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Toward a Global Phylogeny of the “ Living Fossil“
Crustacean Order of the Notostraca
Bram Vanschoenw inkel1* 9, Tom Pinceel19, M aarten P. M . Vanhove2, Carla Denis1, M erlijn Jocque3,
Brian V. Timms4, Luc Brendonck 1
1 Laboratory of Aquatic Ecology and Evolutionary Biology, KU Leuven, Leuven, Belgium, 2 Laboratory of Animal Diversity and Systematics, KU Leuven, Leuven, Belgium,
3 Royal Belgian Institute o f Natural Sciences, Brussels, Belgium, 4 Australian Museum, Sydney, New South Wales, Australia
Abstract
Tadpole shrim p (Crustacea, Notostraca) are iconic inhabitants o f tem porary aquatic habitats w orldw ide. Often cited as prime
examples o f evolutionary stasis, surviving representatives closely resemble fossils older than 200 mya, suggestive o f an
ancient origin. Despite significant interest in the gro u p as 'living fossils' the taxonom y o f surviving taxa is still under debate
and both the phylogenetic relationships am ong different lineages and the tim in g o f diversification remain unclear. We
constructed a m olecular phylogeny o f the Notostraca using model based phylogenetic methods. Our analyses supported
the m onophyly o f the tw o genera Triops and Lepidurus, although for Triops support was weak. Results also revealed high
levels o f cryptic diversity as well as a peculiar biogeographic link between Australia and North America presumably
mediated by historic long distance dispersal. We concluded that, although some present day tadpole shrim p species closely
resemble fossil specimens as old as 250 mya, no m olecular support was found for an ancient (pre) Mesozoic radiation.
Instead, living tadpole shrim p are most likely the result o f a relatively recent radiation in the Cenozoic era and close
resemblances between recent and fossil taxa are probably the result o f the highly conserved general m orphology in this
gro up and o f homoplasy.
C ita tio n : Vanschoenwinkel B, Pinceel T, Vanhove MPM, Denis C, Jocque M, e t al. (2012) Toward a Global Phylogeny o f th e "Living Fossil" Crustacean O rder o f the
Notostraca. PLoS ONE 7(4): e34998. doi:10.1371/journal.pone.0034998
E d ito r: Keith A. Crandall, Brigham Young University, United States of America
R eceiv ed Septem ber 14, 2011; A c c e p te d March 8, 2012; P u b lish e d April 18, 2012
C o p y rig h t: © 2012 Vanschoenwinkel e t al. This is an open-access article distributed under th e term s of th e Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided th e original author and source are credited.
F u n d in g : BV currently holds a postdoctoral fellowship with th e Research Foundation Flanders (FWO-Vlaanderen). This research was funded by th e fund for
scientific research Flanders. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
C o m p e tin g In te re s ts : The authors have declared th a t no com peting interests exist.
* E-mail: [email protected]
9 These authors contributed equally to this work.
opportunistic pred ato rs, surviving unfavorable conditions such as
dro u g h t or frost as d o rm a n t eggs in the sedim ent [15]. W hile b o th
genera can b e m orphologically distinguished b y the presence o f a
supra-anal p late in Lepidurus, tadpole shrim p are know n to display
substantial levels o f w ithin-species phenotypic plasticity. In
c ontrast w ith a highly conserved general m orphology, notostracans typically show considerable phenotypic variation w ithin
lineages a n d populations m aking it difficult to distinguish species
a n d subspecies [13,16], W ith in Triops, for instance, the absence o f
second m axillae is a good diagnostic ch ara cte r to distinguish T.
australiensis (Spencer & H aii, 1895) a n d T. longicaudatus from T.
cancriformis a n d T. granarius (Lucas, 1864). H ow ever, v a riation in
o th er m orphological traits such as telson a rm a tu re , n u m b e r o f
segm ents a n d shape o f the dorsal org an is often less consistent.
C onsequently, various au th o rs suggest th a t m orphological tax o n ­
om y should be h an d led w ith utm ost care, considering large
num bers o f individuals [13,17,18], F u rth e r com plicating system ­
atica are the different m odes o f rep ro d u ctio n th a t evolved w ithin
the notostracans. D e p en d in g on species a n d p opulation, gonochoric (separate sexes), h erm a p h ro d itic as well as androdioecious
populations (containing herm ap h ro d ites a n d a p ro p o rtio n o f
males) a re found [19,20],
In the 1950’s, L inder [17] a n d L onghurst [21] revised the alp h a
taxonom y o f the N otostraca red u cin g the n u m b e r o f accepted
nom inal species from m ore th a n fifty to four in Triops a n d five in
Introduction
T ad p o le shrim p (C rustacea, N otostraca) com prise one living
family, the T riopsidae, including two genera: Triops S chrank, 1803
a n d Lepidurus L each, 1819. M em bers o f this group are often
considered p rim e exam ples o f evolutionary stasis [1-3] w ith the
oldest confirm ed n o to stracan fossils d atin g back as far as the
U p p e r C arboniferous p erio d [4], Alleged to have rem ain ed
virtually u n c h an g e d du rin g a n evolutionary tim efram e o f m ore
th a n 250 m illion years, som e surviving m em bers o f this ancient
crustacean o rd e r are frequently referred to as living fossils. T h e
c o n tem p o rary Triops cancrifornris (Bosc, 1801), for instance, is
regularly cited as the oldest living species because o f its striking
resem blance to late P erm ian [5] a n d early Triassic fossils [6-10],
Sim ilarly, fossils from the late C retaceous have b e en identified as
the living species Triops longicaudatus (le C onte, 1846) while o th er
fossils from sim ilar deposits o f the sam e age w ere classified in the
extant genus Lepidurus [11,12]. T h e long e volutionary history o f the
group, to g eth er w ith its p resu m ed ‘living fossii’ status a n d wide
c u rre n t distribution ranges, a re suggestive o f a n an cien t radiation.
T ad p o le shrim p have a near-w orldw ide distribution w ith
highest abu n d an ces in arid a n d sem i-arid regions [13]. B oth
g enera are typical for freshw ater, a n d occasionally saline,
tem p o rary aquatic habitats although certain Lepidurus species have
b e en recorded in p e rm a n e n t lakes in arctic regions [14], T h ey are
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Lepidurus. Based o n m olecular phylogenies, how ever, it was
recently p ro p o sed to recognise m ore species, even though
m olecular divergence am o n g clades is often quite low [22]. A t
this tim e th ere are six accepted Triops species w ith p resum ably four
ad ditional lineages deserving species status [22] a n d approxim ately
8 Lepidurus species [3,16].
C u rren d y , m olecular phylogenetic research is alm ost exclusively
lim ited to representatives o f Triops, b u t see [3,23-24], a n d largescale studies considering large n u m b ers o f populations over a
significant p ro p o rtio n o f species distributions are ra re [22,25].
E xcept for a first exploratory study by M a n to v an i a n d cow orkers
[26], no a tte m p t has b e en m ade to reco n stru ct the phylogenetic
relationships w ithin this group a t a global scale a n d considering
m ost recognised species a n d subspecies. T h e m ain reason is th a t in
co n trast to the well studied T. cancriformis a n d T. mauritanicus (Ghigi,
1921) populations in E urope, m aterial from less intensively studied
continents such as A frica, S o u th A m erica a n d A ustralia was not
available.
