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The blackburni/murchisona species complex in Australian Pseudotetracha (Coleoptera: Carabidae: Cicindelinae... Article in Journal of Zoological Systematics and Evolutionary Research · August 2012 DOI: 10.1111/j.1439-0469.2012.00659.x
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The blackburni/murchisona species complex in
1 2
Australian Pseudotetracha (Coleoptera:
3
Carabidae: Cicindelinae: Megacephalini):
4
evaluating molecular and karyological evidence
5
Alejandro López-López1, Peter Hudson2, José Galián1*
6 7 8 9 10
1) 2)
Departamento de Zoología y Antropología Física, Universidad de Murcia, Campus de Espinardo, 30100 Murcia (Spain) South Australian Museum, North Terrace, Adelaide, SA 5000 (Australia)
*Corresponding author
11
ABSTRACT
12
Our phylogenetic analysis of three endemic species of the Australian tiger beetle genus
13
Pseudotetracha Fleutiaux, 1864 from South Australia used sequences of two fragments of the
14
mitochondrial genes 16S rRNA and cytochrome oxidase III. A matrix for each gene and two
15
combined matrices were constructed. We compared these three riparian species, together with
16
data from nine taxa of this genus available in GenBank, by using parsimony and bayesian
17
methods. These molecular results are in agreement with the phylogenetic hypothesis for the
18
blackburni/murchisona species complex previously proposed based on morphology, whereas
19
other recent molecular analysis have questioned the existence of this species complex. In all of
20
our analyses, samples of P. blackburni divided into two statistically supported clades, one of
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which is more closely related to P. mendacia and P. pulchra than to the other P. blackburni
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clade. This suggests the existence of a cryptic new species. Additionally we analyzed
23
chromosomes of the second metaphase cells of members of the two clades. The observations
24
showed different karyotypes as blackburni-1 has two types of second meiotic metaphase cells
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with 11 and 12 chromosomes, whereas in blackburni-2 all cells have 12 chromosomes, adding
26
evidence for the putative existence of two species.
27 28
INTRODUCTION
29
The Australian genus Pseudotetracha Fleutiaux, 1894 is a group of tiger beetles included in the
30
Pantropical tribe Megacephalini. It was considered a subgenus of Megacephala by Horn (1910)
31
and Sumlin (1997), whereas Huber (1994) elevated it to a full genus; this has been accepted by
32
Zerm et al. (2007). Currently, 19 species of Pseudotetracha have been described, but only those
33
from southern Australia have been studied. It is likely that additional undescribed species exist.
34
Based on morphological characters, Sumlin (1997) suggested the existence of several closely
35
related species within the blackburni/murchisona complex, including P. blackburni, P.
36
murchisona, P. corpulenta, P. mendacia and P. cuprascens. However, a subsequent analysis
37
based on nuclear 18S, mitochondrial 16S and cytochrome oxidase III genes, of 40 South
38
American and Australian taxa (Zerm et al. 2007) determined that P. blackburni should not be
39
grouped with other members of Sumlin’s blackburni/murchisona complex.
40
Recently, a new Species Delimitation Plugin (Masters et al. 2011) for GENEIOUS (Drummond
41
et al 2010) has become available. This tool calculates statistics that help to determine if each
42
clade obtained in a phylogenetic tree has identity as a distinct species. One of these statistics is
43
the ratio between the mean distance within the members of the clade and the mean distance of
44
those individuals to the nearest clade. The larger the value of this ratio is, the more distinct is
45
the identity of the group. Other statistic is the P ID, which represents the mean probability (95%
46
confidence interval) for a member of the problem species to fit inside (Strict P ID) or at least to
47
be the sister group (Liberal P ID) of the clade made up by the other individuals belonging to this
48
species. These statistics have been successfully used in the gastropod genus Lunella (Williams
49
et al. 2011) where eight cryptic species were recognized, and in the Alveolata genus Eimeria
50
(Ogedengbe et al. 2011) in which the probability of making a correct identification of an
51
unknown specimen or sequence based on partial COI sequences has been used. The
52
implementation of these methods in combination with traditional barcoding analyses could be of
53
great help for the accurate identification of difficult organisms.
