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INDEPENDENT GENE PHYLOGENIES AND MORPHOLOGY DEMONSTRATE A
MALAGASY ORIGIN FOR A WIDE-RANGING GROUP OF
SWALLOWTAIL BUTTERFLIES
EVGUENI V. ZAKHAROV,1,2,3 CAMPBELL R. SMITH,4,5 DAVID C. LEES,4,6 ALISON CAMERON,7,8
RICHARD I. VANE-WRIGHT,4,9 AND FELIX A. H. SPERLING1,10
1 Department
of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
2 E-mail: [email protected]
4 Biogeography and Conservation Laboratory, Department of Entomology, The Natural History Museum, Cromwell Road,
London SW7 5BD, United Kingdom
5 E-mail: [email protected]
6 E-mail: [email protected]
7 Faculty of Biological Sciences, School of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
8 E-mail: [email protected]
9 E-mail: [email protected]
10 E-mail: [email protected]
Abstract. Madagascar is home to numerous endemic species and lineages, but the processes that have contributed
to its endangered diversity are still poorly understood. Evidence is accumulating to demonstrate the importance of
Tertiary dispersal across varying distances of oceanic barriers, supplementing vicariance relationships dating back to
the Cretaceous, but these hypotheses remain tentative in the absence of well-supported phylogenies. In the Papilio
demoleus group of swallowtail butterflies, three of the five recognized species are restricted to Madagascar, whereas
the remaining two species range across the Afrotropical zone and southern Asia plus Australia. We reconstructed
phylogenetic relationships for all species in the P. demoleus group, as well as 11 outgroup Papilio species, using 60
morphological characters and about 4 kb of nucleotide sequences from two mitochondrial (cytochrome oxidase I and
II) and two nuclear (wg and EF-1a) genes. Of the three endemic Malagasy species, the two that are formally listed
as endangered or at risk represented the most basal divergences in the group, while the more common third endemic
was clearly related to African P. demodocus. The fifth species, P. demoleus, showed little differentiation across southern
Asia, but showed divergence from its subspecies sthenelus in Australia. Dispersal-vicariance analysis using cladograms
derived from morphology and three independent genes indicated a Malagasy diversification of lime swallowtails in
the middle Miocene. Thus, diversification processes on the island of Madagascar may have contributed to the origin
of common butterflies that now occur throughout much of the Old World tropical and subtemperate regions. An
alternative hypothesis, that Madagascar is a refuge for ancient lineages resulting from successive colonizations from
Africa, is less parsimonious and does not explain the relatively low continental diversity of the group.
Key words.
Biogeography, dispersal, island colonization, Madagascar, Papilionidae, speciation, vicariance.
Received May 7, 2004.
Accepted September 1, 2004.
Islands can play an important role in both generating and
conserving biodiversity. Species radiations on island archipelagos are well studied (e.g., Grant and Grant 2002; Gillespie and Roderick 2002; Jordan et al. 2003), although the
contribution of such island diversification to surrounding
continents remains poorly documented. Under the principles
of island biogeography, island size and distance from the
mainland are the primary determinants of the accumulation
of species that disperse from larger land masses (MacArthur
and Wilson 1967). The likelihood that island species will
colonize continents seems relatively low, because islands
usually contain both fewer species and smaller total populations than mainlands. Furthermore, island species are likely
to be at a competitive disadvantage when islands are colonized by continental species (Gargominy et al. 1996; Whittaker 1998; Cowie 2001; O’Dowd et al. 2003). However, the
widespread assumption that islands contribute little to continental biotas remains relatively untested, and the development of more rigorous methods of phylogenetic and biogeographic analysis, as well as opportunities for the gener3 Present address: 107 Galvin Life Sciences Center, University
of Notre Dame, Notre Dame, Indiana 46556.
q 2004 The Society for the Study of Evolution. All rights reserved.
ation of large molecular datasets that can contribute to more
robust reconstructions, have greatly improved the opportunities to assess such biogeographic hypotheses.
