Phylogeography and mitochondrial DNA divergence in Dolichopoda cave crickets (Orthoptera, Rhahidophoridae)

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Hereditas 146: 3345 (2009)
Phylogeography and mitochondrial DNA divergence in Dolichopoda cave
crickets (Orthoptera, Rhahidophoridae)
LENE MARTINSEN, FEDERICA VENANZETTI and LUTZ BACHMANN
Natural History Museum, Department for Zoology, University of Oslo, Norway
Martinsen, L., Venanzetti; F. and Bachmann, L. 2008. Phylogeography and mitochondrial DNA divergence in Dolichopoda
cave crickets (Orthoptera, Rhahidophoridae). * Hereditas 146: 3345. Lund, Sweden. eISSN 1601-5223. Received
March 25, 2008. Accepted July 7, 2008
Dolichopoda cave crickets are found in caves in the northern Mediterranean region from the Pyrenees in the west to the
Caucasus in the east. In this study we analysed the phylogeny within the genus Dolichopoda using parts of the mitochondrial
cytochrome oxidase I and 16S ribosomal genes, and explored biogeographic patterns through a dispersalvicariance analysis
(DIVA). Phylogenetic analyses grouped the 15 species into the four geographically restricted main lineages corresponding to
the Caucasus, Greece, the Pyrenees and Italy, respectively. The species occur largely in allopatry. The Caucasian and Greek
species were basal in the phylogeny, as was the clade including the nine Italian species, which grouped into two major
lineages, one mainly including species from western Italian coastal regions and islands, and the other including species with a
predominantly inland distribution. Thus it seems likely that there have been two main immigrations into Italy followed by
multiple consecutive speciation events. The DIVA analysis supported the assumption of an east-west migration route, and
indicated that there have been four major dispersal events. Since the statistical support for the basal node connecting D. remyi
and D. hussoni with the west Mediterranean species is low, alternative interpretations for the colonization of the
Mediterranean, namely parallel colonization of the main areas of the current Dolichopoda distribution, i.e. the Caucasus,
Greece, Italy, and the Pyrenees is also possible. Particular emphasis was put on the D. geniculata-laetitiae species complex. D.
geniculata included several recently diverged lineages and constitutes a paraphyletic species complex, also embracing the
closely related D. laetitiae.
Lutz Bachmann, Natural History Museum, Department for Zoology, University of Oslo, Norway. E-mail: [email protected]
nhm.uio.no
Troglophilous, apterous insects are suitable model
systems for addressing phylogeographic questions. As
they are bad dispersers and dependent on cave
habitats, the extent of gene flow between populations
is expected low compared to other terrestrial species.
The narrow geographic range that is typical for cave
populations, along with their high level of endemism,
usually cause distinct biogeographic patterns (PORTER
2007). In contrast, surface organisms often have
more complex patterns due to recurrent episodes of
extinction, recolonization, and secondary contact
(SBORDONI et al. 2000).
The Orthopteran family Rhaphidophoridae (camel
crickets, cave crickets) consists of hydrophilous, apterous, hump-backed, cricket-like insects, usually with
slender, elongated appendages (RENTZ 1991). The
family has a world-wide distribution and all species
are confined to wet forests, rock outcrops and caves
(RENTZ 1991). Troglophily, the dependence on caves,
is well developed within the family, as more than half
of the 300 species are cave dwellers. The cavernicolous
Rhaphidophoridae are found in the northern Mediterranean region, the Cape region of South Africa,
eastern United States, Central America, Patagonia in
South-America, Australia (including Tasmania), New
Zealand, South-East Asia and Japan (DI RUSSO and
SBORDONI 1998). The genus Dolichopoda Bolivar, I.
1880 (Dolichopodainae, Rhaphidophoridae) (ICZN
2002) is found in caves in the northern Mediterranean
region from the Pyrenees in the west to the Caucasus
in the east (SBORDONI et al. 1987). The highest species
diversity is found in the insular and peninsular areas
of Italy and Greece (SBORDONI et al. 1987).
Many Dolichopoda populations depend strictly
upon natural caves but some populations have also
colonized cave-like habitats, such as rock crevices and
ravines in mesic or moist woods, and also man-made
hypogean environments, e.g. cellars, Etruscan tombs,
Roman aquaducts and catacombs (SBORDONI et al.
1987, 1991). The natural and man-made caves may
differ in their ecological conditions and populations
inhabiting these two habitats can differ in their
phenology and life history traits (BERNARDINI and
DI RUSSO 2004).
Although some individuals are occasionally observed outside their caves in moist or mesic woods,
especially in the northern parts of Italy (CARCHINI
et al. 1983; BACCETTI 1987), there is a high degree of
geographical isolation between the different Dolichopoda populations. Strictly allopatric speciation processes have been assumed for Dolichopoda populations
(SBORDONI et al. 1987), as is also true for many other
DOI: 10.1111/j.1601-5223.2008.02068.x
34
L. Martinsen et al.
cave organisms (SBORDONI et al. 2000; GIBERT and
DEHARVENG 2002), and usually species show vicariant
allopatric ranges (SBORDONI et al. 1991). An exception is the occurrence of D. schiavazzii in a man-made
cave together with D. baccettii at the Argentario
promontory. However, this is thought to be due to
passive dispersal by Passionist Fathers established in
this area since the 18th century (ALLEGRUCCI et al.
1982, 1997).
Studies on several Dolichopoda species such as e.g.
D. geniculata, D. schavaizzii and D. linderi, indicate
that gene flow between populations is dependent
on the habitat structure surrounding the caves
(ALLEGRUCCI et al. 1997; CESARONI et al. 1997).