54
The objective of our study is to resolve the contradictory results of previous studies of the
55
phylogeny and systematics of the blackburni/murchisona complex. To this aim we have
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analyzed a fragment of the cytochrome oxidase III and a fragment of the 16S rRNA of the
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mitochondrial genome of 89 Pseudotetracha samples. A fragment of the 18S nuclear rRNA
58
proved not to be phylogenetically informative. We included a large number of samples of P.
59
blackburni and P. whelani, and some of P. australis (not previously studied molecularly), and
60
we predicted that our analysis of DNA sequences would help to show the existence of cryptic
61
taxa.
62 63
MATERIAL AND METHODS
64
In this work we used samples from Pseudotetracha australis (Chaudoir, 1865), which is found
65
in riparian and saline lake habitats in the central, south and eastern parts of Australia; P.
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blackburni (Fleutiaux, 1895), which is found only in saline lakes in the western half of the
67
continent; and P. whelani Sumlin, 1992 which is only known from saline lake habitats in South
68
Australia. All three species share typical characteristics of tiger beetles, such as being nocturnal
69
predators of other invertebrates, with tunnel-burrowing larvae.
70
The samples analysed in this work (*Table S1) included the Pseudotetracha sequences used by
71
Zerm et al. (2007) and available from the GenBank database, along with those from 89
72
Pseudotetracha samples collected in South Australia in 2004. Vouchers are deposited in the
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Zoology and Physical Anthropology Department insect collection of the University of Murcia
74
and in the South Australian Museum. These samples were identified as P. blackburni, P.
75
whelani and P. australis using the identification key provided by Sumlin (1997). Beetles were
76
preserved in absolute ethanol. DNA was extracted from each sample using a commercial kit
77
provided by Qiagen (Hilden, Germany) or Invitek (Berlin, Germany). Three markers were
78
amplified using a commercial PCR kit and a standard amplification protocol: a) a fragment of
79
the mitochondrial cytochrome oxidase III gene, b) a fragment of the 16S mitochondrial rRNA
80
(both of them using the same primers of the analysis by Zerm et al. 2007, see Table 1) and c) a
81
fragment of the 18S nuclear rRNA (primers by Shull et al. 2001, detailed in Table 1). The PCR
82
products were checked by agarose gel electrophoresis, purified and sequenced using an ABI
83
Prism 3130 from the Molecular Biology Service of the University of Murcia or by Macrogen
84
Inc. (Korea). The 16S rRNA sequence was successfully amplified from all the samples, but only
85
50 cytochrome oxidase III and 74 18S rRNA sequences were obtained.
86
The sequences were edited and aligned using MEGA 4 (Tamura et al. 2007). Sequences of
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Neocollyris sp., Pentacomia discrepans, Odontocheila confusa, Grammognatha euphratica and
88
Megacephala virginica from the work of Zerm et al. (2007) were used as outgroups. We
89
constructed an alignment for each sequence, coxIII and 16S, but, except for separating all
90
individuals of P. whelani from the rest of the species, the 18S alignment was not considered
91
further due to the lack of variation. Two combined matrices were also constructed: i) Matrix 1,
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comprising only the individuals from which both the coxIII and 16S sequences were obtained,
93
and ii) Matrix 2, a concatenated matrix including all the individuals and genes, including those
94
individuals which lack information for one or two sequences. According to Wiens (2006) and
95
Wiens & Moen (2008), the inclusion of more taxa and more sequence sites should improve the
96
phylogenetic inference even if incomplete.
97
For each alignment we performed Maximum Parsimony and Bayesian Inference analyses. The
98
Maximum Parsimony analyses were performed in PAUPUP (Swofford 2002; Calendini and
99
Martin 2005) by an heuristic search. In order to correct for possible “evolutionary noise” caused
100
by the more variable nucleotide positions, i.e. the first and third codon positions, a weighting of
101
2:5:1 to the first, second and third codon positions, respectively, and 5 for the 16S ribosomal
102
fragments, was carried out. Before the Bayesian Inference analyses, we searched the best model
103
in jMODELTEST v0.1 (Posada 2008). The Bayesian Inference analyses were made using
104
MRBAYES v3.1.2 (Ronquist and Huelsenbeck 2003), with 10000000 generations and keeping
105
a tree every 2500 generations. After the analyses, the burn in was determined independently for
106
each matrix.
107
Gaps were treated as a 5th state in all the analyses, as unavailable sequences were treated as
108
missing data in the concatenated matrix. The 16S sequence did not present gaps within the
109
genus Pseudotetracha, so this gap treatment should not affect the ingroup relationships.