H ere, we use D N A sequence d a ta from two m itochondrial
genes (the p ro tein coding C ytochrom e c O xidase subunit I or C O I
a n d 12S rR N A ) to elucidate the evolutionary relationships
betw een notostracans from 60 different p opulations a ro u n d the
globe. A vailable sequence inform ation is com bined w ith a large
n u m b e r o f newly obtain ed sequences, featuring several recendy
discovered A ustralian n o to stracan lineages. D ue to the large scale
o f the study a n d the isolated n a tu re o f the considered populations,
it is reasonable to assum e th a t gene flow will be extrem ely low a n d
m atrilineal m arkers will suffice to gain insight in phylogenetic
relationships a t this scale.
Based on this dataset w e evaluate the m onophyly o f b o th
recognised genera, discuss the biogeography a n d phylogenetic
relations a m o n g e x ta n t lineages a n d evaluate the p otential
presence o f cryptic species in the light o f the often controversial
species delineations in notostracans. Since discussion o f species
status requires a taxonom ic revision including m orphological
studies, ra th e r th a n going into the taxonom ic status o f closely
related species com plexes, we focus o n m ajor evolutionary
lineages. Finally, we use m olecular clocks to investigate w h eth er
gene genealogies are consistent w ith a n a n cien t (pre) M esozoic
rad iatio n suggested b y fossil rem ains.
C a n a d a while the m ax im u m 12S distance o f 27.8% was calculated
betw een South A frican T. granarius a n d T. cancriformis from
Belgium . A n overview o f the average, m inim um a n d m axim um
K 2 P distances w ithin a n d am o n g m ain n o to stracan lineages is
pro v id ed in T ab le 1 a n d T ab le S2. E stim ates o f divergence tim es
betw een m ain lineages are provided in T ab le S3. A n additional
genetic distance m atrix calculated using u n co rre c te d p distances is
pro v id ed in T ab le S4.
O verall, phylogenetic analysis o f b o th m itochon drial genes
using five different m ethods o f phylogenetic reconstruction
resulted in sim ilar topologies (Figure 2) w hich w ere confirm ed in
trees based on com bined analysis o f the tw o genes (Figure SI).
T h e m onophyly o f Lepidurus is confirm ed in all trees, except in
the Q P tree (62%) for the C O I gene a n d in the Q P (66%) a n d Bí
trees (68%) for the 12S gene. F u rth e rm o re , phylogenetic analysis
o f the unique am ino acid sequences tran slated from the full C O I
dataset (carried o u t in P hyM L a n d M rB ayes u n d e r the M tM a m
[27] + I m odel (as selected w ith the help o f P ro tT est v.2.4 [28];
results n o t shown) supported m onophyly o f the genus.
Phylogenetic reconstruction yielded no statistical support for
m onophyly o f the n om inal genus Triops n o r for a n alternative
positioning o f its lineages. O nly M L (52%), N J (40%) a n d Bí
(posterior p robability o f 80) analyses o f the C O I gene provide
w eak sup p o rt for the m onophyly o f Triops. In the absence o f a
resolved topology, we resorted to con strain t analyses to form ally
test the hypothesis o f m onophyly.
C o n stra in t analyses enforcing m onophyly o f all Triops rep resen ­
tatives, w ere c o n d u cted for M P a n d M L trees. B oth the K ishinoH asegaw a test a n d n o n p a ram etric T em p leto n (W ilcoxon signedranks) a n d w inning-sites (sign) tests identified the co nstrained C O I
a n d 12S trees, w ith a length o f 1278 a n d 411 respectively, as
significandy m ore parsim onious (p< 0.0001) th a n the u n co n ­
strained M P tree w ith a length o f 1448 a n d 455 m u tatio n al events.
W h en co m p arin g the con strain ed M L C O I a n d 12S trees (—In
7250 a n d 2478 respectively) to the unco n strain ed M L trees (—In
7258and 2490 respectively) in Paup*, the K ishino-H asegaw a test
significantly (P < 0.05) supported the con strain ed tree as the m ost
likely scenario. K 2 P distances betw een the g enera also largely
exceed those w ithin (T able 1). B ased o n the w hole range o f
confirm ed m olecular clocks in invertebrates a (pre) M esozoic
rad iatio n as suggested by fossil rem ains is highly im plausible.
A ccording to sta n d ard m olecular clocks used for crustaceans (1.4—
2.8% m ya *) initial diversification in the N otostraca started
approxim ately 2 5 .5 -1 2 .7 5 m ya. E ven according to the slowest
m olecular clocks, b o th g en era p resum ably did n o t diverge before
29.75 m ya (based on a C O I clock o f 1.2% m y a - 1 ) o r before
55.6 m ya (based o n a 12S clock o f 0.5% m ya ^
In Lepidurus, the basal position o f L. apus lubbocki is supported by
all phylogenetic searches in the C O I tree. T h e A ustralian Lepidurus
lineage, w hich based on m orphological traits was traditionally
considered a subspecies o f L. apus (L. apus viridis) em erged as a sister
species o f a clade c o ntaining the N o rth A m erican L. couesii, L.
arcticus a n d the E u ro p e a n Lepidurus lineages previously identified as
L. couesii. T h e m onophyly o f the subspecies o f the presum ably
w idespread L. apus, hence, could n o t be confirm ed.
Analyses confirm the m onophyly o f five m ain evolutionary
lineages w ithin the genus Triops'. T. granarius, T. cancriformis, T.
mauritanicus a n d a fourth lineage c o ntaining T. longicaudatus a n d T.
newberryi. T h e fifth lineage com prised haplotypes belonging to a
recendy discovered Triops sp. p o p u latio n from the saline Lake
C arey in W estern A ustralia. T h e m onophyly o f the various
A ustralian lineages identified as T. australiensis, how ever, could not
be confirm ed although there was w eak support for this clade in the
C O I dataset. As a result, this tax o n could be paraphyletic. W ithin
Results
A n overview o f the 89 Triops a n d Lepidurus populations included
in o u r analyses a n d th eir localities is p rovided in T ab le S I a n d
plo tted in Figure 1. D etailed in form ation a b o u t the know n
distribution o f different N o to stra ca n lineages c an be consulted in
T e x t S I.
C h a ra c te ris tic s o f t h e m i t o c h o n d r i a l DNA s e q u e n c e s a n d
align m ent
41 C O I a n d 53 12S sequences w ere aligned to gether w ith 123
ad ditional C O I a n d 74 12S sequences d raw n from G enB ank a n d
trim m ed to a length o f 568 b p a n d 328 bp, respectively. E xcluding
the outgroup, the com plete C O I a n d 12S datasets com prised 78
a n d 72 un iq u e haplotypes, respectively. T h e C O I alignm ent
co n tain ed 240 variable sites (42%) o f w hich 223 (39%) w ere found
to b e p arsim ony inform ative while the 12S alig n m en t con tain ed
111 variable sites (34%) o f w hich 97 (29%) w ere parsim ony
inform ative.
