
a code that consists of a specimen identifier from the project database and a sequence identifier;
followed with its geographic origin. Lineages with high probabilities of representing one or more
candidate species in the PTP results are shown with thick lines and the most recent common ancestor
of each candidate species (the species’ coalescent) is shown with a black spot. The branches within the
candidate species are shown with thin lines.
JOURNAL OF NATURAL HISTORY 275
compared with B. patagiatus (Williams, Brown, et al. 2012). The revised nomenclature
for these species is shown in Table 1.
Discussion
Sampling
Revisionary studies re-assess the variation within a group, so the sampling pattern is crucially
important, requiring consideration of at least two major components. First, all potential
species need to be included so that there are not articially enlarged gaps introduced into
the analysis from missing branches of the tree, otherwise the PTP results could be biased
because some species might then appear more distinct than would actually have been the
Table 1. List of the valid names of species recognised (shown in bold) and synonyms from the named
taxa sampled for COI barcodes within the subgenus Bombus s. str. from Williams, Brown, et al. (2012)
and re-assessed using the PTP results in Figure 2 and morphology (see text). Bombus xanthopus is
accepted as a separate species provisionally (see text).
Bombus ignitus Smith, 1869 Bombus jacobsoni Skorikov, 1912
Bombus sporadicus Nylander, 1848 Bombus hypocrita Pérez, 1905
sapporoensis Cockerell, 1911
Bombus czerskianus Vogt, 1911 STAT. NOV.
malaisei Bischoff, 1930
Bombus terricola Kirby, 1837
?Bombus xanthopus Kriechbaumer, 1870 STAT. REV.Bombus occidentalis Greene, 1858
Bombus terrestris (Linnaeus, 1758)
audax (Harris, 1776)
dalmatinus Dalla Torre, 1882
canariensis Pérez, 1895
terrestriformis Vogt, 1911
lusitanicus Krüger, 1956
africanus Vogt in Krüger, 1956
maderensis Erlandsson, 1979
Bombus mckayi Ashmead, 1902 STAT. REV.
Bombus lantschouensis Vogt, 1908
vasilievi Skorikov, 1913
beickianus Bischoff, 1936
pseudosporadicus Bischoff, 1936
Bombus tunicatus Smith, 1852
gilgitensis Cockerell, 1905
Bombus minshanensis Bischoff, 1936
Bombus affinis Cresson, 1863
Bombus magnus Vogt, 1911
flavoscutellaris G. & W. Trautmann, 1915
luteostriatus Krüger, 1954
Bombus franklini (Frison, 1921) Bombus patagiatus Nylander, 1848
brevipilosus Bischoff, 1936
Bombus longipennis Friese, 1918
Bombus ganjsuensis Skorikov, 1913 STAT. REV.
Bombus minshanicola Bischoff, 1936 STAT. REV.
Bombus reinigi Tkalcu, 1974 STAT. REV.
Bombus lucorum (Linnaeus, 1761)
alaiensis Reinig, 1930
mongolicus Krüger, 1954
Bombus cryptarum (Fabricius, 1775)
albocinctus Smith, 1854
moderatus Cresson, 1863
terrestricoloratus Krüger, 1951
iranicus Krüger, 1954
borochorensis Krüger, 1954
turkestanicus Krüger, 1954
burjaeticus Krüger, 1954
florilegus Panfilov, 1956
reinigianus Rasmont, 1984
armeniensis Rasmont, 1984
Bombus reinigi Tkalcu, 1974 STAT. REV.
276 P. H. WILLIAMS
case had all species been included. Second, variation within species should be represented as
evenly as possible among all species, to avoid over-sampling of just some of the most
common species. This can cause the tting of PTP models to be biased by an imbalance
across the tree in the sampled low-level variation, which can over-emphasise gaps of un-
sampled variation within other less common species so that they are interpreted falsely as
multiple species (Williams et al. 2020). Consequently, although adding more samples before
ltering for unique haplotypes would generally be benecial, unfortunately the increasing
restrictions from national policies on collecting, sequencing, or even the use of COI-barcodes
in some parts of the world, are making sampling this variation more and more dicult
(Williams et al. 2020). However, the international consortium collaborating for the 2012 study
was able to represent all known species world-wide more evenly (Figure 1) than in any other
study so far.