110
The Bayesian Inference matrices and trees were imported to GENEIOUS v5 (Drummond et al.
111
2010), where we calculated the statistics included in the Species Delimitation Plugin (Masters et
112
al. 2011), which calculates a series of statistics that help to decide if an obtained clade can be
113
considered as a distinctive species.
114
A meiotic chromosomal survey was performed in individuals of P. blackburni to test the
115
differences found in the mitochondrial analysis with an independent marker such as the
116
chromosome number. Gonads previously fixed in ethanol-acetic acid 3:1 were squashed in 45%
117
acetic acid and the coverslip removed after immersion in liquid nitrogen. The slides were
118
stained with Giemsa in phosphate buffer pH=6.8, washed and analyzed in a Zeiss Primo Star
119
phase contrast microscope.
120
Moreover, a topology test was performed for this group importing the concatenated Matrix 2 to
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RaxML 7.0.3 (Stamatakis 2006) where two Maximum Likelihood analyses were performed: a)
122
a standard analysis with a rapid hill-climbing search algorithm and b) the same analysis but
123
forcing all the P. blackburni samples into a monophyletic group excluding the other species.
124
The best trees obtained were imported to CONSEL 1.19 (Shimodaira and Hasegawa 2001;
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Shimodaira 2002) in order to perform the topology test.
126 127
RESULTS
128
After editing, the cytochrome oxidase III sequences included 288 nucleotides, of which 77 were
129
variable and 69 phylogenetically informative. The 16S sequence included 265 nucleotides, of
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which 52 were variable and 46 phylogenetically informative. The 18S fragment had a length of
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442 nucleotides, and it was conserved across the samples: only one substitution and a two-bases
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insertion were shared by the P. whelani samples. All the obtained sequences were submitted to
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the GenBank database under accession codes JF772233-JF772320 (16S), JF772321-JF772394
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(18S) and JF772395-JF772444 (coxIII).
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In all cases, the model chosen for the Bayesian Inference analysis was the General Time
136
Reversible (GTR) model considering the invariant sites frequency (not for the concatenated
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matrices) and the Gamma distribution.
138
The trees obtained by Maximum Parsimony and Bayesian Inference with the coxIII alignment
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articulate into three clades. One of them includes P. pulchra, P. mendacia, P. australis, P.
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cuprascens, P. corpulenta and the samples we identified as P. blackburni. A second clade
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contains P. whelani samples, including the sample studied by Zerm et al. (2007). The third clade
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contains P. oleadorsa, P. ion, P. helmsi and the sample identified as P. blackburni by Zerm et
143
al. (2007). The Bayesian Inference tree presents a similar topology, with some differences in the
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internal nodes.
145
In the trees obtained with the 16S alignment (Figure 1), the samples are arranged in the same
146
clades as the coxIII analysis, although the clades including P. australis and P. blackburni are
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better resolved, with three subclades: i) P. australis, ii) P. cuprascens + P. corpulenta and iii) P.
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blackburni + P. pulchra + P. mendacia. The samples identified as P. blackburni are split into
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two clades, with the P. blackburni of Zerm et al. (2007) separate from the P. blackburni
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samples analyzed in the present work.
151
In all trees obtained with the combined Matrix 1 and with the concatenated Matrix 2 (Figure 2)
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with Maximum Parsimony and Bayesian Inference, Pseudotetracha samples grouped into four
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principal clades. One of these (A) includes all the samples identified as P. whelani together with
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the P. whelani sample studied by Zerm et al. (2007). P. whelani is shown to be the sister group
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of the blackburni/murchisona complex. A second clade (B) included all the samples identified
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as P. australis, which is the sister group of the third principal clade (C), including P. cuprascens
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+ P. corpulenta. The remaining principal clade (D, inserted in Figure 2), although not
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completely resolved in the Bayesian Inference analysis with Matrix 2, contains the samples
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identified as P. blackburni along with P. mendacia and P. pulchra. Within this clade, two
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subclades are apparent, i) blackburni-2 with P. mendacia and P. pulchra, and ii) blackburni-1.
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Both P. blackburni subclades are clustered inside the blackburni/murchisona complex as
162
defined by Sumlin (1997), but separated from the P. blackburni sequenced by Zerm et al.
163
(2007) (see below). Sample no. 163 was separated from both clades of P. blackburni.