These studies show that groups of populations from
caves surrounded by xeric vegetation in coastal
Mediterranean habitats are genetically more structured than groups of populations living in montane
mesophilous forest. The latter show, at the same scale,
higher values of Nm, i.e. higher levels of gene flow. It
has been reported that populations as distant as 50 km
apart might have recently exchanged migrant individuals (SBORDONI et al. 2000).
Dispersal of Dolichopoda by man has occurred on
several occasions. Unintentional transplantation of
eggs, nymphs or even adults, is common in Dolichopoda (BERNARDINI et al. 1996). One example is the
occurrence of D. schiavazzii in the Argentario Promontory. Another example is D. laetitiae in the Poscola
cave, which is quite isolated from the geographic range
of the Dolichopoda genus (BERNARDINI et al. 1996).
The phylogenetic relationship among several western Mediterranean Dolichopoda species have been
studied by applying a variety of markers such as
epiphallus morphology, egg chorion structure, allozyme variability, single copy DNA-DNA hybridization, and RFLPs of mtDNA (ALLEGRUCCI et al.
1992; VENANZETTI et al. 1993). ALLEGRUCCI et al.
(2005) recently published a mitochondrial DNA
phylogeny of the Mediterranean species of Dolichopoda genus based on sequences of the cytochrome
oxidase I (COI) and the ribosomal 16S rRNA (16S)
genes. The authors suggested that the genetic diversity
reflects the geographical distribution pattern, with the
geographically close species being the most related
ones, and that the present-day geographical distribution appears to have been influenced by glacial and
interglacial cycles during the Pleistocene.
The present study is a phylogenetic evaluation of
Dolichopoda, using COI and 16S, significantly expanding the data set from previous studies (Allegrucci
et al. 2005). Our analysis included samples from a
broader geographic range, including the westernmost
species D. bolivari, the Sardinia species D. muceddai
Hereditas 146 (2009)
and the Greek species D. hussoni. In addition, a
possible historical biogeographic scenario was reconstructed by a dispersal-vicariance analysis using the
DIVA software (RONQUIST 1996).
MATERIAL AND METHODS
Material
Our study included 45 specimens of Dolichopoda spp.
representing 35 populations and 12 species. Additional
sequences from 43 specimens and 26 populations
available in GenBank from ALLEGRUCCI et al.
(2005), were also included in the analyses. In total 16
species and 45 populations were included. Troglophilus
cavicola, Ceutohilus gracipiles, Hadenoecus cumberlandicus and Euhadenoecus insolitus were used in the
phylogenetic analyses as outgroup taxa. All the species
and populations included in the analysis are listed in
Table 1.
DNA extractions, PCR and sequencing
DNA extractions were done with the Pure Gene Kit
(Gentra Systems). The mitochondrial 16S rRNA and
COI genes were PCR amplified using the primers LRJ-12887 (5?-CCGGTCTGAACTCAGATCACGT-3?)
and LR-N-13398 (5?-CGCCTGTTTATCAAAAACAT-3?) for the 16S region (SIMON et al. 1994) and
C1-J-1751 (5?-GGATCACCTGATATAGCATTYCC3?) (SIMON et al. 1994) and C1-N-2700 (5-AATATA
ACAATAAATTGTATTTT-3?) (KNOWLES and OTTE
2000) for the COI region. The internal primers (5?GCTATTATAGCATAAATTATTC-3?) and (5-GAG
ATCCAATTTTATATCAACA-3?) were used as sequencing primers for COI. The PCR conditions for
the 16S region were as follows: 948C for 5 min,
followed by 30 cycles of denaturation at 948C for
45 s, annealing of primers at 458C for 30 s, elongation
of primers at 728C for 30 s, and one final extension
step at 728C for 5 min. The PCR conditions for the
COI region were as follows: 948C for 5 min, followed
by 30 cycles of denaturation at 948C for 45 s, annealing of primers at 458C for 30 s, elongation of primers
at 728C for 1 min, and one final extension step at 728C
for 5 min. PCR products were purified using the
QIAquick PCR Purification Kit (Qiagen). Sequencing
was done on an ABI automated sequencer (3100),
using BigDye for cycle sequencing (Applied Biosystems). GenBank accession numbers are given in
Table 1.
Statistical and phylogenetic analyses
The sequences were aligned using Clustal X
(THOMPSON et al. 1997) followed by manual adjustments. Nucleotide composition, number of variable
Taxon
Ingroup taxa
Dolichopoda
D. schiavazzii
Pop.
Locality
Necropoli di Vetulonia, Grosseto, Toscana, Italy
Grotta dei Pipistrelli, Montorsaio, Grosseto, Toscana, Italy
CPS
VET1,VET2
ORS1,ORS2
ORS A
CPS
CIS
BDO
CIS
BDO1, BDO2
POP
MRC
POP
MRC
MRC A
BSC
FIC
FIC1, FIC2 A
CAM
VET
ORS
D. schiavazzii caprai
BSC
FIC
D. aegilion
CAM
D. linderi
MTB
BNP
VMY
SIR
FRN
BRA
CAM A
MTB
BNP
VMY
SIR1,2 A
FRN1,2
BRA1,BRA2
BRA1, 2 A
SIS
SIS1-3 A
D. muceddai
D. baccettii
SAR
PST
SAR1,2 A
PST1, 2
D. laetitiae
PSC
PST1, 2 A
PSC1, 2
GDP
FOR
PSC1, 2 A
GDP1, 2 A
FOR
D. bolivari
D. bormansi
Monastero dei Fratelli Passionisti, Orbetello, Grosseto,
Toscana, Italy
Acquedotto di Cisternino, Livorno, Toscana, Italy
Grotta di Buca dell’oro, Isola d’Elba, Grosseto, Toscana,
Italy
Populonia, Grosseto, Toscana, Italy
Marciana, Isola d’Elba, Grosseto, Toscana, Italy
GenBank accession no.