G e n e tic d i s t a n c e s a n d m i t o c h o n d r i a l DNA d iversity
A C O I m axim um K 2 P distance o f 35.7% was recorded
betw een a n A ustralian T. australiensis haplotype a n d L. couesii from
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.81
.8 2
.2 9
7 2 7.17 3 x 74
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T. m a u rita n icu s
T. g ra n a riu s
T. a u stra lie n sis
T. lo n g ica u d a tu s
T. n e w b e rry i
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Figure 1. O verview o f th e general habitus and th e geographic d istrib ution o f Notostraca taxa and populations included in this
study. (A-B) E x am p les o f t a d p o le s h rim p re p re s e n ta tiv e s b e lo n g in g to th e g e n e r a L ep id uru s a n d Triops, re sp ec tiv e ly , illu stra tin g th e s u p ra a n a l p la te :
a p o s te rio r e x te n s io n o f th e te ls o n c h a ra c te ris tic fo r Lepidurus. (A) Lep id uru s a pu s (p h o to : J a c q u e s Pages), (B) Triops c a n c rifo rm is (p h o to : Aline
W aterk ey n ), sca le b a r = 2 cm ; (C) G e o g ra p h ic d is trib u tio n o f in v e s tig a te d N o to s tra c a p o p u la tio n s . Locality n u m b e rs c o rre s p o n d w ith p o p u la tio n
e n trie s in T a b le S1.
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 3 4 9 9 8 .g 0 0 1
Triops, T. cancriformis a n d T. mauritanicus em erged as two sister
groups. T h e m in im u m genetic distance b etw een these two clades
(11.0%) was sm aller th a n the genetic distances to the o th er m ain
Triops lineages (17.9-23.8% ). T h e Triops p o p u latio n from Lake
C arey in W estern A ustralia did not cluster together w ith o th er
A ustralian populations b u t instead em erged as a distinct lineage.
C O I a n d 12S sequences diverged 12.3-17.9% a n d 7.4—11.1%
betw een haplotypes from Lake C arey a n d T. australiensis
specim ens, respectively. In the 12S analysis, B í, M L , N J a n d Q P
trees place the A m erican Triops clade, w hich contains specim ens
m orphologically identified as T. longicaudatus a n d T. newberryi, as an
evolutionary sister o f the A ustralian T. sp. clade from Lake C arey.
K 2 P values furth er justify this position. M ax im u m genetic
divergences b etw een Lake C arey a n d T. australiensis haplotypes
o f 17.9% a n d 11.1% in the C O I a n d 12S gene, respectively, w ere
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higher th a n the divergences o f 16.3% a n d 8.6% identified betw een
the L ake C a rey species a n d T. longicaudatus.
Discussion
W e reco n stru cted the first large-scale m olecular phylogeny o f
the prim itive crustacean o rd e r N otostraca, w hich is ch aracterised
by m orphological stasis th ro u g h o u t its fossil re co rd [1,4,8,9], Based
on results from the analysis o f two m itochondrial genes (C O I a n d
12S rD N A ), w e discuss the phylogenetic relationships w ithin this
enigm atic group in w hich m orphological tax o n o m y is com plicated
by phenotypic variability w ithin a n d low variability am o n g
nom inal species.
A p relim in ary a tte m p t to resolve phylogenetic relations in the
N o to straca based o n 12S a n d 16S rD N A m arkers was p erform ed
by M a n to v an i a n d cow orkers [26], Splitting the genus Triops as
suggested b y these authors, how ever, is likely to be unjustified since
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freshw ater invertebrates in o th er regions [37,38] are likely to have
b e en involved.
O verall, o u r m olecular d a ta suggest th a t the taxonom y o f
E u ro p ea n a n d A ustralian Lepidurus lineages is in need o f revision.
Results confirm the suggestion o f Fryer [39] th a t the A ustralian
Lepidurus lineage is not a subspecies o f L. apus a n d m ost likely
deserves species status, as the two tax a are paraphyletic a n d differ
14.7-17.5% at the C O I gene. T h e re m a in in g lineages in L. apus (L.
apus lubbocki a n d L. apus apus), p ro b ab ly also represent different
species as they are separated b y a genetic distance o f 2 2 .1 -2 3 .6 %
a n d do n o t form a m onophyletic group. A ccording to accepted
m olecular thresholds, the species status o f L. arcticus a n d the
E u ro p ea n L. sp. clade, w hich was previously considered conspecific to the A m erican L. couesii, [3] is confirm ed.
Table 1. M olecular divergence (m inim um , m axim um and
average Klmura 2-param eter distances) w ith in main
notostracan lineages based on COI and 12S rRNA genes.
S p e c ie s
COI (%)
12S (%)
71 australiensis
0.4-14.2 (9.6)
0.0-8.0 (3.6)
T. cancriform is
0.0-0.9 (0.4)
0.0-2.3 (0.8)
T. m a urita n icus
0.9-10.4 (7.0)
0.3-5.1 (2.4)
T. lon g icau d atu s
2.2-5.0 (3.9)
0.7
T. n e w b e rryi
0.2-1.6 (0.8)
T. gran a rius
21.1
L. a. apus
0.2
L. sp
0.2-1.6(0.7)
4.1-11.3 (8.5)
-
P h y l o g e n e t i c r e la tio n s in T riop s
0.6
L. virid is
Triops
0.0-30.5 (17.1)
1.0-25.6 (14.7)
Lepidurus
14.6-25.9 (16.8)
5.3-10.8 (7.7)
B oth analyses o f a relatively ra p id (CO I) a n d a m ore slowly
evolving m itochondrial m ark e r (12SrD NA), consistently recovered
a com b-like tree depicting hypothetical phylogenetic relations
am o n g the four m ain Triops lineages ( T. granarius, T. australiensis, T.
cancrformis-mauritanicus, T. longicaudatus-newberryi). T h e possibility o f
radiation, as suggested for o th er b ra n ch io p o d crustaceans [40] a n d
ra p id diversification in Triops early in its evolutionary history,
hence, c an n o t be excluded. In terc o n tin e n ta l dispersal a n d
subsequent isolation follow ed by genetic differentiation u n d e r
lim ited gene flow alm ost certainly led to spéciation in the four
m ain Triops lineages, w hich are largely restricted to different
biogeographic regions. D ivergence o f the fifth lineage, T. sp., in
tu rn , p resum ably results from a un iq u e h a b ita t shift from
freshw ater to saline habitats.