Using COI-barcode trees
It has often been claimed that trees for individual genes such as COI may not agree with
species’ phylogeny, which has indeed been observed for bumblebees (Williams et al.
2016, 2019, 2020). However, for bumblebees it is uncommon for these dierences in trees
to aect the near-terminal groups that aect species recognition (Williams et al. 2019),
with the dierences more often aecting the older deeper relationships in the trees. But
the possibility of dierences underlines the need for integrative taxonomy.
Changes in interpretation
The most recent global revision of all species of the subgenus Bombus s. str. by Williams,
Brown, et al. (2012) lacked a statistically signicant single transition from the Yule to the
coalescent models to show a clear threshold indicating species (their table 2). A choice
was made of a best-supported threshold (their gs 2‒4), which gave results that agree for
17 of the 23 of the species supported here by PTP.
Given the uncertainty in the location of a GMYC threshold in the 2012 analysis, some of
the present results might be obtained from the previous tree by a small adjustment in the
position of the GMYC threshold. For example, branching between B. sporadicus and
B. czerskianus as recognised by PTP is very close to the 2012 GMYC threshold (their
g. 2). Branching to the species B. longipennis, B. minshanicola, B. mckayi and
B. ganjsuensis is slightly closer to the terminals in the 2012 tree and is actually closer to
the terminals than are the most recent common ancestors for some other groups not
recognised here as separate species, e.g. for additional splits within the species B. ignitus,
B. terrestris, and B. cryptarum (Williams, Brown, et al. 2012, g. 2, cf. Figure 2). Recognition
of the species B. longipennis, B. minshanicola, B. mckayi, and B. ganjsuensis by the PTP
procedure is therefore likely to be due in part to dierences in the MrBayes models used
to build the shape of the tree as well as to the PTP models used to interpret its shape (as
for B. czerskianus). This dierence in species recognised might be attributed in part to
a greater distortion of the tree to t the data when tting an ultrametric tree with BEAST
(used in 2012 for GMYC) compared with tting a simpler metric tree with MrBayes (used
here for PTP). This would make the metric tree and the PTP procedure preferable.
JOURNAL OF NATURAL HISTORY 277
The PTP procedure used here shows local variations on the tree in the support values
for species, quantifying these uncertainties and mapping them onto the tree (Figure 2).
Some of these support values appear low, but they are still the highest in their local areas
of the tree and so represent the most likely interpretations at present. Uncertainties might
be reduced in the future by obtaining representation of more of the variation within these
species from further sampling (see comments on sampling above).
Species recognised
None of the taxa revised to species here in Table 1 was unknown: all have been described
and named previously (only B. czerskianus had been described but not recognised before
with the status of a separate species) so it is only the interpretation of their status as
species that has changed here since the 2012 analysis. Bombus sporadicus and
B. czerskianus have distributions widely separated between northern Europe and north-
eastern Asia (Tkalců 1967).
Of particular importance is the splitting of B. mckayi from B. occidentalis s. str. in
western North America, both of which were originally distinguished as separate species
on the basis of colour pattern. Bombus occidentalis s. str. is of particular conservation
concern (Colla and Ratti 2010; Hateld et al. 2015b; Sheeld et al. 2016). Recognising the
two taxa as separate species is likely to further increase the conservation priority of
B. occidentalis s. str. where it occurs in the western USA (Colorado, Washington etc.) and
western Canada (British Columbia, Alberta etc.). Bombus mckayi, which occurs in Alaska
and western Canada (British Columbia, Yukon), appears to be less threatened (Sheeld
et al. 2016), but should nonetheless be protected from the introduction of closely related
species.
Bombus xanthopus was originally described as a species separate from B. terrestris
because of its distinctive lack of a yellow thoracic band and possession of an orange-
tailed colour pattern within this group in Europe (Kriechbaumer 1870). It is endemic to
Corsica, Capri, and Elba (Estoup et al. 1996; Rasmont et al. 2008). From the broadest
global sample of Bombus s. str. to date, Williams, Brown, et al. (2012) interpreted the
taxon xanthopus from GMYC results as part of B. terrestris s. l. (note under their table 4),
an interpretation shared previously by others (Estoup et al. 1996; Rasmont et al. 2008).