COI
AY507638, AY507639
AY507635, AY507636
AY793633
AY507632
16S
AY507601, AY507602
AY507585, AY507584
AY793573
AY507575
AY507631
AY507629, AY507630
AY507573
AY507567, AY507568
AY507637
AY507634
AY793635
Buca sopra cimitero, Orbetello, Grosseto, Toscana, Italy
AY507643
Caverna di Fichino, Cascianna Terme, Pistoia, Toscana, Italy AY507633
AY793634, AY793636
Miniera di Campese, Isola del Giglio, Grosseto, Toscana,
AY507652
Italy
AY793600
Grotte de Montbolo, Montbolo, Eastern Pyrenees, France
AY507627
Grotte de Bon Repaux, Bon Repaux, Ariege,Pyrenees, France AY507626
Grotte de Valmanya, Vinca, Eastern Pyrenees, France
AY507628
Sirach Cave, Eastern Pyrenees, France
AY793598, AY793599
Forat negre cueva, Serradel, Llerida, Pyrenees, Spain
AY507648, AY507649
Grotta di Brando, Bastia, Corsica, France
AY507646, AY507647
AY793631, AY793632,
AY793627
Grotta di Sisco, Corsica, France
AY793625, AY793626,
AY793628
MonteLimbara, Sardinia, Italy
AY793629, AY793630
Grotta di Punta degli Stretti, Orbetello, Grosseto, Toscana, AY507650, AY507651
Italy
AY793639, AY793640
Grotta della Poscola, Monte di Malo, Priabona, Vicenza,
AY507641, AY507642
Veneto, Italy
AY793611, AY793613
Grotta della Piana, Umbria, Italy
AY793612, AY793610
Ruderi di Villa Chigi, Formello, Roma, Lazio, Italy
AY507589
AY507582
AY793572
AY507571
AY507577
AY793574
AY507572
AY793570
AY507583
AY507565
AY507603
AY793567
AY507579, AY507580
AY507605, AY507606
AY793578
AY793579
AY793575, AY793575
AY507594, AY507593
AY793571
AY507591, AY507592
AY793581
AY793582
Phylogeography and mitochondrial DNA divergence in Dolichopoda
Individual
Hereditas 146 (2009)
Table 1. Species, populations, localities and accession numbers in GenBank for the Dolichopoda sequences included in this study. (The abbreviations for
individuals that are followed by an A is from ALLEGRUCCI et al. 2005).
35
36
Table 1 (Continued)
D. laetitiae etrusca
Pop.
DIA
D. palpata
TRE
D. capreensis
CPR
D. geniculata
PIL
PAS
CLP
TUS
ISC
D. ligustica
D. ligustica
septentrionalis
DIA
DIA1, 2 A
TRE1, 2
TRE1, 2 A
CPR
CPR1, 2 A
PIL
PAS
CLP
AUS
FON
PNZ
TUS
ISC
ISC A
PRA
VAL
VAL1, 2 A
AUS
FON A
PNZ 1
CON
PNZ1 A
PNZ2 A
CON
SFL
BOS
PUG
CON1, 2 A
SFL
BOS1, 2
PUG1, 2
PRA
VAL
D. geniculata
pontiana
Individual
Locality
Grotta del diavolo, Semproniano, Grosseto, Toscana, Italy
AY507640
AY793614,
Grotta di Tremusa, Scilla, Reggio di Calabria, Calabria, Italy AY507610,
AY793608,
Grotta San Michele, Isola di Capri, Napoli, Campania, Italy AY507644
AY793606,
Grotta la Pila, Poggio Moiano, Rieti, Lazio, Italy
AY507620
Grotta di Pastena, Pastena, Frosinone, Lazio, Italy
AY507621
Grotta Regina Margherita, Collepardo, Frosinone, Lazio,
AY507609
Italy
Cunicolo dell’acquedotto, Frascati, Roma, Lazio, Italy
AY507624
Fontana cunicoli, Isola di Ischia, Napoli, Campania, Italy
AY507622
AY793595
Grotta delle Praie, Lettomanoppello, Perugia, Umbria, Italy AY507618
Grotta Valmarino, Monte S. Biagio, Latina, Lazio, Italy
AY507625
AY793616,
Grotta degli ausi, Prossedi, Latina, Lazio, Italy
AY507611
Grotta di Fontanella, Vico Equense, Campania, Italy
AY793594
Le Forme, Isola di Ponza, Latina, Lazio, Italy
AY507619
Buco del Corno, Valle Cavallina, Zandobbio, Bergamo
lombardia, Italy
D. hussoni
D. remyi
VLT1, 2 A
SAB 2 A
NAO
EDE A
POZ A
VOR A
GOL A
AY793607
AY793617
AY793596
AY793597
AY507614
Grotta di Valletto, Corsica, France
Grotta di Sabara, Corsica, France
Naoussa cave, Naoussa, Macedonia, Greece
Edessa Cave, Macedonia, Northern-East Greece
Pozarska Cave, Macedonia, Northern-East Greece
Vorontzovskaya Cave, Caucasus, Russia;
Golova Otapa Cave, Caucasus, Russia
AY793601, AY793602,
AY793603
AY793620, AY793621
AY793619
AY507615
AY793637
AY793638
AY793622
AY793623
AY507578
AY793580
AY507564 AY507598
AY793588
AY507574
AY793587
AY507587
AY507586
AY507563
AY507599
AY507581
AY793585
AY507590
AY507600
AY793583
AY507566
AY793584
AY507588
AY793586
AY507576
AY793568
AY507597
AY507569, AY507570
AY507595, AY507596
AY793569
AY793577
AY793576
AY507607
AY793589
AY793590
AY793566
AY793565
Hereditas 146 (2009)
D. euxina
VLT
SAB
NAO
EDE
POZ
VOR
GOL
AY793615
AY507645
AY793609
AY793604, AY793605
Grotta Selva, Zandobbio, Bergamo, Lombardia, Italy
AY507623
Grotta di Bossea, Frabosa Soprana, Cuneo, Piemonte, Italy AY507612, AY507613
Grotta del Pugnetto, Val di Lanzo, Torino, Piemonte, Italy AY507616, AY507617
PUG1, 2, 3 A
D. cyrnensis
GenBank accession no.