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The relationships of the species included in the blackburni/murchisona complex varied between
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the different analyses, and are only completely resolved in the Bayesian Inference analyses with
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the two combined matrices. The analysis with Matrix 2 resolved the basal relationships inside
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this group but did not resolve the P. blackburni clades. However, the analysis with the Matrix 1
168
(Figure 2) resolved the relationship within the P. blackburni clades showing the samples
169
assigned to the clade blackburni-1 as a sister group to the clade that included blackburni-2 + (P.
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mendacia + P. pulchra).
171
The statistics calculated using the Species Delimitation Plugin for P. australis and P. whelani
172
and for the two clades of P. blackburni based on Matrix 2 are shown in Table 2 (a detailed table
173
for all matrices can be found in supporting information *Table S2). From these data it was
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concluded that all the groups are monophyletic, except for blackburni-1 in two cases: i) if
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sample number 163 is considered as part of it, and ii) in the Matrix 2 analyses due to the low
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resolution. The values of the ratio between the average distance between the samples of one
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group (Intra Dist) and the average distance between those samples and the closest clade (Inter
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Dist) is small, below 0.2, meaning that genetic differences within these clades are small relative
179
to the differences between clades, so that there is a well defined separation of clades. Only the
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clade blackburni-2 shows a slightly higher value than the others. The probability (P ID) of a
181
new sequence fitting inside (Strict) or at least as sister group (Liberal) of its clade is high in all
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cases except for blackburni-2 in the Matrix 1 analysis, where the Strict P ID value decays to
183
0.45.
184
The results of the topology test show that the topology obtained by the standard analysis has a
185
higher likelihood value than the constrained topology, but none of the analyses performed by
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CONSEL are significant (Table 3).
187
Results on karyotype analysis of meiotic cells showed that 4 individuals from the blackburni-2
188
clade have metaphase II cells with 12 chromosomes (2n=24), whereas cells from 8 members of
189
the blackburni-1 clade have either 11 or 12 chromosomes (2n=23) (Figure 3). These differences
190
are indicative of chromosomal rearrangements occurring in parallel with the separation of the
191
two lineages.
192 193
DISCUSSION
194
This work supports the blackburni/murchisona complex hypothesized by Sumlin (1997).
195
Additionally, this work shows molecular data of P. australis for the first time. This species was
196
not analysed by Zerm et al. (2007) and Sumlin (1997) did not include it in the
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blackburni/murchisona species complex. P. pulchra was grouped by Zerm et al. (2007) together
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with P. mendacia, and this is consistent with the clustering obtained in this work showing these
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two species as part of the blackburni/murchisona species complex. If we accept the proposal of
200
Sumlin (1997) about the members of this complex, then it should be considered that the
201
blackburni/murchisona species complex is made by clades B+C+D (Figure 2). In this case P.
202
australis (clade B) should be included within this group. Alternatively, if we consider that the
203
blackburni/murchisona species complex is only represented by the clade D (Figure 2), then P.
204
corpulenta and P. cuprascens (clade C) should be excluded from this complex. In any case, P.
205
pulchra should be considered as a member of this group. The position of P. murchisona could
206
not be tested due to the lack of molecular data for this species.
207
Zerm et al. (2007) studied a single specimen of P. blackburni from Western Australia (the main
208
area of distribution of this species). It is remarkable that it did not cluster together with our 21
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samples of this species collected from Eyre Peninsula. This fact suggests three possibilities: i)
210
this sample was misidentified by Zerm et al. (2007), ii) there was a methodological error (e.g.
211
DNA contamination with other Pseudotetracha sample), or iii) a still undescribed, ‘cryptic’
212
taxon is involved. The analysis of the sequences obtained by Zerm et al. (2007) for each of the
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two genes in this putative P. blackburni specimen showed that they correspond to taxa of the
214
oleadorsa/ion/helmsi group, rather than to P. blackburni. This unexpected result is more
215
probably due to a misidentification rather than a contamination event, as this would have
216
occurred in each of the three sequenced genes. These problems are not uncommon (Vilgalys
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2003), and make up the rationale for studying more than one sample of each taxon. The
218
sequence of the P. blackburni sample was critical to refute the existence of the
219
blackburni/murchisona complex (Zerm et al. 2007). This conclusion is quite opposite to that
220
reported here.