Based on m olecular clocks, T. cancriformis a n d T. mauritanicus
m ost likely diverged betw een 2 .6 -1 2 .4 m ya confirm ing the
estim ate by K o rn a n d cow orkers [41] based o n 16S rD N A
suggesting a poten tial link w ith the M essinian Salinity Crisis at the
e n d o f the M iocene (5-6 mya). T ectonic activity a ro u n d the
G ib ra ltar straight, isolating the M e d ite rran e a n from the A tlantic
O cean , a n d low rainfall resulted in strong v a riation in sea level
including n e ar com plete drying o f the basin [42]. C lim ate
fluctuations, due to loss o f the buffering capacity o f the
M ed ite rran e a n , m ay have led to c o n tractio n o f suitable Triops
h a b ita t a n d a split b etw een T. mauritanicus a n d T. cancriformis
th ro u g h vicariance. T h e clade form ed by T. cancriformis, w hich,
a p a rt from its m ostly E u ro p ea n origin, also encom passes a
Ja p a n e se p opulation, is characterised b y a large n u m b e r o f closely
related haplotypes. As a result, M a n to v an i a n d cow orkers [43]
c oncluded th a t this tax o n did n o t c ontain cryptic species. Low
nucleotide a n d haplotype diversity over a w ide geographical range
(E urope a n d Asia) suggests a relatively recent postglacial
colonisation o f its c u rre n t distribution a re a [25]. A grow ing
n u m b e r o f studies show th a t postglacially colonised regions are
c haracterised by low er genetic diversity [44,45]. F ro m the
b eginning o f the Q u a te rn a ry (2.4 m ya) until 10 kya ice sheets
cyclically e x p an d e d a n d receded [45]. D u rin g cold periods,
E u ro p ea n T. caliciformis populations w ere m ost likely restricted
to refugia southw ards o f the ice shelf. In contrast, cryptic diversity
was d e m o n stra ted in its sister species T. mauritanicus, found in
Ib e ria a n d N o rth A frica [22]. T h e m ore southern distribution o f
this species c an explain w hy it appears to have b e en less affected
by the Pleistocene glaciations th a n T. cancriformis in term s o f
surviving lineages. K o rn a n d cow orkers [22] recognised six
m orphologically distinguishable lineages (five o f w hich occur in
Iberia). As arg u ed by these authors, clim ate fluctuations in
southern E urope associated w ith the Pleistocene glaciations m ay
have co n trib u ted to fragm entation o f species ranges facilitating the
Statistics are only provided for taxa for which multiple sequences were
available.
doi:10.1371 /journal.pone.0034998.t001
in o u r results the m onophyly o f b o th g enera is confirm ed. As a
result, the m ain m orphological difference betw een Lepidurus a n d
Triops, the presence o f a supra-anal p late (a posteriorly directed
m edian extension o f the telson w hich is presen t in Lepidurus b u t
never in Triops; Figure IA , B), is supported as a system atically
inform ative character.
In o rd e r to discuss the poten tial species status o f the m ain
n o to stracan lineages, we will focus m ainly on C O I, w hich is the
sta n d ard m ark e r for b a rco d in g [29]. In b ra n ch io p o d crustaceans
average sequence divergences > 7 - 1 0 % at C O I [30,31,32] a n d 4 5% a t 12S [33,34] are typically considered indicative for species
level differentiation, a lthough in com bination w ith m orphological
support, species status has som etim es b e en a ttrib u te d to m o n o ­
phyletic clades w ith low er sequence divergences. M u ru g a n a n d
cow orkers [35], for instance, p ro p o sed to p ro m o te six T.
longicaudatus lineages to species level a lthough m ax im u m diver­
gence betw een these lineages observed at the 12S rR N A gene
a m o u n te d to only 1.6% . In a m ore recent p a p er, K o rn a n d
cow orkers [22] ascribed species status to six m orphologically
distinguishable T. mauritanicus lineages w hich differed only 2.9—
5.1% at the sam e gene. D e p en d in g o n the species concept used
a n d w ithout in form ation from hybridization trials confirm ing
reproductive isolation, these decisions can be considered c o n tro ­
versial.
P h y l o g e n e t i c re la tio n s in L e p id u ru s
C o m p a re d to Triops, Lepidurus has a m ore restricted distribution.
T ypical for subarctic a n d tem p erate clim ate zones, the genus is
generally replaced b y Triops in w arm er, sem i-arid a n d a rid regions.
D espite a confirm ed presence in the fossil re co rd o f the Triassic,
th ere are no reports o f cu rre n t populations in S u b -S ah a ran A frica
[36], In contrast to the situation in Triops, E u ro p ea n a n d N o rth
A m erican Lepidurus lineages did not em erge as m onophyletic
groups. T his could im ply secondary con tact b etw een N earctic a n d
P alearctic Lepidurus lineages possibly facilitated by the closer
proxim ity o f Lepidurus species ranges to n o rth e rn m igration
corridors such as the B ering Strait. Efficient dispersal in the
(sub) arctic is also suggested b y the circum arctic distribution o f the
recent L. arcticus [23]. Birds o r m am m als (e.g. caribou, moose)
w hich have b e en show n to b e im p o rta n t dispersal vectors o f
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M o lecu lar P h y lo g e n y o f a P re s u m e d Living Fossil
A COI
B 12S rRNA
C yclestheria h islo pi
77 9 3 80
94100
62 4 7 46
6 2 9 0 81
30100
L. viridis, B ro o k to n , A u s tra lia (7 7 )
L. couesii. C a n a d a (82)
iL s p .T ric a s e , Italy (8 3 )
31100
i L s p ., V eglie,Italy (8 5 )
100100
9110 1
L. s p . S a n D o n a ci, Italy (8 4 )
s p ., F ra n c a v illa F o n ta n a , Italy ( 8 6 )
s p .; V e g lie,Italy (83)
f i L arcticus, T n jo rs a rv e r, Ic e la n d (81)
L. lem m oni, M exico (8 9 )
i l L. apus apus, M a rc h e g g , A u stria (7 9 )
^ L apus apus, B erlin, G e rm a n y ( 8 8 )
■ N L apu s lu bb o cki, C a s te l P o rz ia n o , Italy (8 0 )
■ II B ain ’s Vlei, S o u th A frica (6 7 )
54100163
759195
66 0083
98100 —
ou mm
rW
V a lg a R o ck , A u s tra lia (3)
T a ustraliensis
62100
eB a la n R o ck , A u s tra lia (5)
■ ^ 5 2 87
W a lg a , B alan & B. m a n y a R ock. A u s tra lia (5)
8 2 9 9 | r■ E
B .m a n y a R o ck , A u s tra lia (1 4 )
[[(a
6 7 8 7 8 6 /* G
C ib b & T w in e R o ck , A u s tra lia (11)
T w ine R o ck , A u s tra lia (1 2 )
M arsilea , P a ro o , A u s tra lia (7)
-e iP a v n e s F ind c la y p a n , A u s tra lia (4 )
M arsilea , P a ro o , A u s tra lia (7)
r W o n b e rn a R o ck , A u s tra lia (2)
É
B a la d o n ia R o ck , A u s tra lia (6 )
99100
A fg h a n R o c k s , A u s tra lia (1 2 )
— ei U luru, A u s tra lia (1 0 )
___________ _____
T. gra na riu s
O s a k a , JJaappaa n (6 6 )
84 87 7 7 T| B a la n R o ck , A u s tra lia (5)
83 9 9 9 9
_ 981 00
80100 99
t , - 52 22
*'
40 80
1 0 0 1 0 0 99
99100
T. austra lie n sis
l006S|fn
7-
H |W a lg a R ock, A u s tra lia (3)
r i B .m a n y a R ock, A u s tra lia (1 3 )
_ r G ib b R o c k , A u s tra lia (10)
~ T w ine R o c k A u s tra lia (11)
B lo o d w o o d , P a ro o , A u s tra lia ( 8 )
;i P a y n e s F ind c la y p a n , A u s tra lia (4)
.M a r s ile a , P a ro o , A u s tra lia (7 )
r B a l a d o n ia R ock, A u s tra lia ( 6 )
I- - - - - - - l- W o n b e m a R o ck , A u s tra lia Ï2 )
—I
1 A fg h an R o c k s , A u s tra lia (12)
1
U luru, A u s tra lia (1 0 )
• M arsilea , P a ro o , A u s tra lia (7)
L ak e C a re y , A u s tra lia (1 )
S3 56 9 5
Z a c a t e c a s , M exico (6 8 )
L ak e C a re y , A u s tra lia ( 1 )
- 97 9 8
99100
T sp
7 lo ng ica u da tus
100100
51 8768
73 71
T sp .