L. Martinsen et al.
Taxon
AY793563
AY793591
IND A
IND
Indian Grave Point Cave, De Kalb Co., TN, USA
AY793592
BAT A
BAT
Bat Cave, Carter Cave State Park, Carter Co., KY, USA
AY793561
AY793593
Hamden, CT, USA
CEU A
AY793564
AY793624
TRO A
CEU
Ceuthophilus
C. gracilipes
Hadenoecus
H. cumberlandicus
Euhadenoecus
E. insolitus
AY507604
Outgroup taxa
Troglophilus
T. cavicola
TRO
TRO
Grotta della Poscola, Monte di Malo, Priabona, Vicenza,
Veneto, Italy
Covoli di Veroli Cave, Veneto, Italy
AY507653
GenBank accession no.
Locality
Individual
Pop.
Taxon
Table 1 (Continued)
AY793562
Phylogeography and mitochondrial DNA divergence in Dolichopoda
Hereditas 146 (2009)
37
and parsimony informative sites, and the transition/
transversion ratio were calculated using MEGA version 3.1 (KUMAR et al. 2004).
To assess whether nucleotide substitutions reach
saturation in the comparison of ingroup species,
transitions and transversions were plotted against
uncorrected genetic distances (p-distances) for all
pairwise comparisons in both fragments.
DnaSP (ROZAS et al. 2003) was used to estimate
nucleotide diversity (Pi), average number of nucleotide
differences (k), and haplotype diversity (Hd).
Phylogenetic analyses were conducted using Bayesian analysis, maximum likelihood (ML) and maximum parsimony (MP). The analyses were conducted
on each data set separately, as well as a concatenated data set. A partition homogeneity test was
performed in PAUP (SWOFFORD 1998) to test the
congruence between the 16S and COI genes. The
appropriate substitution model for the two alignments
were determined using the program MrModeltest
(NYLANDER 2004). The general time reversible substitution model (GTRGI) including invariable
sites (I) and rate variation across sites (G) obtained
highest likelihood score for both COI and 16S rRNA
data sets, and was implemented in the Bayesian and
maximum likelihood analyses.
The Bayesian phylogenetic analysis was conducted
using MrBayes (HUELSENBECK and RONQUIST 2001).
Each analysis was run with two million generations,
four chains (one cold, three heated) and a sample
frequency of 100. A 50% majority rule consensus tree
was made from each analysis with the first 4000 trees
ignored as burn-in.
The maximum likelihood analysis was done using
PAUP (SWOFFORD 1998) through the Bioportal Website at the Univ. of Oslo, Norway (/<http://www.
bioportal.uio.no//>).
Maximum parsimony analysis was done using the
program TNT-Tree analysis using New Technology
(GOLOBOFF et al. 2008) made available online with the
sponsorship of Willi Hennig Society (/<http://www.
cladistics.org//>).
Uncorrected p-distances between populations were
calculated in MEGA (KUMAR et al. 2004) for species
represented by more than three populations and an
analysis of variance (ANOVA) with subsequent Posthoc tests Tukey honest significant difference test for
unequal sample size (SPJOTVOLL and STOLINE 1973)was conducted in Statistica (STATSOFT 2005) to
compare the variation within these species.
The program DIVA (dispersal vicariance analysis)
(RONQUIST 1996) was used to reconstruct hypothetical
past biogeographic patterns of the Dolichopoda taxa
during the colonization of the northern Mediterranean
38
L. Martinsen et al.
region. This program gives geographic locations for
common ancestors employing an approach similar to
character optimization. The distributional data for the
Dolichopoda genus were taken from relevant literature
(BACCETTI and CAPRA 1959; SBORDONI et al. 1976,
1985, 1987, 1991, 2004; CARCHINI et al. 1983). The
areas were defined as congruent areas shared by two or
more species (sympatric distribution) according to the
definition used by SANMARTIN (2003). The exceptions
are the areas two and six (below) that are inhabited by
only one Dolichopoda species each, but because of the
distance to the other areas it would be artificial to
include them in any of the other categories. The islands
off the coast of Italy were assigned to the same
category as the mainland. This is because each island
inhabits only one species, and because the islands can
be considered as a unit together with the continental
areas due to territorial continuity as a result of drop in
sea level in upper Miocene and marine regression in
glacial Pleistocene periods (LA-GRECA 1990). Corsica
and Sardinia are grouped together because of their
closeness and their geological history occurring on the
same microplate.