The island of Madagascar is widely known for its unique
and diverse flora and fauna. It has been isolated from Africa
since the late Jurassic and from other Gondwanan elements
such as India since the late Cretaceous (Rabinowitz et al. 1983;
Storey et al. 1995; Briggs 2003). These conditions have led
to the evolution of a very large number of endemic species,
although the processes that have contributed to the biotic diversity within Madagascar are still poorly understood. Many
endemics represent high-ranking, very distinct groups, such as
lemurs and tenrecs. As a result, and because much of the
island’s biodiversity is now under threat of extinction, Madagascar is considered one of the world’s 10 most important
regions for biodiversity conservation (Myers et al. 2000).
In spite of the proximity of Madagascar to Africa since its
separation from that continent about 165 million years ago,
the Malagasy flora exhibits remarkably high affinity with
Indo-Australo-Malesian floras far to the east (Schatz 1996).
Such phytogeographic connections are especially prevalent
among eastern humid forest taxa, and in some cases may
represent relictual Cretaceous Gondwanan disjunctions, as
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well as repeated long-distance or stepping-stone dispersal
events across the Indian Ocean. Although the fauna of Madagascar shows more connections with Africa (Paulian 1972;
Warren et al. 2003), some Oriental affinities are still present.
For example, several Malagasy birds show clear affinities
with Oriental groups that are unknown in Africa (Dorst 1972).
The presence of some lineages of plants and animals in Madagascar may be explained by Cretaceous dispersals to the
island from India or South Africa, and floral and faunal exchange with the remains of Gondwana via Antarctica during
the time of the initial radiation of the angiosperms (Koechlin
1972; Schatz 1996; Vences et al. 2001). This scenario may
account for the evolution of seven endemic plant families
and three recently extinct ratite species (Cooper et al. 2001).
In addition, recent phylogenetic evidence and fossils suggest that part of the modern vertebrate fauna of Madagascar
had an independent origin from that of the Cretaceous fauna
of Madagascar (Krause et al. 1999). There is growing evidence for nontectonic dispersal to Madagascar (and Africa)
from Laurasia and western Malesia via India along Lemurian
stepping-stones in the western Indian Ocean during the Eocene-Oligocene (McKenzie and Sclater 1973; Schatz 1996;
Jansa et al. 1999). Late Tertiary introduction by rafting has
been suggested for an endangered endemic tortoise in Madagascar (Caccone et al. 1999). Neogene colonization of Madagascar by African ancestors has been reported in Malagasy
oscine songbirds (Cibois et al. 2001) and sunbirds in the
Pliocene (Warren et al. 2003). Yoder et al. (1996) found that
the Malagasy lemuriforms comprise a monophyletic group
that probably dispersed from Africa in the early Tertiary and
a single African Miocene origin is also posited for carnivores
(Yoder et al. 2003). Thus, multiple overseas dispersals to and
from Madagascar have been proposed both for plants and
animals (see also Renvoise 1979; Fisher 1996; Raxworthy et
al. 2002; Treutlein and Wink 2002; Nagy et al. 2003; Vences
et al. 2003, 2004). Nonetheless, such hypotheses of overseas
dispersal remain tentative in the absence of more and better
supported phylogenetic evidence for the relationships of different elements of the Malagasy biota.
Complex patterns of vicariance and dispersal, along with
long isolation of the island, account for much of the endemism of the Malagasy biota. At the species level, at least
80% of the Malagasy flora and 75% of its fauna is endemic
(Schatz 2001), and this is also the level of species endemism
for butterflies (Lees et al. 2003). For the conservation of
invertebrates, butterflies of the family Papilionidae have been
championed as a flagship group (Collins and Morris 1985).
Thirteen species of the family are found on Madagascar (Paulian and Viette 1968), of which 10 are endemic to mainland
Madagascar and the Comoros (Ackery et al. 1995). Three of
the endemics are currently classified by the IUCN as threatened (IUCN 2003), and one of these belongs to the Papilio
demoleus group, the subject of this study.
The P. demoleus group, or lime swallowtails, comprises
five butterfly species from the Old World tropics that share
a distinctive black and yellow pattern. Three of them, P.
erithonioides Grose-Smith, 1891, P. grosesmithi Rothschild,
1926, and P. morondavana Grose-Smith, 1891, are found only
on Madagascar, while the other two have wide distributions
in the Afrotropical (P. demodocus Esper, 1798) and IndoAustralian (P. demoleus Linnaeus, 1758) regions.