Z a c a t e c a s . M exico (6 8 )
T. lo ng ica u da tus
F re s n o . U è A (.71)
S a n t a R o s a , U S A (7 2 )
K a n s a s , U S A (7 4 )
K a iro u an , T u n is ia (6 4 )
“ T l B a in 's Vlei, S o u th A frica (67)
andis, N a m ib ia (6 3 )
T. cancriform is
9 9 100 97
vD ¡cr96 9 0 4 4
6294 \
533356
E u ro p e
I C h ih u a h u a n d e s e r t, U S A (7 3 )
93 28100
10094
52 79 64
77 6 0
C yclestheria h islo p i
i L. viridis. B ro o k to n , A u s tra lia (77)
L. vindis, M in e g a rra , A u s tra lia (7 6 )
apu s lubbocki, C P o rz ia n o , Italy (8 0 )
•L. arcticus, T h o rs ja rv e r, Ic e la n d (8 1 )
L. apu s apus. M arkthof, A u stria (78)
L. apu s a p u s , M a rc h e g g , A u stria (7 9 )
L. lem m oni, M ex ico (89)
98 6 3 -
C o m m e rc ia l kit. U S A (6 9 )
C o m m e rc ia l kit, K a n s a s , U S A (7 4 )
98 63 99
96100
C h ih u a h u a n d e s e r t , U S A (7 5 )
H uelva, S p a in (3 4 )
x M Ä37 V illam an riq u e , S p a in (3 5 )
4356
— n i i r u c i i ii n a u t a i , ijl OTOCCO (6 1 )
_H- J e n d o u b a , T u n e s la (5 9 )
L K a iro u an , T u n e s ia (60)
r S a g r e s , P o rtu g a l (3 2 )
f “ M A4 F a ro , P o rtu g a l (36)
S a g r e s , T u n e s & Vila d o B ispo, P o rtu g a l
r E x tre m a d u ra , S p a in (3 3 )
(32, 3 7 & 38)
iT l L a A lb u era, S p a in (39)
r El P u e rto d e S a n t a M aria & P u e rto R e a l,
M A9 P u e rto R e a l, S p a in (41 ) S p a in (4 0 8 41
\ MA11 B e n a lu p , S p a in (4 2 )
L M A12 T ahivilla, S p a in (4 3 )
E x tre m a d u ra , S p a in (3 1 )
I
T. cancriform is
96 6 6 69
E u ro p e
_„,j, Japan (18)
ki Sicily, Italy (21, 24)
98100
QP ML MP
NJ Bl
- 8 9 81
r Youssoufia. Morocco (I
9598
_ n .E s s a o u ir a , Morocco ( f
" 9 9 98 r i Casablanca, Moroccc
■ 76 67 9 8 1 0 0 1 j — El-Haieb. Morocco If
IjLTim ahdite, Morocco (l_
3595
T. m auritanicus
5861 45
47 70
7 7 8 9 94
95100
- - 56
3 4 52
•— High atlas, Morocco (57)
A del maestre, Spain (30)
r Jendouba. Tunisia (59)
—» Ain-8 enimatfiar Morocco (61)
-Kairouan. Tunisia (60)
~ S a ares, Portugal (32)
- 9196
10094
B61Q0
D o n a n a N a tio n a l P a rk , S p a in (4 4 )
C aceres. Spam (52)
Villamanrfque, Spain (35)
-0.1 substitutions/site
— 0.1 substitu tio n s/site
M A25 O u riq u e , P o rtu g a l (4 5 )
M A28 El C u e rv o , S p a in (4 7 )
. M A29 C a s tr o M arim , P o rtu g a l (4 8 )
M A30 B a d a jo z , S p a in (4 9 )
MA31 N avalvillar d e P e la , S p a in (50)
■ O u riq u e , P o rtu g a l (45)
- F u e n t e s d e A n d a lu c ía , S p a in (5 1 )
C l C a s tr o V e rd e, P o rtu g a l (46)
Figure 2. Bayesian inference phylogram based on (A) COI and (B) 12S rRNA sequences. N u m b e rs a t n o d e s re p r e s e n t b o o ts tr a p v a lu e s o f
q u a r te t p u z zlin g , m a x im u m lik elih o o d , m a x im u m p a rs im o n y , n e ig h b o u r jo in in g a n d p o s te r io r p ro b a b ility o f B ayesian in fe re n c e . U n su p p o r te d
g r o u p in g s a re in d ic a te d u sin g a
No v a lu e is p ro v id e d w h e n a n a lte rn a tiv e p la c e m e n t o f th e c la d e in th e p h y lo g e n y is s u g g e s te d . N u m b e rs
b e tw e e n b ra c k e ts a re lo cality n u m b e rs c o rre s p o n d in g to p o p u la tio n e n trie s in T a b le S1.
d o i:1 0 .1 3 7 1 /jo u rn a l.p o n e .0 0 3 4 9 9 8 .g 0 0 2
this is subject to furth er m orphological investigation, genetic
distances suggest th a t the Ja p a n ese , the T un esian , the S outh
A frican a n d the N a m ib ian clades p ro b ab ly represent four different
species.
T h e T. australiensis clade, in tu rn , com prises several m on o p h y ­
letic groups a n d endem ic haplotypes exclusive to specific localities.
F o u r clades are restricted to rock pools on granite inselbergs, while
the rem ain in g lineages inh ab it clay pans. A ustralian Triops are
currently gro u p ed into a single species, T. australiensis [18,49] b u t
this m ay be unjustified since the m onophyly o f this n om inal species
is not strongly supported in o u r analyses. W h at is m ore, K 2 P
genetic distances u p to 14.2% a t the C O I gene are well in range o f
those used by o th er researchers to distinguish b etw een species in
o th er Triops lineages [22,31,35]. F or exam ple, the clade com pris­
ing rock pool populations from W alga R ock, B alan R ock a n d
B ullam anya R ock in W estern A ustralia, m inim ally diverged 9.4—
11.8% a t the C O I gene from a clay p a n p o p u latio n in the sam e
a rea a n d 12.0-14.0% from the clade th a t inhabits the rock pools
on the sandstone m onolith U lu ru in the N o rth e rn T errito ry . T h e
em ergence o f different lineages in the Ib e ria n Peninsula th ro u g h
founder effects a n d genetic drift [46],
C o m p a re d to the relatively m odest genetic distances in T.
cancriformis a n d T. mauritanicus, the T. granarius clade was show n to
h a rb o u r m ore divergent haplotypes. T. granarius has a highly
scattered distribution including J a p a n , C h in a [47] a n d b o th
n o rth ern - a n d southern A frica [48]. G iven the vast size o f its range
it is n o t surprising th a t the m ost distant populations J a p a n ,
southern Africa) are substantially differentiated, w ith a m inim um
genetic distance b etw een th em o f 21.1% . B oth C O I a n d 12S
datasets suggest th a t A frican-E urasian T. granarius consists o f
different lineages including a S outh A frican, N am ibian, N o rth
A frican a n d Ja p a n e se clade. ETnexpectedly, the two southern
A frican lineages did not cluster together. In stead the S outh A frican
pop u latio n was show n to be m o re closely related to lineages from
T u n isia th a n to N a m ib ian populations (min. K 2 P distance at 12S
o f 4.1% vs. 11.3% , respectively). E x p an d in g on the findings by
K o rn a n d H u n d sd o erfer [48], the S outh A frican haplotypes
represent a fourth m onophyletic lineage in T. granarius. A lthough
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M o lecu lar P h y lo g e n y o f a P re s u m e d Living Fossil
relatively large genetic distances betw een rock pool a n d clay p a n
Triops populations in W estern A ustralia co n trast w ith the
geographic p roxim ity o f these populations p roviding a firm
indication o f h a b ita t specialization. O verall, it is clear th a t T.
australiensis contains a lot o f cryptic diversity. A detailed
m orphological revision o f T. australiensis including a discussion o f
the potential species status o f different lineages is cu rre n d y u n d e r
p re p a ra tio n (B.V. T im m s, unpublished data).