Based on the extant distribution pattern of Dolichopoda we defined six areas: (1) the Pyrenees, (2)
continental Italy north (north of Emilia Romana, in
connection with the Italian Alps), (3) Corsica and
Sardinia, (4) continental Italy central and south (south
of Emilia Romana), (5) Greece, (6) the Caucasus.
Since the DIVA program only accepts fully resolved
trees, we chose the maximum likelihood tree in our
DIVA analyses. The ML tree included only one
polytomy, a trichotomy within the D. geniculatalaetitiae complex, compared to three polytomies in
the maximum parsimony and Bayesian trees. All taxa
within the trichotomy have the same distribution
according to our area classification, so we randomly
chose one of the three possible solutions of the
trichotomy for the DIVA input file.
DIVA does not perform well on the most basal
nodes, because the input tree is only a small part of the
tree of life, and the optimal state for the basal node
depends heavily on the rest of the tree of life. This
usually results in the root node distribution being large
and including most or all of the areas occupied by the
terminals (RONQUIST 1996). This can be solved by
incorporating outgroups and/or by imposing constraints on the number of unit areas allowed in
ancestral distributions. We included one of the outgroups, T. cavicola, in the DIVA analysis. The other
outgroups used in the phylogenetic analyses are
Nearctic species, so they cannot be used to restrict
the ingroup distribution. SANMARTIN (2003) argues
that one can constrain the maximum number of areas
Hereditas 146 (2009)
in ancestral distributions to the number of areas in the
most widespread extant descendant. Since no extant
Dolichopoda species are widespread among all of the
defined regions, we consider it likely that the common
ancestors of the genus also had a restricted distribution. We therefore tried two different values for the
maxareas option, two and four, to impose a constraint
on the number of unit areas allowed in ancestral
distributions.
We also conducted a spatial analysis of molecular
variance using the software SAMOVA (DUPANLOUP
et al. 2002). This program aims at clustering geographically homogeneous populations into user-defined
numbers of groups (K) in order to maximize the total
genetic variance observed between groups (FCT index).
At the same time, the program also identifies genetic
barriers between grouped populations. SAMOVA
analyses were computed for K-values ranging from 4
to 15. For each K-value, 10 000 simulated annealing
steps were performed starting from each of 250 sets of
initial conditions.
RESULTS
Sequence alignments
The mitochondrial DNA matrix included 94 sequences representing 45 populations and 16 species
of Dolichopoda cave crickets, of which 43 sequences
were taken from ALLEGRUCCI et al. (2005). A total of
1439 nucleotide characters, 496 from the 16S gene and
943 from the COI gene, were included in the alignment. There were 362 variable sites (25.2%), of which
296 (20.6%) were parsimony informative. The COI
region included 282 variable sites (225 parsimony
informative) and the 16S gene 80 variable sites (71
parsimony informative). The transition/transversion
(ti/tv) ratios for 16S ranged from 0.27, which is in
agreement with the range calculated by ALLEGRUCCI
et al. (2005). The ti/tv ratios for COI ranged from 0.5
11.5, which is a broader range than reported by
ALLEGRUCCI et al. (2005).
Phylogenetic analyses
The congruence of the 16S and COI datasets were
evaluated using a partition homogeneity test, which
yielded no significant difference between the data
partitions (p 0.52). Thus, the 16S and COI data
sets were combined and analyzed simultaneously. The
phylogeny (Fig. 1) is derived from the Bayesian
analysis, showing bootstrap support values from the
MP and ML analyses as well. The three different
phylogenetic inferences used (MP, MrBayes and ML)
gave very similar tree topologies and only a few
incongruences occurred. The parsimony analysis
Hereditas 146 (2009)
Phylogeography and mitochondrial DNA divergence in Dolichopoda
39
Fig. 1. Bayesian inference of the phylogeny of the genus Dolichopoda. The terminal taxa are populations following the
abbreviations listed in Table 1. Numbers in parenthesis indicate the codes for the predefined geographic areas used in the DIVA
analyses. Posterior probabilities and bootstrap support 50 in the MP analysis are given for each node.
40
L. Martinsen et al.
Hereditas 146 (2009)
yielded 4083 trees of equal length (1441 steps) and the
50% majority-rule consensus tree was congruent with
the MrBayes phylogeny (Fig. 1).
Almost all Dolichopoda species were found monophyletic with high statistical support. Only D. geniculata was found paraphyletic in relation to D. laetitiae.
The Caucasian D. euxina is the sister group to a clade
consisting of all other species included in this study. D.
remyi and D. hussoni from Greece are found in a
trichotomy with the species from Italy/Spain/France.
Within this clade, a monophyletic group of the
Pyrenean species (D. linderi and D. bolivari) forms a
sister group to the Italian species. In the clade
including the Italian Dolichopoda species, two well
supported groups can be found, one including species
from central and west (D. laetitiae and D. geniculata)
and north Italy (D. ligustica), and the other species
from central and northern Italy including Sardinia
and Corsica (D. schiavazzi, D. aegilion, D. baccetti, D.
bormansi, D. muceddai and D. cyrnensis) and south
Italy (D. capreensis and D. palpata). The geographic
distribution of the species and populations is depicted
in Fig. 2.
Intrapopulational sequence variation
In most instances sequences from the same species
cluster together in the tree, though not always with the
sequences from the same population. This variation
SFL
PUG
can be either due to lineage sorting, stochastic
extinction of ancestral haplotype lineages within a
population, or spatial proximity of isolated populations and gene flow between them. As the number of
sequences per population is low, the intrapopulational
sequence variation is not discussed in this paper.