221
The 21 samples of P. blackburni analysed in this work and identified following the
222
morphological criteria of Sumlin (1997), collected from two different lakes separated by 120
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km, formed two clades in all analyses, blackburni-1coming from Lake Gilles and blackburni-2
224
from Lake Yaninee. The study of type material would help to assess which of these clades
225
corresponds to current P. blackburni taxon, and which should be described as a new species
226
(both clades might even correspond to undescribed taxa). Alternatively, an ancestral
227
introgression of an ancestor of P. mendacia + P. pulchra in P. blackburni, eventually resulting
228
in blackburni-2, could not be rejected. The possibility that one of these clades is actually P.
229
murchisona (a species for which no molecular data are available) is rejected because the main
230
character separating these two species, the elytral punctuation pattern, does not correspond to
231
the murchisona pattern in any of them, compared to figure 1f in Sumlin (1997). Although the
232
topology tests showed that the possibility of the two groups of P. blackburni actually fitting in a
233
single monophyletic group has a smaller likelihood, the topology tests are not significant, and so
234
we could not discard that possibility.
235
The species delimitation statistics (see below) provide adequate support to consider each clade
236
of P. blackburni as a different species, a conclusion that is corroborated by observation of
237
metaphase II cells from members of the two clades. The only chromosomal data so far known
238
for Pseudotetracha, is P. whelani (Galián & Hudson 1999) with 12+XY meioformula. Our
239
preliminary data for P. blackburni (Figure 3) indicate a reduction of one pair of chromosomes in
240
blackburni-2, and a reduction of three chromosomes in blackburni-1. The availability of only
241
second metaphase plates does not allow us to accurately determine the meioformula of this
242
species, but it allows inferring a different karyotype in both clades and presumably a sex
243
chromosome system in blackburni-1 different from the XY observed in the close species P.
244
whelani and very likely that in blackburni-2. The nature of these chromosomal differences needs
245
to be clarified with further analysis of metaphase I and mitotic metaphase plates.
246
The analysis of the cytochrome oxidase III sequence in sample 163 showed that it made up a
247
separate branch from supposedly related samples, but in the analysis based on the 16S sequence
248
it fitted inside the blackburni-1 clade. We interpret this incongruence as the result of homoplasy
249
in the cytochrome oxidase III fragment due to the high nucleotide substitution rate in this gene
250
(Pons et al. 2010) and we suspect that sample number 163 is actually a member of the
251
blackburni-1 clade as the analysis of the 16S sequence shows. Analysis of additional nuclear
252
and mitochondrial genes is needed to more reliably ascertain the relationships of this sample
253
and to understand the nature of this incongruence. The possibility that this sequence represented
254
a nuclear copy of a mitochondrial gene was discarded by determining that all the entire
255
sequence codes the same amino acid sequence as other blackburni-1.
256
The 16S sequence has been shown to be a reliable marker for differentiating species in
257
Pseudotetracha (Figure 1), as well as in other groups of animals where Vences et al. (2005)
258
indicates that 16S is sufficiently variable to unambiguously identify most species in different
259
groups of vertebrates. This marker has also been successfully used in insects (Han et al. 2010,
260
Hosoya and Araya 2005, Yeh et al. 2004), and other invertebrates (Barr et al. 2009). The
261
analysis of the cytochrome oxidase III gene resulted in less robust conclusions (for example in
262
the position of sample 163), probably due to the fact that cytochrome genes showed extremely
263
high substitution rates mainly due to the third codon sites as reported by Pons et al. (2010) for
264
the order Coleoptera.
265
The species delimitation statistics indicate that these four taxa (Table 3 and Table *S1) are
266
distinct. If P. whelani is considered as a reference, the Intra/Inter distances ratio values for
267
blackburni-1 are similar to this species, whereas the values for blackburni-2 are slightly higher,
268
perhaps due to the reduced number of available samples. Also, the Strict PI D value for this
269
clade is lower than the same value found for the other clades, especially with Matrix 1, because
270
of the reduced number of samples again. Nevertheless, the P ID values for this clade increased
271
when we considered more samples (Matrix 2) or the P ID is Liberal. In fact, the Liberal P ID
272
value for blackburni-2 is over 0.90, which provides strong support for the separation of this
273
clade from the others as a distinct species.