These taxa were subsequently shown to dier in cephalic labial gland secretions and
interpreted as separate species (Lecocq et al. 2014, 2016). Because the PTP results here
are inconsistent depending on which outgroup is used to root the tree, more data on
population variation and possibly from improved analytical methods are needed to
assess its status. Until that can be achieved, I conclude that B. xanthopus is interpreted
as a separate species, but only provisionally (Table 1), to recognise explicitly this
uncertainty. This seems preferable to introducing another taxonomic category (of sub-
species: Lecocq et al. 2016) that has been interpreted inconsistently (Wilson and Brown
1953; Zink 2004). But whether or not the taxon xanthopus is interpreted as a species
separate from B. terrestris s. str., it is important for the conservation of its unique genetic
complement that B. terrestris s. str. is not introduced for commercial pollination within
its area of distribution.
Recognising B. ganjsuensis as a species separate from B. patagiatus may be unsurpris-
ing because the two are separated by the Gobi Desert. Bombus ganjsuensis is restricted to
278 P. H. WILLIAMS
the hills of North China (Gansu, Ningxia, Shaanxi, Shanxi, Hebei, Beijing, but also within
central Neimenggu at the top of isolated mountains like Huanggangling: Williams, An,
et al. 2012), whereas B. patagiatus is much more widespread in the taiga forest zone of
north-eastern Russia, Mongolia, north-eastern China (e.g. Liaoning, Jilin, Heilongjiang, and
north-eastern Neimenggu: Williams, An, et al. 2012), and Korea. The important distinction
for the trade in pollinators and conservation is that B. ganjsuensis remains a species
separate from the similar-looking far eastern and Japanese B. hypocrita and should be
protected from the introduction of closely related species
The other problematic species complex is centred on the high mountains at the east
and south of the Qinghai-Tibetan plateau (QTP): including B. reinigi (western Himalaya:
Pakistan to Nepal), B. longipennis (eastern Himalaya: Bhutan, Sikkim, Xizang), and
B. minshanicola (on the eastern plateau and Hengduan mountains of western Shaanxi,
Ningxia, Gansu, Sichuan, Yunnan, Qinghai). Although the last decade has seen a lot more
sampling of these bees from the eastern QTP in China by the Chinese Academy of Sciences,
much more information is still needed in order to assess their status more reliably from the
high Himalaya and trans Himalaya, where these bees are not abundant (Williams in prep.).
Conclusion
This re-assessment of the COI-barcode data from Williams, Brown, et al. (2012) does not
give a radically dierent interpretation – most of the same species are recognised
(although these results are from the same data). However, the improved methods ne-
tune the interpretation by ‘splitting’ ve species that will be important for conservation.
Ideally an entirely new set of samples would be required in order to test these patterns by
replication of the study. But unfortunately, if anything, it is becoming more dicult to bring
new samples together for global reviews, emphasising the value of the earlier broadly
collaborative international study (Williams et al. 2020). We look forward still to having the
quest to understand these bumblebee species informed by better data and methods,
although these must always be selected carefully to be appropriate to the ideas on how
species can be recognised. Keys to help identify the newly supported species will be
included in forthcoming identication guides to the bumblebees of North America (a
revised edition of Williams et al. 2014) and to the bumblebees of the Himalaya (Williams
in prep.).
Acknowledgements
I am especially grateful to all of those who have collected the specimens used in Williams et al.
(2012) and re-analysed here, including M. Brown, J. Carolan, J. An, D. Goulson, M. Aytekin, L. Best,
A. Byvaltsev, B. Cederberg, R. Dawson, J. Huang, M. Ito, A. Monfared, R. Raina, P. Schmid-Hempel,
C. Sheeld, P. Sima, and Z. Xie. Thanks to L. Bailey, S. Colla, and M. Orr for discussion. Thanks to an
anonymous referee for suggestions on the manuscript.
Disclosure statement
No potential conict of interest was reported by the author.
JOURNAL OF NATURAL HISTORY 279
Funding
No additional funding was obtained for this study.
ORCID
Paul H. Williams http://orcid.org/0000-0002-6996-5682
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