Further analysis is required to resolve these issues.
Intraspecific genetic diversity
The number of polymorphic (segregating) sites (S),
nucleotide diversity (p) and average number of nucleotide differences (k) for species with N]7 is presented
in Table 2 (N number of individuals).
The genetic variation within D. geniculata was high
when compared to other Dolichopoda species. In order
to test whether this intraspecific variation in D.
geniculata was significantly higher than in other
Dolichopoda species, i.e. D. geniculata, D. laetitiae, D.
ligustica, D. linderi and D. schiavazzii, we performed
an ANOVA using the calculated p-distances obtained
from comparisons between individuals within species.
The ANOVA revealed a highly significant difference
in intraspecific genetic variation between species
(F34.77, p B0.001). Post-hoc tests showed that
all comparisons with D. geniculata were significant
(pB0.01), and that the genetic variation within D.
geniculata was significantly higher than in the other
species.
PSC
CON
BOS
CIS
FIC
GDP
ORS
POP
SIS
VET DIA FOR
BRA
CAM
PIL PRA
SAB
VLT CPS PST TUS
CLP
BSC
AUS
PAS
SAR
VAL
PNZ
FON
ISC
CPR
MRC
BNP
VMY
FRN
SIR
MTB
BDO
VOR, GOL
(Caucasus)
POZ
NAO
TRE
Fig. 2. The geographical distribution of the Dolichopoda species and populations included in this study.
EDE
Phylogeography and mitochondrial DNA divergence in Dolichopoda
Hereditas 146 (2009)
41
Table 2. Intraspecific genetic variation of the COI and 16S target regions in various Dolichopoda species. Only
species represented by n]7 individuals have been included. S number of polymorphic (segregating) sites, p nucleotide diversity, k average number of nucleotide differences.
Species
D.
D.
D.
D.
D.
laetitiae
geniculata
schiavazzii
ligustica
bormansi
No. of individuals
10
15
16
11
7
COI
16S
COI16S
S
p
k
S
p
k
S
p
k
16
71
31
22
10
0.0066
0.0225
0.0067
0.0085
0.005
6.2
24.193
6.4
7.636
4.667
3
22
2
1
2
0.0041
0.0139
0.001
0.0005
0.0021
1.429
6.333
0.476
0.250
1.000
19
90
32
23
12
0.006
0.0198
0.0047
0.0057
0.0037
7.667
26.819
6.617
7.718
5.238
DIVA analyses
When the maxareas option was not used, and the
analysis was done without the outgroup taxa, the
analysis gave three solutions for the ancestral area
reconstruction at the root node: i) all the areas except
northern Italy (area 2) and Corsica (area 3); ii) all the
areas except Corsica (area 3); and iii) all the areas in
the analyses. When we included the outgroup, but still
not restricting the maxareas, the analysis gave five
different options for the root node: i) 16, ii) 146,
iii) 56, iv) 156, and v) 1456. When
restricting the maximal number of areas (maxareas)
to two and four, and keeping the outgroup, the
analysis gave only one solution for the root node,
Greece and Caucasus (56). The two analyses gave
the same results for all the nodes, except the node
between D. hussoni and the Italian and Pyrenean
species. Maxareas 2 gives two MP solutions: i) 15
or ii) 45; while maxareas 4 gives three MP
solutions: i) 15, ii) 45, or iii) 145. The optimal
reconstruction from both these analyses required four
dispersal events: 1) to the Pyrenees (area 1), 2) to
continental Italy central and south (area 4), 3)
to continental Italy northern part (area 2), and 4) to
Corsica/Sardinia (area 3).
SAMOVA
As expected, the FCT values increased with increasing
number of user-defined groups (K), while FSC decreased (DUPANLOUP et al. 2002). The FCT estimate
did not reach a clear plateau value when plotted
against the K values, though the slope of the curve
decreased slightly when reaching K 9 (data not
shown). Not surprisingly, the Greek species D. rymei
and D. hussoni group first against all other species
included in this study. Already for the lowest K value
(K 4), all D. schiavazzii populations are grouped.
With increasing K, the new groupings followed the
phylogenetic and geographic patterns. With K 7, the
species assigned to the predefined biogeographic areas
in the DIVA analysis group together. However,
biogeographic areas 3 (Corsica and Sardinia) and 4
(continental Italy, central and south) do not group
separately. Instead, there was a group including D.
geniculata and D. laetitiae, a second one including D.
caprensis and D. palpata, and a group containing the
remaining species from biogeographic areas 3 and 4.
With further increasing K more species group separately until at K15 basically all species are found in
separate groups with only D. linderi and D. bolivari
kept together.
DISCUSSION
This study focuses on the phylogeography and
mtDNA variation in the Mediterranean cave cricket
genus Dolichopoda. By means of a dispersal vicariance
analysis we further assessed the importance of vicariance and dispersal in shaping the current distribution
of the genus Dolichopoda in the northern Mediterranaen area.
Phylogenetic considerations
The recovered phylogenetic relationships of Dolichopoda species (Fig. 1) are to a large extent in
good agreement with previously published data
(ALLEGRUCCI et al. 2005). However, geographically
close species did not always turn out as closest
relatives. We recovered a southern Italian clade consisting of D. palpata and D. capreensis that is the sister
group to the coastal western clade consisting of D.
schiavazzii, D. baccettii and D. aegilion, and a clade
including D. bormansi, D. muceddai and D. cyrnensis.
This is in contrast to previous analyses that accepted
D. palpata and D. capreensis as sister group to the
central-Italian clade consisting of D. geniculataD.
laetitiaeD. ligustica (ALLEGRUCCI et al. 2005).