Papilio demodocus occurs throughout sub-Saharan Africa,
extending into Saudi Arabia, Yemen, and Oman. Its existence
on Madagascar, Mauritius, Réunion, and the four Comoro
Islands is likely to be the result of recent and perhaps even
deliberate introductions (Paulian 1951; Paulian and Viette
1968; Turlin 1994), though it is also possible that the species
arrived naturally in Madagascar as two of us (D. C. Lees and
A. Cameron) have observed it occasionally in natural habitats. The species is abundant in anthropogenic habitats and
is not threatened.
The larvae feed mostly on Rutaceae but with no record on
native species and the species is considered to be a pest of
Citrus crops (Paulian and Viette 1968; Collins and Morris
1985; Ackery et al. 1995). Although some populations in
South Africa have switched to larval feeding on Apiaceae
(van Son 1949; Clarke et al. 1963), there is no evidence that
they have done this in Madagascar. Occasional use of Anacardiaceae, Asteraceae, Ptaeroxylaceae, and Sapindaceae as
larval host plants has been reported (Ackery et al. 1995),
though these records may represent oviposition mistakes because larvae of P. demodocus are not known to develop adequately on host plants in these families. Two subspecies are
recognized, P. demodocus demodocus Esper, 1798, from virtually the entire range, and the distinctive P. demodocus bennetti Dixey, 1898, originally described as a separate species
from Socotra Island (Yemen).
The distribution of P. demoleus extends from Iran through
India, Sri Lanka, Malaysia, southern China, and Japan, and
from the Lesser Sunda Islands to mainland New Guinea and
Australia. The species was absent from the Philippines, Sumatra, Java, and Borneo until its recent invasion of the islands
after their large-scale deforestation by man (Hiura 1973; Corbet and Pendlebury 1978, 1992). Most recently, the species
is now established on the northern tip of Sulawesi (VaneWright and de Jong 2003). Six subspecies are currently recognized: demoleus Linnaeus, 1758 (China through South Asia
and Pakistan to the Arabian Peninsula); malayanus Wallace,
1865 (Malay Peninsula and Sumatra); libanius Fruhstorfer,
1908 (Philippines, Talaud, Sula, Taiwan); sthenelus Macleay,
1826 (Sumba and Australia); novoguineensis Rothschild,
1908 (Papua New Guinea); and sthenelinus Rothschild, 1895
(Flores and Alor). In Papua New Guinea and in Australia,
the larvae develop on wild leguminous plants of the genus
Cullen (Fabaceae; Braby 2000), but no host record is available for the Lesser Sunda island populations (Parsons 1999).
All Asian subspecies feed on Citrus, which is commonly
planted as a crop or ornament in towns and smaller settlements and probably facilitates range expansion of P. demoleus in Asia (Matsumoto 2002).
The rarest of the three Malagasy endemics, P. morondavana, is currently classified as potentially ‘‘endangered’’
while ‘‘data deficient’’ (IUCN 2003). It is confined to dry
deciduous forests of west-southwestern and northern Madagascar and is threatened by loss of habitat (Collins and Morris 1985; Conservation International 2003). Our field observations and museum collections suggest that P. grosesmithi
(lower risk: near threatened) is more commonly encountered
than P. morondavana. It is found over a very similar geo-
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graphic range below about 800 m in dry deciduous and gallery
forests of western and northern Madagascar, ranging east as
far as Fianarantsoa, and slightly further southwest than P.
morondavana, while P. morondavana is known from the far
north. According to field observations in 2001–2002 (A.
Cameron), P. morondavana can be abundant at the beginning
of the wet season at some sites, but appears to be more localized than P. grosesmithi.