U nexpectedly, the Triops sp. po p u latio n from the saline Lake
C arey in W estern A ustralia did n o t cluster together w ith o th er
A ustralian populations, b u t instead em erged as a distinct lineage.
K 2 P distances b etw een T. sp. a n d its closest relatives T. australiensis
(min.: 12.3-17.9% ) a n d T. longicaudatus (min.: 15.8-16.3% )
indicate th a t this lineage represents a species new to science
aw aiting form al description (B.V. T im m s, in prep.). T ree
topologies suggest th a t the species m ay have evolved d u rin g the
initial ra d ia tio n th a t gave rise to all p resent-day lineages coinciding
w ith a uniq u e h a b ita t shift from freshw ater to saline systems.
C urrently, it is the only n o to stracan po p u latio n know n from saline
habitats (105 g L *). Finally, acco rd in g to 12S tree topologies T.
sp. could have closer affinities to A m erican th a n to o th er
A ustralian Triops lineages a n d m ay reflect a biogeographic link
m ed iated b y historic long distance dispersal. C onsidering the
C enozoic origin o f living N otostraca, this biogeographic link
betw een A ustralian a n d A m erican lineages m ost likely reflects
historic long-distance dispersal. M igratory birds a n d particularly
w aders, w hich often feed o n b ra n ch io p o d crustaceans a n d have
b e en show n to carry propagules, are prim e can d id ate vectors [38].
T h e bar-tailed godw it subspecies Limosa lapponica baueri, for
instance, m igrates back a n d forth from A laska to A ustralia each
year, often in a 11000 km nonstop flight [50] illustrating the
potential o f long distance dispersal b etw een N o rth A m erica a n d
A ustralia.
T h e m onophyletic T. longicaudatus-newbenyi clade is largely
endem ic to the A m ericas, while p resu m ed T. longicaudatus
populations o n pacific islands such as the G alápagos, H aw aii
a n d N ew C aledonia [21,36] m ay reflect efficient long distance
dispersal, p resum ably by avian vectors, as discussed above.
J a p a n e se records o f T. longicaudatus, o n the o th er h a n d , are
a ttrib u te d to re ce n t anth ro p o g en ic introductions as a biological
control ag en t in rice fields [48]. Based o n o u r analyses we confirm
the m onophyly o f N o rth A m erican Triops populations b u t n o t the
m onophyly o f the species T. newberryi a n d T. longicaudatus. T.
newberryi differed only by 0 .0 -5 .2 % a t C O I a n d 1.0% a t 12S from
T. longicaudatus. T h e sequenced specim ens, hence, should p ro bably
be considered conspecific. T hese findings support the need for a
m orphological taxonom ic revision o f Triops across N o rth A m erica
E v id e n c e fo r a n a n c i e n t ra d ia tio n ?
A lthough fossils suggest th a t som e living tadpole shrim p species
closely resem ble fossils as old as 250 m illion years, b o th sta n d ard
a n d extrem e m olecular clocks for m itochondrial genes in
invertebrates consistently date the m ost recen t co m m o n ancestor
o f all living T riopsidae in the C enozoic era, w ith estim ates o f
divergence tim es am o n g the basal lineages ra nging betw een 29.75
a n d 55.6 m ya (Paleogene period). A n a n cien t (pre) M esozoic
rad iatio n as suggested by fossil rem ains, o n the o th er h and, w ould
explain to d ay ’s distribution o f lineages by vicariance ra th e r th an
long distance dispersal o f several lineages. If w e w ould assum e th a t
Lepidurus a n d Triops indeed existed as separate lineages in the
m iddle T riassic (220 m ya) th en this w ould im ply a m u ta tio n ra te at
the C O I gene o f a b o u t 0.16% p e r m ya w hich is highly unrealistic
since the lowest ra te o f evolution observed a t this gene in
invertebrates is 1.2% p e r m ya [53]. C o n tem p o ra ry tadpole shrim p
species thus alm ost certainly are the result o f a m ore recent
rad iatio n from a single ancestral lineage surviving into the T ertiary
ra th e r th a n a gro u p o f relict lineages from a n earlier (pre-)
M esozoic rad iatio n th a t p resum ably gave rise to a n u m b e r o f
extinct a n cien t lineages know n from the fossil reco rd [4], T h e
scenario o f a recen t radiation, dispersal a n d spéciation in isolation
a dequately explains why, despite a (pre-) P a n g a ea n origin o f the
T riopsidae, a n u m b e r o f lineages are linked to biogeographic
regions (e.g. T. australiensis a n d the A ustralian Lepidurus sp. in
A ustralia, T. longicaudatus-newbenyi in the A m ericas, a n d T.
cancriformis in the Palearctic).