Previous morphological studies of Dolichopoda have
led to the delineation of four subgenera: Capraiacris
Baccetti, 1977; Chopardina Uvarov, 1921; Dolichopoda
Bolivar, 1880; and Petrochilosina Boudou-Saltet, 1980.
42
L. Martinsen et al.
This subgeneric division of Dolichopoda is not reflected in the mtDNA phylogeny. However, evolution
in subterranean habitats is believed to be influenced by
convergence and molecular phylogeny may not necessary mirror morphological differentiation (LEFEBURE
et al. 2006). Bioclimatic factors may be the major
determinants of the morphometric patterns observed
in Dolichopoda (ALLEGRUCCI et al. 1987), and consistent with this one may expect a high degree of
convergence in Dolichopoda.
There is little resolution of the basal nodes in the
phylogeny of Dolichopoda. The relationships between
the Caucasian clade, the two Greek species, and the
major clades of the Italian and Pyrenean Dolichopoda
species, are not properly resolved. The split of the
Caucasian species from the rest of Dolichopoda is only
weakly supported.
The topology within the two Italian groups mainly
supported earlier findings (ALLEGRUCCI et al. 2005),
but provides additional information regarding intraspecific relationships of D. schiavazzii, the Corsica
Sardinian species, and the D. geniculatalaetitiae
complex as well. Little genetic structure was found
within D. schiavazzii which might have been caused by
relatively recent dispersal or gene flow between the
cave populations. Accordingly, all D. schiavazzii populations are grouped in the SAMOVA already with
K4, and kept as a separate group with increasing K.
Previous allozyme analyses have revealed substantial
genetic differentiation between populations of D.
schiavazzii (ALLEGRUCCI et al. 1997).
It was previously suggested that D. baccettii and D.
aegilion were sister taxa, but statistical support values
were low (ALLEGRUCCI et al. 2005). In the phylogenetic analysis presented here, D. aegilion is a sister
group to D. schiavazzii, though also with low statistical support. However, there is strong evidence from
the phylogenetic analyses that D. baccettii, D. aegilion
and D. schiavazzii, form a monophyletic group. However, this is not reflected in the SAMOVA, which
grouped D. aegilion and D. baccetti together with D.
bormansi, D. cyrnensis and D. muceddai (K 46) after
D. schivazzii grouped separately.
The Corsican species D. bormansi is more closely
related to the Sardinian species D. muceddai than to
the other Corsican species D. cyrnensis. This was also
reported earlier by SBORDONI et al. (2004), but was
not addressed in other analyses (ALLEGRUCCI et al.
2005). This may indicate that either two Dolichopoda
lineages evolved in allopatry on Corsica and Sardinia,
and the Sardinian lineage later recolonized Corsica, or
that two species developed on Corsica with one
subsequently spreading to Sardinia.
Hereditas 146 (2009)
We noticed significantly higher sequence diversity
within the D. geniculatalaetitiae species complex than
in the other species. The D. geniculatalaetitiae complex is characterized by high degree of dispersal and
subsequent isolation of populations. D. schiavazzii is
also isolated in several populations, but the sequence
divergence is much lower among these populations.
However, genetic drift may have been a stronger force
in D. geniculataD. laetitiae than in other Dolichopoda
species. The D. geniculatalaetitiae lineage is found in
parts of Italy with a very heterogenous landscape that
is fragmented by mountain ranges and bordered by
the sea. As recently suggested, such landscapes can
foster organismal diversity even though they were
more climatically stable than regions frequented
by glaciers and tundra-like conditions (WEISS and
FERRAND 2007). Earlier studies of the D. geniculata
D. laetitiae complex have shown that peripheral
populations or groups of populations that are found
close to the Tyrrhenian and the Adriatic coast, show
higher degrees of genetic differentiation among
each other and from the central inland populations
than is observed among central inland populations
(SBORDONI et al. 1987). However, this is not reflected
in our mtDNA phylogeny.
Phylogeography
The present distribution of Dolichopoda is congruent
with what is known about refugia for temperate
European species during climatic fluctuations of the
Pleistocene. The three main refugia that have been
deduced from phylogenetic studies for most temperate
species in Europe are Iberia, Italy and the Balkans
(HEWITT 2000). This is exactly where most Dolichopoda species are distributed today. In addition there
are four Dolichopoda species in Turkey and two in the
Caucasus (DI RUSSO et al. 2007).
The DIVA analyses indicated that there has been an
east-west migration of Dolichopoda (or its ancestor)
into the Mediterranean area, since the most eastern
lineage from Caucasus branches off basally in the tree
and is most closely related to the outgroup species.
However, the support value for this is low in the tree
topology. On the other hand this result is in agreement
with previous observations on the migration of
Dolichopoda from the East reviewed by HUBBEL and
NORTON (1978). The authors claimed that no rhaphidophorid species survived the Pleistocene glaciations
north of the Alpine-Carpathian barrier, and that the
post-Miocene immigration of Dolichopodini into
the northern Aegeid and Tyrrhenid areas came from
the region north of the Caspian Sea (HUBBELL and
NORTON 1978). The center of origin for Dolichopoda
seems to be in the eastern part of its range, probably in
Phylogeography and mitochondrial DNA divergence in Dolichopoda
Hereditas 146 (2009)
the Caucasian area or in Greece. Although not
included in this study, the number of Dolichopoda
species in Greece is very high indicating a center of
origin in this area with spreading westward and
eastward to Turkey and the Caucasus.