Papilio erithonioides (not classified as threatened) occurs
in spiny forests of the far southwest as well as deciduous
and gallery forests in the southwestern, western, and northern
parts of the island, also penetrating marginally into northwestern and southwestern rainforests. Our recent observations show it to be by far the most common endemic. Significantly, all three species can be found sympatrically at
some sites, notably the unprotected Kirindy Forest near Morondavana. Papilio demodocus is found in anthropic habitats
throughout the island including the plateau. Further details
of the nomenclature, distribution, and bionomics will be provided in a companion publication (C. R. Smith and R. I. VaneWright, unpubl. ms.).
Although the life history and geographic variability of the
P. demoleus species group is relatively well known (at least
outside Madagascar), their phylogenetic relationships remain
unknown and pose questions about their origin and evolution.
Carcasson (1964) considered P. demodocus to be the ancestral
species, from which the three species endemic in Madagascar
were isolated, one after another, as the result of multiple
invasions of a mainland form. This model corresponds to the
concept of duplex species, in which the rare species, with a
more restricted range, owes its origin to isolations and reunions with the broader range of a sister species (Zeuner
1943; Corbet 1944). However, in this case the vicariance that
is normally caused by sea-level rise is replaced by dispersal
of a relatively mobile species across a fixed sea level gap.
In contrast to a sequential colonization scenario, speciation
of Malagasy endemics solely on Madagascar, whether in different parts of the island or in contact with each other, remains
a possibility for the P. demoleus group. Endemic Malagasy
radiations have been reported for a variety of taxa such as
tortoises, songbirds, butterflies, and colubrid snakes (Caccone et al. 1999; Cibois et al. 2001; Torres et al. 2001; Nagy
et al. 2003). However, only a handful of recent studies have
suggested that the Malagasy biota has contributed to the surrounding continents.
Here, we report results of phylogenetic analyses based on
morphology and nucleotide sequences of two mitochondrial
genes, cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII), and two nuclear genes, wingless (wg),
and elongation factor 1a (EF-1a). We use inferred phylogenies combined with estimated divergence times to reconstruct ancestral areas for the P. demoleus species group and
to identify patterns of species dispersal that have contributed
to the enigmatic biodiversity of Madagascar. Moreover, any
improvement in our understanding of the systematics of these
butterflies provides a better foundation for appropriate conservation action (Vane-Wright 2003).
MATERIALS
AND
METHODS
Sampling Strategy
Sampled species and GenBank accession numbers are given in Table 1. Ingroup taxa included all five recognized species of the P. demoleus group, with sampling across species
ranges where possible. Four out of six described subspecies
of P. demoleus were sampled; P. d. novoguinensis and P. d.
sthenelinus were not available for this study. Both subspecies
of P. demodocus were sampled, including P. d. demodocus
and P. d. bennetti (a taxon restricted to Socotra Island, Yemen). However, only two legs from two old pinned specimens
collected in 1967 (British Museum of Natural History Entomology Department, specimen register nos. 220150 and
220151) were available for DNA extraction for P. d. bennetti.
Eleven outgroup taxa were chosen, representing major
monophyletic lineages within the genus based on a recent
reconstruction of Papilio phylogeny (Zakharov et al. 2004)
to test the monophyly of the P. demoleus group and to relate
its phylogenetic position within Papilio. Trees were rooted
with a single neotropical species, Papilio thoas. The original
data and tree files are available from www.treebase.org (accession number SN1870).
Morphology
A total of 60 characters (Fig. 1, Appendix) were scored
by C. R. Smith from wing patterns and male and female
genitalia for the five species of lime swallowtails (including
both subspecies of P. demodocus) and all outgroup taxa.
Where material was available, male and female genitalia were
examined in several specimens of each species. Dissections
were made using standard techniques, after abdomens were
soaked in cold 10% KOH solution overnight and subsequently stored in glycerol. Voucher information for specimens
scored for morphological characters is available as supplementary online materials at http://dx.doi.org/10.1554/04293.1.s1.
Although all species of the P. demoleus group share the
distinctive wing pattern of the group, some problems in homology assessment of wing pattern elements were encountered in scoring outgroup species. Certain elements of wing
pattern in some species of Papilio are fused or lost completely, making the wing pattern very different from a generalized swallowtail groundplan of black and yellow stripes,
spots, and patches. To help hypothesize homology, we evaluated the position of pattern elements on the wing with respect to wing venation and examined series of related species.