274
ACKNOWLEDGEMENTS
275
Thanks are due to Eduardo Díaz for helping in sample collection, to Carlos Ruiz and José
276
Serrano for their valuable suggestions and advice on the manuscript, and to three anonymous
277
referees for their valuable comments. A López-López is granted with a FPU grant (AP2009-
278
1184) from the Ministerio de Educación of Spain. This work has been funded by project
279
CGL2008-03628 of the Ministerio de Ciencia e Innovación, Spain.
280 281
SECOND SUMMARY
282
El complejo de especies blackburni/murchisona en el género australiano Pseudotetracha
283
(Coleoptera:
284
citogenéticas
285
Nuestro análisis de tres especies del género endémico australiano Pseudotetracha Fleutiaux,
286
1864 recolectadas en Australia del Sur usa secuencias de dos fragmentos de los genes
287
mitocondriales ARNr 16S y citocromo oxidasa III. Fue construida una matriz para cada gen y
288
otras dos matrices combinadas. Comparamos estas tres especies riparias junto con datos de
289
nueve taxones de este género disponibles en la base de datos GenBank, por medio de métodos
290
de parsimonia y bayesianos. Los resultados moleculares se muestran de acuerdo con la hipótesis
291
filogenética acerca del complejo de especies blackburni/murchisona propuesto previamente en
292
base a la morfología, mientras que otros estudios moleculares más recientes han cuestionado la
293
existencia de este complejo de especies. En todos los análisis, las muestras de P. blackburni se
294
dividen en dos clados con soporte estadístico, separados por las secuencias de P. mendacia y P.
295
pulchra, hecho que sugiere la existencia de una nueva especie críptica. Adicionalmente se han
296
analizado cromosomas en células de la segunda división de la meiosis en miembros de ambos
297
clados. Las observaciones mostraron cariotipos diferentes, así blackburni-1 presentó dos tipos
298
de células en la segunda división meiótica con 11 y 12 cromosomas respectivamente, mientras
299
que en blackburni-2 todas las células mostraron 12 cromosomas, proporcionando así evidencias
300
adicionales que apoyan la posible existencia de dos especies.
301
Cicindelinae:
Megacephalini):
evaluando
evidencias
moleculares
y
302 303 304
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373
374 375
Figure 1: Bayesian Inference tree based on the 16S rRNA alignment. Numbers in the nodes
376
correspond to the Posterior Probability (upper number) and bootstrap values (lower number,
377
only in the nodes also present in the Maximum Parsimony tree).
378 379
Figure 2: Bayesian Inference tree based on the Matrix 2 analysis. The blackburni/murchisona
380
species complex (encircled by the dotted line) is drawn after the Matrix 1 analysis, which
381
provides a better resolution for this group. Numbers in the nodes correspond to the Posterior
382
Probability (upper number) and bootstrap values (lower number, only in the nodes also present
383
in the Maximum Parsimony tree).
384 385
Figure 3: Haploid cells at meiotic metaphase II stage showing chromosomes of blackburni-1
386
(a,b,c, sample 32) and blackburni-2 (d,e,f, sample 49). A) Two cells, with n=11 chromosomes
387
(left) and with n=12 chromosomes (right) in blackburni-1. B) Meiogram of a cell with n=11
388
chromosomes. C) Meiogram of a cell with n=12 chromosomes. D) Two cells with n=12
389
chromosomes in blackburni-1. E) Meiogram of a cell with n=12 chromosomes, including the Y
390
heterosome. F) Cell with 12 chromosomes including the X heterosome. The images have been
391
modified by the GNU Image Manipulation Program (GIMP 2.6.8, http://www.gimp.org) with
392
the aim of producing a clearest image.