Since the statistical support for the basal node
connecting D. remyi and D. hussoni with the west
Mediterranean species is low, alternative scenarios for
the colonization of the Mediterranean area also need
to be considered. Parallel colonization of the main
areas of the current Dolichopoda distribution, i.e. the
43
Caucasus, Greece, Italy, and the Pyrenees may also be
possible. The phylogenetic analyses suggests two
possible scenarios for the invasion of Italy. The most
parsimonious is that Italy was colonized once, since all
the Italian species are found in one monophyletic
clade. Within Italy an early vicariance would then have
split the ancestor into two lineages. These two lineages
would have led to the two present Italian groupings in
the phylogenetic tree; one group mainly including
species from western Italian coastal regions and
islands, and the second group including species with
T. cavicola Greece (5), Continental
Italy North (2), Continental Italy
central and south (4)
D. euxina Caucasus (6)
D. remyi Greece (5)
D. hussoni Greece (5)
D. baccettii Mainland Italy Central (4)
D. aegilion Mainland Italy Central (4)
4
4
3+4
D. schiavazzii Mainland Italy Central (4)
D. bormansi Corsica (3)
4
3
D. muceddai Sardinia (3)
D. cyrnensis Corsica (3)
D. palpata Mainland Italy South (4)
4
5+6
D. capreensis Mainland Italy
D. ligustica Mainland Italy North (2)
5
D. geniculata pontiana (PNZ)
Mainland Italy Centra, Ponza Islandl (4)
D. geniculata (VAL, AUS, TUS, PAS)
Mainland Italy Central (4)
4
2:1+5/4+5
4:1+5/4+5/1+4+5
1+4
2+4
4
4
4
4
4
4
D. geniculata (CLP, PIL) Mainland
Italy Central (4)
D. geniculata (PRA) Mainland Italy
Central (4)
D. laetitiae laetitiae Mainland Italy
Central (4)
D. laetitiae etrus c a Mainland Italy
Central (4)
D. geniculata (FON) Mainland Italy
Central (4)
D. geniculata (ISC) Mainland Italy
Central, Ischia Island (4)
D. linderi Pyrenees/France (1)
D. bolivari Pyrenees/Spain (1)
Fig. 3. The results from the dispersal-vicariance analysis of the genus using the program DIVA. The pre-assigned distribution
areas used in the analysis were: 1 The Pyrenees, 2 continental Italy north, 3 CorsicaSardinia, 4 continental Italy
central and south, 5 Greece, 6 Caucasus. The optimal reconstructions at each node are shown for two different values, i.e.
for the maxareas options 2 and 4. The dispersal events are depicted on the figure as blue spots. The analysis is based on the
maximum likelihood tree. Distribution areas are given in parentheses. This tree depicts only the tree topology, branch length is
not informative.
44
L. Martinsen et al.
a predominantly inland distribution. The other possible scenario is that Italy was colonized twice by two
different lineages. Although not most parsimonious,
this scenario more easily explains (i) the close relationship of D. capreensis with D. palpata, but not with D.
geniculatalaetitiae, and (ii) that D.capreensisD. palpata is associated with the D. schiavazzii group but not
with D. geniculatalaetitiae, and (iii) that D. ligustica is
sister group to the D. geniculatalaetitiae complex.
Support for the latter scenario can be found in the
SAMOVA results and the phylogenetic trees. Area 4 as
defined in the DIVA analysis (continental Italy central
and south) does not correspond to a monophyletic
clade in the phylogeny (Fig. 3). In the SAMOVA, area
4 was never recognized as a separate group.
It is unlikely that the last common ancestor of
Dolichopoda had a wide distribution with all populations choosing similar cave habitats at the beginning of
the glacialinterglacial periods of Pleistocene. One can
assume that the last common ancestor for the genus
Dolichopoda was already to a certain degree adapted
to caves. Note, the outgroup taxa in our study are also
cave dwellers, though two of the four species in
Euhadenoecus and several of the Ceuthopilus species
are epigean. It seems reasonable to assume that the
climatic fluctuations during Pleistocene forced the
ancestor of the genus to be more dependent on a
cavernicolous lifestyle. The caves evidently provided an
environment that protected their inhabitants not only
against the rigors during the ice ages but also against
the aridity of the Mediterranean climate that supervened during the interglacials and in post-glacial time
(HUBBELL and NORTON 1978). The dry Mediterranean climate is, together with distance, the major
factor that limits the movement of the cave crickets
outside the caves and therefore gene flow between
populations.
In summary, we suggest that a combination of
vicariance and dispersal events can explain the distribution of Dolichopoda species and populations.
Dispersal can explain the main distributional range
of the genus as demonstrated by the dispersal events in
the DIVA analysis, while vicariance due to habitat
fragmentation may have been the main force for
splitting the species into their current populations
during the glacial-interglacial cycles. The DIVA analysis suggests an eastwest migration of the Dolichopoda into the Mediterranean area. An alternative
explanation is a parallel invasion to the Mediterranean
region from a northern ancestor population that was
forced southward due to changing climate.
Acknowledgements We want to thank Gunnhild Marthinsen, Eirik Rindal, Tor Arne Carlsen, HaĚŠvard Kauserud and
Fahri Saatcioglu for advice when analyzing data and writing
Hereditas 146 (2009)
the manuscript. The project was supported by the ‘National
Centre for Biosystematics’ (Project no. 146515/420), cofunded by the Norwegian Research Council and the Natural
History Museum, University of Oslo, Norway.
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Phylogeography of southern European refugia: evolutionary perspectives on the origins and conservation of
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