Some genitalic homologies were likewise problematic. Further details of the wing patterns and male and female genital
anatomy, and the character hypotheses derived from them,
will be provided in a companion publication (C. R. Smith
and R. I. Vane-Wright, unpubl. ms.).
Molecular Techniques
Selection of genes for molecular phylogenetic reconstruction was based on previous successful studies in Lepidoptera
(Sperling 2003) and other insects (Caterino et al. 2000). The
two most commonly used mitochondrialDNA protein-coding
genes, COI and COII, which are generally most useful in
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delineating taxa at the species level (Sperling 2003; but see
Wahlberg et al. 2003), also represent the maternal history of
speciation in the P. demoleus species-group. Two other loci,
wg and EF-1a, are the most commonly used protein-coding
nuclear genes in phylogenetic studies in Lepidoptera and are
usually considered useful at higher taxonomic levels due to
their lower substitution rates (Sperling 2003). Both nuclear
genes appear to have only a single copy in lepidopteran genome and have either no introns (EF-1a) or a 450-bp exon
between introns (wg).
Mitochondrial genes COI, tRNA-leu, COII and nuclear
genes wg and EF-1a were sequenced new by E. V. Zakharov
or retrieved from GenBank for all 11 outgroups and two
specimens per species (10 total) in the ingroup. For EF-1a,
an additional fragment of about 245 bp was sequenced from
the 39-end to extend previously published sequences that were
incomplete for the gene (Reed and Sperling 1999). An 825bp fragment comprising the 39-end of the COI gene was sequenced for an additional 40 ingroup individuals, except the
two specimens of P. demodocus bennetti, which produced
only highly degraded DNA.
Material of varying quality was used for DNA extraction,
ranging from alcohol-preserved freshly caught samples to
dried nonrelaxed specimens and legs of museum pinned butterflies. Total genomic DNA was extracted either by standard
phenol-chloroform procedure as in Sperling and Harrison
(1994) or using a Qiagen (Valencia, CA) DNeasy tissue kit.
Polymerase chain reactions (PCR) were performed in Biometra (Goettingen, Germany) TGradient or TPersonal thermal
cyclers using reaction and cycling conditions described previously (Brower and DeSalle 1998; Caterino et al. 2001).
PCR products were cleaned using a Qiagen QIAquick PCR
purification kit when only a single DNA band was visible in
a gel or, when more than one band was observed, a combination of gel-separation and subsequent purification with a
Qiagen QIAEX II gel extraction kit. Purified PCR products
were directly sequenced using DYEnamicTM ET terminator
cycle sequencing (Amersham Pharmacia Biotech, Cleveland,
OH) or Applied Biosystems (ABI, Foster City, CA) Big Dye
terminator cycle sequencing, under manufacturer’s recommendations. Fluorescently labeled sequencing products were
filtered through Sephadex-packed columns, dried, resuspended, and fractionated on an ABI 377 automated sequencer.
All fragments were sequenced in both directions using the
same primers that were used for PCR (Appendix 2, available
online at http://dx.doi.org/10.1554/04-293.1.s2). Sequences
were assembled into contiguous arrays using Sequencher,
version 4.1 (GeneCode Corp., Ann Arbor, MI).
Phylogenetic Analysis
For morphological data, most parsimonious cladograms
were inferred from the equally weighted and unordered data
matrix using a branch-and-bound search in PAUP* version
4.0b10 (Swofford 1998). Bootstrap analysis was done in
PAUP using 1000 replicates and used heuristic searches with
tree bisection-reconnection (TBR) branch swapping with no
additional random replicates. The results of maximum parsimony analysis were checked using a heuristic search algorithm (with 10 random additional replicates and TBR
branch swapping) of NONA 2.0 (Goloboff 1999) spawned
with the aid of WinClada (Nixon 2002). Bayesian analysis
of morphological dataset was performed in MrBayes version
3.04b (Huelsenbeck and Ronquist 2001) with the matrix treated as standard binary characters (DATATYPE 5 STANDARD), we ran four chains with 5.0 3 105 generations sampling every 100th tree. The first 2500 sampled trees were
discarded before computing a consensus tree.
