Download Supplementary materials Information on primers PCR amplifications

Supplementary materials
Information on primers
PCR amplifications were performed with four different combinations of seven primers. In
two of these combinations, universal primers were used, whereas in the other two, the
primers were elephantid-specific. The universal primers were L14724, L14979 and
H15149 (Irwin et al. 1991). Two of the elephantid-specific primers involved were
Elcytb65 and Elcytb320 (Yang et al. 1996), while the other two elephant-specific primers
(Elcyt-4L and Elcyt-4R: see Fig. 1) were designed for this study using elephant and
mammoth sequences available in GenBank. The Elcytb65/Elcytb320 combination
amplified a 228 bp fragment (see Fig. 1) residing within the 425 bp fragment produced by
the universal primer pair L14724/H15149, while the Elcyt-4L/Elcyt-4R pair amplified a
56 bp fragment (see Fig. 1) residing within the 170 bp fragment produced by the
universal primer pair L14979/H15149. The 228 bp fragment and the 56 bp fragment
overlap over a stretch of 16 bp (see Fig. 1). Thus, the length of the combined amplified
DNA fragments is 268 bp.
PCR strategy, sequencing and length of sequences retrieved
Table 1 summarizes the number of DNA extractions performed and the number of
successful WGA-treatments and PCR amplifications. Slightly more than 50% of the
DNA extractions were successful in the WGA-treatments as documented by the presence
of DNA smears, when aliquots were run on a 1% agarose gel stained with Ethidium
Bromide.
DNA extracts with or without WGA treatment were amplified using nested PCR. For
each DNA extract, one PCR amplification was initially performed with the primer
combination L14724/H15149 and another with the primer combination L14979/H15149.
The PCR product (425 bp) of the L14724/H15149 combination served as a template in
the nested PCR using the Elcytb65/Elcytb320R (228 bp) pair of primers. The PCR
product (170 bp) of the L14979/H15149 combination served as the template in the PCR
using the pair of primers Elcyt-4L/Elcyt-4R (56 bp). The non-WGA treated DNA extracts
produced no positive results. For the WGA-treated Tilos, Cyprus, and Iraq DNA extracts,
both the nested PCR targeting the 228 bp fragment and the one targeting the 56 bp
fragment produced positive results. For the WGA-treated DNA extract from Crete only
the nested PCR targeting the 56 bp fragment was positive. In the sequencing procedure,
both strands of the PCR products were sequenced. Since the 228 bp and the 56 bp
fragments overlap with each other (see Fig. 1), and the poor quality stretches of the
retrieved sequences were discarded, the final sequence lengths were 258, 252, and 256 bp
for Tilos, Cyprus and Iraq, respectively. For the Cretan sample, only the 56 bp fragment
was sequenced, yielding 43 bp of quality sequence.
Exclusion from analysis of two published Mammuthus sequences:
Two M. primigenius sequences, U23738 (ht3 in Orlando et al. 2006) and U23739, were
not included in the phylogenetic analysis of Poulakakis et al. (2006) because of concerns
regarding their authenticity. The M. primigenius U23738 sequence terminates only 4
bases after the diagnostic position G315 (see Fig. A in Orlando et al. 2006) and this raises
doubts about its quality since the ends of PCR-sequenced fragments often sequence
poorly. Moreover, the two sequences, although they are supposed to have originated from
Mammuthus, do not cluster together in the analysis of Yang et al. (1996). One of them
(U23738) is placed within the Elephas clade, while the other (U23739) is placed within
the Mammuthus clade.
Exclusion of Loxodonta sequences for the taxonomic diagnosis:
There is a long tradition of paleontogical work on the pygmy elephantid fauna from the
Mediterranean islands that dates back to the late 1800’s (Osborn 1942; Maglio 1973). On
these islands, many elephantid fossil remains have been found, but none of them have
been assigned to the genus Loxodonta. It is true that elephant species are often difficult to
distinguish, but it is not difficult to distinguish fossil remains belonging to the three
genera, Elephas, Loxodonta, and Mammuthus using molars and cranial features (Osborn
1942 and Maglio 1973). Since crania are not known from Crete, only molars are relevant
in this context. No molars from Crete or any of the Mediterranean islands were ever
assigned to Loxodonta (Bate 1907; Mol et al. 1996). This genus reached North Africa
only in the Middle-Late Pleistocene (Ambrosetti 1968, Mol et al., 1996) and was never
found in Europe or Asia (Maglio 1973). The only North African sample (L. atlantica) has
been found in Algeria, which is a long way from Crete. The geographic distribution of
this genus coupled with the fact no Loxodonta remains have ever been found in any
Mediterranean islands, although there are many elephantid fossils from these islands,
suggests that Loxodonta is not a plausible contender for ancestry of the pygmy
Mediterranean elephantids.