393 394
Table 1: List of primers used in this work Fragment coxIII 16S 18S
395
Primer name coxIII-F coxIII-R 16S-F 16S-R 18S5’ 18Sb5.0
Sequence CTGTAGAAYTWGGRAGAACTTGRCC TACTATATAAATTATCTACCTCATC CCGAGTATTTTGACTGTGC TAATCCAACATCGAGGTCGCAA GACAACCTGGTTGATCCTGCCAGT TAACCGCAACAACTTTAAT
Reference Zerm et al. (2007) Shull et al. (2001)
Excluding 163 blackburni-1 yes 0.08 0.84 blackburni-2 no 0.04 0.22 blackburni-1 yes 0.05 0.22 blackburni-1 yes 0.16 1.07 Considering 163 as blackburni-1 blackburni-1* yes 0.08 0.83 blackburni-2 no 0.05 0.23 blackburni-1* yes 0.05 0.23 blackburni-1* yes 0.16 1.07
P. australis blackburni-1 blackburni-2 P. whelani P. australis blackburni-1* blackburni-2 P. whelani
0.96 0.91 0.84 0.94
0.99 0.97 0.94 0.98
0.10 0.23 0.23 0.15
0.96 0.90 0.84 0.94
0.99 0.97 0.94 0.98
(Liberal)
0.10 0.19 0.23 0.15
P ID
P ID (Strict)
Closest Species
Species
Intra/Inter
MATRIX
Closest
species with more than one sample available are shown.
Inter Dist -
397
Intra Dist
Table 2: Species Delimitation results above the Bayesian Inference trees for the Matrix 2. Only
Monophyly
396
398 399
Table 3: Results of the topology test performed by CONSEL. Obs: observed likelihood
400
difference. p-au: p-value of the approximately unbiased test calculated from the multiscale
401
bootstrap. Bootstrap
Topology Not constrained Constrained
Rank Obs
p-au
Tests Kishino- Shimodaira- Weighted Weighted Multiscale Usual Hasegawa Hasegawa K-H S-H
1
-2.2
0.602
0.600
0.599
0.607
0.607
0.607
0.607
2
2.2
0.398
0.400
0.401
0.393
0.393
0.393
0.393
402 403 404
E-MAIL ADRESSES
405
A. López-López:
[email protected]
406
P. Hudson:
[email protected]
407
J. Galián:
[email protected]
408
409
Table S1: List of samples used in this work, showing the collecting data and which sequences
410
were obtained from each sample.
Code
Lake
Coordinates
Date of collection
Species
16S
18S
coxIII
31
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
32
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
yes
33
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
yes
34
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
yes
35
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
36
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
37
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
38
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
yes
39
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
yes
40
Lake Gilles (south)
33º 02’ S, 136º 36’ E
11/03/2004
P blackburni
yes
yes
41
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
yes
43
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
44
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
yes
49
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
yes
52
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
yes
55
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
yes
56
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
57
Lake Yaninee
32º 58’ S, 135º 16’ 20’’ E
12/03/2004
P blackburni
yes
yes
76
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
77
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
78
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
79
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
80
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
81
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
yes
82
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
yes
83
Lake Finniss
31º 43’ S, 136º 49’ 30’’ E
14/03/2004
P whelani
yes
yes
93
near Lake Bumbunga
33º 55’ 50’’ S, 138º 10’ E
27/03/2004
P australis
yes
yes
97
near Lake Bumbunga
33º 55’ 50’’ S, 138º 10’ E
27/03/2004
P australis
yes
98
near Lake Bumbunga
33º 55’ 50’’ S, 138º 10’ E
27/03/2004
P australis
yes
100
near Lake Bumbunga
33º 55’ 50’’ S, 138º 10’ E
27/03/2004
P australis
yes
yes
101
near Lake Bumbunga
33º 55’ 50’’ S, 138º 10’ E
27/03/2004
P australis
yes
yes
102
near Lake Bumbunga
33º 55’ 50’’ S, 138º 10’ E
27/03/2004
P australis
yes
yes
118
near Lake Boggy
35º 18’ 30’’ S, 139º 15’ E
29/03/2004
P australis
yes
yes
134
near Pelican Lagoon
35º 19' 20'' S, 139º 17' 30'' E
29/03/2004
P australis
yes
yes
138
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
yes
yes
yes
yes
yes
Code
Lake
Coordinates
Date of collection
Species
16S
18S
coxIII
139
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
yes
140
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
141
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
yes
142
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
yes
143
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
yes
144
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
145
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
146
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
147
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
148
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
149
Lake Albert
35º 35’ 20’’ S, 139º 23’ E