T h e supra-anal plate, w hich is the key diagnostic ch ara cte r to
distinguish Lepidurus from Triops, is a trait w hich m o d ern Lepidurus
species share w ith a n u m b e r o f T riassic a n d C retaceous fossils
[11,12,36]. G iven the re ce n t origin o f Lepidurus, M esozoic tadpole
shrim p w ith supra-anal plates p ro b ab ly should n o t b e classified in
the sam e genus. T h e supra-anal plate, as such, can be a prim itive
c h ara cte r w hich has b e en lost b o th in a n u m b e r o f fossil
T riopsidae as well as in the e x tan t Triops representatives. O n the
o th er h a n d , considering the fact th a t the oldest know n triopsid
fossils lack a supra-anal p late [5], it is also possible th a t it is a
derived trait w hich has evolved m ultiple tim es b o th in M esozoic
triopsids and, again, in the c om m on ancestor o f m o d ern Lepidurus
lineages. E vidently, c u rre n t tadpole shrim p species h aving evolved
quite recently are n o t living fossils a n d the m yth th a t T. cancriformis
w ould b e the oldest species on the p lan e t m ust be firmly
discredited. “ L iving fossil” is undo u b ted ly a n attractive ta g to
draw a tten tio n to p eculiar tax a exhibiting p rim itive traits. Y et, this
term can b e m isleading a n d the intrinsic scientific value o f such a
label is n o t uncontested. D ifferent definitions are in use a n d
particularly in p o p u lar scientific literatu re “living fossil” is often
used over-sim plistically as a term to designate a n a n cien t species
w hich has p resum ably survived relatively u n c h an g e d until presen t
day. N o t surprisingly, creationist lobbyists eagerly en u m erate
exam ples o f m orphological stasis [54] alth o u g h these b y no m eans
provide evidence against evolution by n a tu ra l selection. N o n eth e­
less, the “living fossil” concept, w hich was originally coined by
D arw in [55], can also be m ore stringently a n d realistically defined
as a tax o n w hich belongs to a group w ith a long evolutionary
history, has re ta in e d a n u m b e r o f prim itive characters a n d has few
living relatives. A ccording to this definition the m em bers o f the
o rd e r N otostraca, in general, can b e considered living fossils. A t
least two m ain factors are likely to have c o n trib u ted to
m orphological stasis in tadpole shrim p: the sim ple body plan
consisting o f a dorsal a rm o r a n d serially re p ea te d structures; traits
w hich are also p re sen t in o th er “ living fossils” such as horseshoe
crabs [56] a n d chitons [57], a n d the very specific h a b ita t type in
w hich these organism s have persisted du rin g th eir evolutionary
[51]-
C ryptic diversity a n d c o n s e r v a t i o n im p lic a tio n s
Present-day T riopsidae consist o f a lim ited n u m b e r o f core
evolutionary lineages w ith generally large distributions co rre ­
sponding to n om inal species. H ow ever, a com plex genetic
substructure was show n in certain lineages, such as T. granarius
a n d T. australiensis, w ith m onophyletic lineages inhab itin g different
parts o f species ranges or co ntrasting h a b ita t types (e.g. large clay
pans versus small ephem eral rock pools). F ro m a conservation
p o in t o f view, these lineages can be considered evolutionary
significant units [52]: ap p ro p ria te conservation units o f w hich
preservation can be recom m ended. W h e th e r these clades should
be raised to species level, despite som etim es m odest levels o f
genetic divergence, is o p en to discussion a n d will likely d e p en d on
w h eth er reliable diagnostic m orphological features can b e form ally
identified.
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M o lecu lar P h y lo g e n y o f a P re s u m e d Living Fossil
history. Since the ap p ea ran c e o f planktivorous fish in the
D evonian a n d C arboniferous, large p re d atio n sensitive b ra n ch io ­
p o d crustaceans such as N o to straca are restricted to extrem e
aqu atic systems th a t lack fish such as tem p o rary pon d s a n d saline
lakes: a very specific niche [4,58] in w hich they still persist today.
m ed iated b y vectors such as w ater birds is a realistic possibility
[63,64]. In addition, the highly conserved general m orphology in
N o to straca th ro u g h o u t th eir evolutionary history im pedes the use
o f fossils to calibrate m olecular clocks. A likelihood ratio test [65]
p erfo rm ed in T R E E -P U Z Z L E [66] rejected clock-like evolution
for b o th the C O I a n d 12S datasets.
E ven th o u g h this m eans th a t we c a n n o t linearly calculate
divergence tim es for individual splits in the phylogenetic trees
based o n genetic distance, we can b ro ad ly estim ate the tim ing o f
diversification a n d the likeliness o f a n an cien t rad iatio n by using
the range o f m olecular clocks know n for invertebrates. A lthough
this a p p ro ac h w hich is used due to the im possibility o f fossil
calibration is relatively coarse, a t the very least it allows
distinguishing b etw een a n ancien t (pre) M esozoic ra diation
suggested b y fossil rem ains a n d a m o re recen t T ertiary or
Q u a te rn a ry radiation. A prerequisite, how ever, is th a t sequences
a re n o t oversatu rated in term s o f accu m u lated m utations. As a
result, substitution saturation for the th ird codon position was
tested for b o th the C O I d a ta in D A M B E 5.2.13 [67]. T h e index o f
substitution satu ratio n (Iss) was found to be significandy sm aller
th a n the critical index o f substitution saturation (Iss c), indicating
little saturation. G enerally accep ted C O I evolution rates for
arth ro p o d s are in the range o f 1.40-2.6% m y a -1 [68-69]. Slowest
a n d fastest rates o f C O I evolution in invertebrates are re p o rte d in
bathysciine beetles (1.2% m ya *; [53]) a n d barnacles (4.9%
m ya *; [70]), respectively. F o r the 12S rR N A coding region, we
apply a n evolutionary ra te o f 0.5% m ya 1 [71] w hich is
com m only used in b ra n ch io p o d crustaceans [72].
G enetic distances w ere com p u ted in M E G A v.4.1. [73] using
K im u ra 2 -p a ra m ete r (K2P) distances [74] allow ing for com parison
w ith earlier studies. Betw een haplotype, w ithin a n d betw een
species a n d w ithin a n d betw een genus divergences w ere
calculated.
Phylogenetic reconstruction was p erfo rm ed for b o th m ito ch o n ­
drial D N A datasets independently, using n e ighbor jo in in g (NJ),
m ax im u m p arsim ony (MP), m ax im u m likelihood (ML), q u a rte t
puzzling (QP) m ethods a n d Bayesian inference (Bí). M P analyses
w ere cond u cted in P aup* v .4 .0 b l0 [75] using the P a u p U p
graphical interface [76]. F or the M L analyses P hyM L [77] was
used. M L analyses in P hyM L (1000 b o o tstrap replicates, NN I)
w ere ru n a ccording to the evolutionary m odel a n d p a ram ete rs as
selected b y jM odeltest. N J analyses w ere p erfo rm ed in M E G A
using the follow ing settings: m ax im u m com posite likelihood,
T am u ra -N e i substitution m odel, defined G a n d 1000 b ootstrap
replicates. Q u a rte t puzzling m ax im u m likelihood analyses w ere
p erfo rm ed in T R E E -P U Z Z L E a ccording to the m odel a n d
p a ram ete rs selected by jM odeltest. B ayesian analyses w ere
cond u cted in M rB ayes v.3.1.2 [78] according to the evolutionary
m odel a n d p a ram ete rs suggested b y jM odeltest. M rB ayes ra n for
5 x l 0 6 generations (lset n u m b e r o f substitution types = 6, rates = invgam m a, n u m b e r o f rate categories for the gam m a
distribution = 4, sam pling frequency = 100 generations) until a
sta n d ard deviation o f split frequencies o f 0.0078 was a ttained. A n
o u tgroup (C. hislopi) was defined a n d in o rd e r to only include trees
in w hich convergence o f the M arkov ch ain h a d b e e n reached, we
chose a b u rn -in o f 25% . T h e rem ain in g trees w ere used to
construct a 50% m ajority consensus tree.
Finally, in o rd e r to integrate the inform ation p rovided b y b o th
genes, phylogenetic analyses w ere also c o n d u cted on a com bined
dataset co n tain in g b o th C O I a n d 12S sequences. P aram eters for
b o th genes w ere estim ated in d ep en d en d y in M rB ayes using the
‘u nlink’ c o m m a n d (partition tw ogenes = 2: 12S, C O I, lset
applyto = (1), nst = 6, rates = invgam m a, n g a m m a ca t = 4, lset
applyto = (2), nst = 6, rates = invgam m a, n g a m m a ca t = 4, unlink
C o n c lu s i o n s
A lthough som e p re sen t day tadpole shrim p species closely
resem ble fossil specim ens as old as 250 m ya, no m olecular support
was found for a n ancien t (pre) M esozoic radiation. Instead, living
tadpole shrim p are m ost likely the result o f a relatively recent
rad iatio n in C enozoic a n d close resem blances b etw een re ce n t a n d
fossil tax a are p ro b ab ly the result o f the highly conserved general
m orphology in this group a n d o f hom oplasy. It is clear th a t m ore
a n d m ore evidence is accum ulating in dicating th a t a lack o f readily
observable phenotypic change (m orphological stasis) d u rin g the
evolutionary history o f a certain lineage does n o t necessarily im ply
evolutionary stasis [59]. As show n in this study, recen t species
w hich are virtually identical to fossils in term s o f their m orphology
m ay rep resen t very different evolutionary lineages.