Phylogenetic analyses
Following the suggestions of Binladen et al. (2006) and Orlando et al. (2006) a new set of
sequences was compiled and additional phylogenetic analyses (Bayesian Inference and
Maximum Likelihood) were performed. Besides including all previously used elephantid
haplotypes (Poulakakis et al. 2006), the new data set included recently published
Mammuthus sequences, the Elephas and Loxodonta haplotypes suggested by Orlando et
al. (2006), and all other elephantid haplotypes available in GenBank. There are 96 entries
in Genbank that contain sequence homologous to the 43 bp fragment of the Cretan
sample. In total, 103 sequences (including three outgroups) were used in the analysis
(Table 2, Fig. 2). The full length DNA sequence for each of these 96 entries was used,
therefore, the sequence length of the haplotypes included in the analyses ranged from
approximately 17,000 bp (whole mtDNA sequence of two Elephas, three Loxodonta, and
three Mammuthus entries) to 43 bp (Cretan bone sequence). The outgroups in all
phylogenetic analyses were those used in Poulakakis et al. (2006).
The best-fit model of DNA substitution and the parameter estimates used for tree
reconstruction were chosen by performing Akaike Information Criterion (AIC, Akaike
1974) in Modeltest v.3.06 (Posada & Crandall 1998), since this method is able to
simultaneously compare multiple nested or non-nested models, assess model selection
uncertainty, and allow for the estimation of phylogenies and model parameters using all
available models (Posada and Buckley 2004). This test indicated that the General Time
Reversible (GTR) model + I (Rodriquez et al. 1990) (I=0.7530) was a significantly better
fit than the other models.
Bayesian inference (BI) was performed with the software MrBayes (v3.1;
Ronquist and Huelsenbeck 2003). The analysis was run with four chains for 2*106
generations and the current tree was saved every 100 generations. A majority rule
consensus tree ('Bayesian' tree) was then calculated (after discarding 103 trees as burnin)
from the posterior distribution of trees, and the posterior probabilities calculated as the
percentage of samples recovering any particular clade. One more independent Bayesian
analyses was run so that global likelihood scores, individual parameter values, topology,
and nodal support could be compared to check for local optima.
Maximum Likelihood (Felsenstein 1981) phylogenetic analysis on the newly
compiled data set was conducted using PAUP* v.4.0b10 (Swofford 2002). Statistical
support of nodes was assessed with 100 bootstrap (Felsenstein 1985) replicates. The
Shimodaira–Hasegawa test was used statistically to compare alternative phylogenetic
hypotheses. Where appropriate, topological constraints were generated and compared
with our optimal topology using the Shimodaira–Hasegawa (1999) SH-test. The
assignment of the Cretan pygmy elephantid within the Mammuthus lineage was avouched
by the results of the SH test (p=0.003)
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Table 1
Summary of the laboratory procedures. The table gives the number of samples (each
sample is a different portion of a single bone) from each locality, the number of
successful DNA extracts of which WGA treatment (WGA column), and the number
of successful PCRs for each elephantid-specific primer combination (PCR column).
Extraction1
WGA
Phenol
Extracts of phenol
Elcytb65/Eclyct320R
Elcyt-4L/Elcyt-4R
length2
Tilos
6 samples
3
3
3
258 bp
Cyrpus
6 samples
5
5
5
252 bp
Iraq
6 samples
3
3
3
256 bp
Crete
6 samples
2
0
2
43 bp
Positive PCRs
Sequence
Specimens
1
Phenol extraction protocol: see Hagelberg & Clegg 1991.
The products of two different PCRs were sequenced for each sample. The sequences retrieved for
each sample from the two different PCRs were identical.
2
Table 2
List of haplotypes used in phylogenetic analyses [species names,
abbreviations used in the tree in Fig. 2, sequence length (bp) and GenBank
accession numbers].