30/03/2004
P australis
yes
yes
161
Lake Gilles (north)
32º 44’ S, 136º 54’ 20’’ E
02/04/2004
P blackburni
yes
yes
162
Lake Gilles (north)
32º 44’ S, 136º 54’ 20’’ E
02/04/2004
P blackburni
yes
yes
yes
163
Lake Gilles (north)
32º 44’ S, 136º 54’ 20’’ E
02/04/2004
P blackburni
yes
yes
yes
164
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
yes
166
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
167
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
168
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
169
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
yes
171
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
yes
172
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
yes
175
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
176
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
yes
177
Lake Gairdner (south)
32º 18’ 20’’ S, 135º 51’ E
02/04/2004
P whelani
yes
yes
yes
178
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
180
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
185
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
190
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
194
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
197
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
199
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
200
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
201
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
202
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
203
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
204
Lake Everard (south)
31º 36’ S, 135º 25’ 40’’ E
03/04/2004
P whelani
yes
yes
yes
206
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
yes
yes
207
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
yes
yes
yes
yes
yes
yes
Code
Lake
Coordinates
Date of collection
Species
16S
208
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
209
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
yes
210
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
yes
213
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
215
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
yes
220
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
yes
222
Lake Everard (north)
31º 07’ S, 135º 19’ 20’’ E
03/04/2004
P whelani
yes
yes
yes
225
Lake Hart
31º 013’ 30’’ S, 136º 24’ E
03/04/2004
P whelani
yes
yes
yes
229
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
yes
230
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
231
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
yes
232
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
yes
233
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
yes
234
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
yes
235
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
yes
236
Lake Finniss
31º 43’ S, 136º 50’ E
04/04/2004
P whelani
yes
411 412
18S
coxIII yes
yes yes yes
yes
yes yes
CoxIII excluding 163 P. australis corpulenta yes 0.02 0.52 0.04 blackburni-1 blackburni-2 yes 0.04 0.22 0.20 blackburni-2 blackburni-1 yes 0.09 0.22 0.42 P. whelani pulchra yes 0.11 0.63 0.18 COXIII considering 163 as blackburni-1 P. australis corpulenta yes 0.02 0.52 0.04 blackburni-1* blackburni-2 no 0.09 0.23 0.40 blackburni-2 blackburni-1* yes 0.09 0.23 0.42 P. whelani pulchra yes 0.11 0.63 0.18 16S P. australis corpulenta yes 0.16 0.96 0.16 blackburni-1 blackburni-2 yes 0.07 0.45 0.16 blackburni-2 mendacia yes 0.07 0.37 0.18 P. whelani corpulenta yes 0.26 1.46 0.18 MATRIX 1 excluding 163 P. australis corpulenta yes 0.01 0.24 0.04 blackburni-1 blackburni-2 yes 0.01 0.10 0.13 blackburni-2 blackburni-1 yes 0.03 0.10 0.27 P. whelani corpulenta yes 0.04 0.34 0.12 MATRIX 1 considering 163 as blackburni-1 P. australis corpulenta yes 0.01 0.24 0.04 blackburni-1* blackburni-2 no 0.03 0.10 0.27 blackburni-2 blackburni-1* yes 0.03 0.10 0.27 P. whelani corpulenta yes 0.04 0.34 0.12 MATRIX 2 excluding 163 P. australis blackburni-1 blackburni-2 P. whelani
blackburni-1 yes 0.08 0.84 0.10 blackburni-2 no 0.04 0.22 0.19 blackburni-1 yes 0.05 0.22 0.23 blackburni-1 yes 0.16 1.07 0.15 MATRIX 2 considering 163 as blackburni-1
P. australis blackburni-1* blackburni-2 P. whelani 415
View publication stats
blackburni-1* blackburni-2 blackburni-1* blackburni-1*
yes no yes yes
0.08 0.05 0.05 0.16
0.83 0.23 0.23 1.07
0.10 0.23 0.23 0.15
(Liberal)
P ID
P ID (Strict)
Intra/Inter
Closest
Closest Species
Species
Inter Dist -
MATRIX
Intra Dist
Table S2: Species Delimitation results above the Bayesian Inference trees. Only species with more than one sample available are shown.
Monophyly
413 414
0.93 0.85 0.37 0.93
0.99 0.95 0.72 0.98
0.93 0.78 0.38 0.93
0.99 0.93 0.72 0.98
0.94 0.92 0.86 0.93
0.98 0.97 0.95 0.98
0.93 0.88 0.45 0.95
0.99 0.96 0.81 0.98
0.93 0.84 0.45 0.95
0.99 0.95 0.81 0.98
0.96 0.91 0.84 0.94
0.99 0.97 0.94 0.98
0.96 0.90 0.84 0.94
0.99 0.97 0.94 0.98