Methods
C O I a n d 12S rR N A genes w ere sequenced for u p to six tadpole
shrim p specim ens p e r p opulation. D N A extraction, polym erase
ch ain reaction a n d sequencing protocols are p rovided in T e x t S2.
All new sam ples w ere collected by the a u thors in the field betw een
2008 a n d 2010, using a sim ple d ip n et (5 m m mesh). Exceptionally,
T. newberryi specim ens from a po p u latio n in K ansas, U S A w ere
la b o ra to ry -h a tch e d from sedim ent in distilled w ater a t 20°C.
Ethics s t a t e m e n t
C ollected anim als w ere an aesthetized in c arbon ized w ater
before transfer to ethanol. C ollection a n d ex p o rt perm its w ere
g ra n te d by the Free State Province D e p a rtm e n t o f T ourism ,
E n v ironm ental a n d E conom ic affairs (South Africa): p e rm it no.:
H K /P 1 /0 7 3 7 5 /0 0 1 a n d b y the A ustralian governm ent: perm it
no. SF 007548 a n d SF005789.
G e n e tic d a t a a n a ly s e s
Sequences w ere aligned (ClustalW m ultiple alignm ent: [60])
a n d trim m ed in B ioE dit S equence A lignm ent E d ito r v.7.0.0 [61].
120 ad ditional C O I a n d 74 12S sequences w ere d raw n from
G enB ank a n d aligned to the new ly o b tain ed D N A fragm ents
(T able S I provides ad ditional details a n d G enB ank accession
codes). T h e cyclestherid con ch o stracan Cyclestheria hislopi (Baird,
1859) was selected as outgroup. Finally the alignm ent was
inspected by eye for any anom alies a n d found to be straightfor­
w ard. All new sequences w ere deposited in G enB ank u n d e r
accession codes JN 1 7 5 2 2 3 -2 6 7 ; JN 1 9 0 3 9 6 -3 9 8 ).
F o r the C O I a n d 12S datasets, jM o d e lte st v.0.1.1 [62]
respectively selected the T IM 1 + I+ G (with a p ro p o rtio n o f
invariable sites o f 0.496 a n d a g am m a-shape p a ra m e te r o f
0.775) m odel, w ith nucleotide frequencies A = 0.32, C = 0.18,
G = 0.10 a n d T = 0.38 a n d ra te m atrix (1.00, 23.28, 2.06, 2.06,
14.10, 1.00) a n d the T P M 2 u f+ G (with a g am m a-shape p a ra m e te r
o f 0.33) m odel, w ith nucleotide frequencies A = 0.37, C = 0.17,
G = 0.11 a n d T = 0.35 a n d rate m atrix (11.35, 46.47, 11.35, 1.00,
46.47, 1.00) as b est fitting m odels o f evolution. M odel averaged
phylogeny analyses w ere p erfo rm ed in the sam e software,
indicating th a t all 88 tested m odels re n d ere d nearly identical trees
for b o th the C O I a n d 12S data.
D a tin g splits b etw een passively dispersed aquatic invertebrates is
problem atic since long distance a n d even interco n tin en tal dispersal
PLoS ONE I w w w .p lo s o n e .o rg
7
April 2 0 1 2 I V o lu m e 7 | Issue 4 | e 3 4 9 9 8
M o lecu lar P h y lo g e n y o f a P re s u m e d Living Fossil
shape = all). M rB ayes ra n for 8x10® generations w ith a sam pling
frequency o f 100 a n d a defined o u tgroup (C. hislopi).
In case phylogenetic analyses did n o t unequivocally support
m onophyly o f the tw o N o to stracan genera, con strain t analysis
using K ishino-H asegaw a- [79] a n d S him odaira-H asegaw a [80]
tests for the M L tree a n d K ishino-H asegaw a as well as T em p leto n
- a n d w inning site tests for the M P tree w ere c o n d u cted in Paup*
to test w h eth er enforcing m onophyly o f g en era led to a statistically
significant increase in tree likelihood.
C O I m olecular clock (1.40% m ya *; below diagonal) a n d 12S
m olecular clock (0.5% m ya *; above diagonal) for crustaceans.
(D O C X )
T a b le S4 U n c o rrec ted p distance m atrix (m in.-m ax.) betw een
investigated noto stracan lineages based on C O I (below diagonal)
a n d 12S rR N A (above diagonal) genes. E m p ty cells indicate th a t
sequence inform ation was unavailable.
(D O C X )
T e x t S I O verview o f cu rre n d y accepted N otostraca species a n d
their know n distributions.
(D O C)
Supporting Inform ation
F ig u re S I B ayesian inference phylogram based on com bined
C O I a n d 12s rR N A sequences. N um bers a t nodes represent
bo o tstrap values o f m ax im u m likelihood (ML), m axim um
p arsim ony (MP) a n d posterior p robability values o f Bayesian
inference (Bí). U n su p p o rte d groupings are indicated using a
No
value is p rovided if this m eth o d o f phylogenetic inference w ould
suggest a n alternative p lac em e n t o f the corresponding clade in the
phylogeny.
(D O C X )
T a b le S I
(D O C)
T e x t S2 D N A isolation, m olecular m arkers, polym erase chain
reaction a n d sequencing.
(D O C)
Acknowledgments
T he m anuscript was improved by valuable discussions and com m ents by
Bart Hellemans, T ine Huyse, Aline W aterkeyn, M aarten H .D . Larm useau
and Pascal I. H ablützel. K eith A. C randall, D. C hristopher Rogers and
two anonym ous reviewers provided m any helpful com m ents on the
m anuscript. W e thank the D epartm ent of Environm ent and Conservation
o f W estern Australia for issuing perm its and Jacques Pages (Mousses et
lichens du H au t Languedoc) and Aline W aterkeyn for contributing
pictures.
O verview o f investigated N o to straca samples.
T a b le S2 K im u ra 2 -p a ra m ete r distance m atrix (m in.-max.)
betw een investigated n o to stracan lineages based o n C O I (below
diagonal) a n d 12S rR N A (above diagonal) genes. E m p ty cells
indicate th a t sequence in form ation was unavailable.
(D O C X )
Author Contributions
Conceived and designed the experim ents: BV T P. P erform ed the
experiments: BV T P C D . Analyzed the data: BV T P M PM V . C ontributed
reagents/m aterials/analysis tools: BV T LB. W rote the paper: BV T P BVT
M J LB.
T a b le S3 D ivergence tim es b etw een m ain T riops clades
(m inim um -m axim um ) based o n the standardly used average
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April 2 0 1 2 I V o lu m e 7 | Issue 4 | e 3 4 9 9 8
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