A/A
Species
Code
Length (bp)
Acc No
1
E. maximus
Em1
1112
AF132522
2
E. maximus
Em2
1137
AB002412
3
E. maximus
Em3
1137
D50844
4
E. maximus
Em4
1005
D83048
5
E. maximus
Em5
1100
AF132526
6
E. maximus
Em6
1137
D50846
7
E. maximus
Em7
1116
AF132520
8
E. maximus
Em8
340
Y13886
9
E. maximus
Em9
340
Y13887
10
E. maximus
Em10
340
Y13884
11
E. maximus
Em11
340
Y13888
12
E. maximus
Em12
340
Y13882
13
E. maximus
Em13
340
Y13883
14
E. maximus
Em14
340
Y13885
15
E. maximus
Em15
340
Y13881
16
E. maximus
Em16
294
DQ236095
17
E. maximus
Em17
307
AF298855
18
E. maximus
Em18
852
AF132523
19
E. maximus
Em19
837
AF132525
20
E. maximus
Em20
16902
NC005129
21
E. maximus
Em21
16902
DQ316068
22
E. maximus
Em22
228
AY424307
23
E. maximus
Em23
1117
AF132521
24
E. maximus
Em24
849
AF132524
25
L. cyclotis
Lc1
1961
AY359266
26
L. cyclotis
Lc2
1961
AY359278
27
L. cyclotis
Lc3
1961
AY359267
28
L. cyclotis
Lc4
1961
AY359269
29
L. cyclotis
Lc5
1961
AY359268
30
L. cyclotis
Lc6
1961
AY359272
31
L. cyclotis
Lc7
1961
AY359270
32
L. cyclotis
Lc8
1961
AY359279
33
L. cyclotis
Lc9
1961
AY359265
34
L. cyclotis
Lc10
1961
AY359271
35
L. cyclotis
Lc11
1961
AY359273
36
L. cyclotis
Lc12
1961
AY359274
37
L. cyclotis
Lc13
1961
AY359275
38
L. cyclotis
Lc14
1961
AY359276
39
L. cyclotis
Lc15
1961
AY359277
40
L. cyclotis
Lc16
1110
AF132527
41
L. cyclotis
Lc17
1089
AF132528
42
L. cyclotis
Lc18
1119
AF132529
43
L. cyclotis
Lc19
1124
AF132530
44
L. cyclotis
Lc20
1135
AF517566
45
L. cyclotis
Lc21
1961
AY741079
46
L. africana
La1
1961
AY742802
47
L. africana
La2
1961
AY742799
48
L. africana
La3
1960
AY742800
49
L. africana
La4
1960
AY742801
50
L. africana
La5
16866
AJ224821
51
L. africana
La6
340
AJ132952
52
L. africana
La7
340
AJ132954
53
L. africana
La8
340
AJ132955
54
L. africana
La9
16866
NC 000934
55
L. africana
La10
307
AF298857
56
L. africana
La11
1137
D84150
57
L. africana
La12
1137
D84151
58
L. africana
La13
1137
D84152
59
L. africana
La14
16909
DQ316069
60
L. africana
La15
1961
AY741067
61
L. africana
La16
1961
AY741068
62
L. africana
La17
1961
AY741069
63
L. africana
La18
1961
AY741070
64
L. africana
La19
1961
AY741071
65
L. africana
La20
1961
AY741072
66
L. africana
La21
1961
AY741073
67
L. africana
La22
1961
AY741074
68
L. africana
La23
1961
AY741075
69
L. africana
La24
1961
AY741076
70
L. africana
La25
1961
AY741077
71
L. africana
La26
1961
AY741078
72
L. africana
La27
1961
AY741080
73
L. africana
La28
1961
AY741081
74
L. africana
La29
1960
AY741320
75
L. africana
La30
1960
AY741321
76
L. africana
La31
1960
AY741322
77
L. africana
La32
1960
AY741323
78
L. africana
La33
1960
AY741324
79
L. africana
La34
1960
AY741325
80
L. africana
La35
1960
AY741326
81
L. africana
La36
1961
AY741327
82
L. africana
La37
1961
AY741328
83
L. africana
La38
1961
AY741329
84
M. primigenius
Mp1
1137
D50842
85
M. primigenius
Mp2
1005
D83047
86
M. primigenius
Mp3
306
AF517567
87
M. primigenius
Mp4
331
U79411
88
M. primigenius
Mp5
307
AF154864
89
M. primigenius
Mp6
139
AF309074
90
M. primigenius
Mp7
139
AF309073
91
M. primigenius
Mp8
16842
DQ316067
92
M. primigenius
Mp9
128
DQ318543
93
M. primigenius
Mp10
140
DQ318541
94
M. primigenius
Mp11
139
NC 007596
95
M. primigenius
Mp12
16770
DQ188829
96
M. primigenius
Mp13
242
S72502
97
M. creticus
CRETE
43
n/c
98
Elephas sp.
TILOS
258
DQ319147
99
E. cyrpiotes
CYPRUS
252
DQ319148
100
E. m. asurus
IRAQ
256
DQ319149
101
Diceros bicornis
Outgroup
1140
X56283
102
Trichechus manatus
Outgroup
1005
D83050
103
Dugong dugong
Outgroup
1140
U07564