Alignment of nucleotide sequences for all genes was trivial
due to lack of indels and was done by eye with the aid of
BioEdit version 5.0.9 (Hall 1999). MEGA version 2.1 (Kumar
et al. 2001) was used to calculate gene statistics such as transition/transversion ratios. PAUP* was used for all parsimony,
bootstrap, and decay analyses of molecular and combined data.
All parsimony analyses of nucleotide data used heuristic
searches (exhaustive search was applied to morphological
data) with 100 random addition sequence replicates and TBR
branch swapping. All base positions were treated as unordered
equally weighted characters. Gaps were treated as missing
data, and multistate characters as polymorphisms. Nonparametric bootstrap probabilities (BP) were based on 500 repetitions of heuristic searches with only 10 random addition
replicates and TBR branch swapping. To determine the number
of additional steps needed to accommodate alternative phylogenetic hypotheses, constraint searches were carried out for
each dataset (Bremer 1988). Decay indices were extracted using the program TreeRot (Sorenson 1999).
Bayesian phylogenetic analyses were conducted for combined and partitioned datasets in MrBayes version 3.04b
(Huelsenbeck and Ronquist 2001) with the general-time reversible model with gamma distributed rates and proportion
of invariable sites applied to the molecular portion of data.
Values of specific parameters for the nucleotide substitution
model were estimated as part of the analysis and were allowed
to vary for individual genes in the combined analyses. We
ran four chains simultaneously, three heated and one cold.
Each Markov chain was started from a random tree and run
for 1.0 3 106 generations, sampling the chains every 100th
cycle. The log-likelihood scores of sample points were plotted against generation time to determine when the chain became stationary. The first half of the sampled trees was discarded as burn-in samples. We ran each analysis three times,
each beginning with different random starting trees, and compared their apparent stationarity levels for convergence
(Huelsenbeck and Bollback 2001). Data remaining after discarding burn-in samples were used to generate a majorityrule consensus tree, where percentage of samples recovering
any particular clade represented the clade’s posterior probability (Huelsenbeck and Ronquist 2001). Probabilities of
95% or higher were considered as significant support. The
mean, variance, and 95% credibility interval were calculated
from the set of substitution parameters.
Dispersal-Vicariance Analysis
To reconstruct the distribution history of the P. demoleus
group we used the dispersal-vicariance approach implemented
in program DIVA (Ronquist 1996). In DIVA a fully bifurcated
phylogeny is used to optimize the distribution of ancestral
species with parsimony. The method is based on optimization
Locality
malayanus
malayanus
malayanus
malayanus
Indonesia: Bali Island
Thailand: Chiang Mai
Thailand: Chiang Mai
Vietnam: Bach Ma Nat.
Park
Iran: Hormozgan Province
Taiwan
Taiwan
Taiwan
Australia: New South
Wales
Australia: New South
Wales
Kenya (ex pupa)
Kenya (ex pupa)
Cameroon: Buea
Cameroon: Buea
Cameroon: Buea
Central African Republic
South Africa: Nelspruit
South Africa: Nelspruit
South Africa: Nelspruit
South Africa: Nelspruit
Madagascar: Antsalova
Madagascar
Madagascar: Tsimelahy
Madagascar: Mahavelo
Madagascar: Mahafalay
Madagascar
Madagascar
Yemen: Socotra Island
Yemen: Socotra Island
Madagascar: Ankitsanga
Madagascar: Mahavelo
Madagascar: Tsimelahy
Madagascar: Bealoka
Madagascar:Bemanateza
Madagascar: Tsifota
Madagascar: Mitoho
Madagascar: Beroboka
Madagascar: Kirindy
Nord
Madagascar: Ambahibe
Madagascar: Sept Lac
Madagascar: Tsimelahy
Madagascar: Zombitse
11.
12.
13.
14.
Papilio
Papilio
Papilio
Papilio
demoleus
demoleus
demoleus
demoleus
libanius
libanius
libanius
sthenelus
15. Papilio demoleus sthenelus
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus demodocus
demodocus bennetti
demodocus bennetti
erithonioides
erithonioides
erithonioides
erithonioides
erithonioides
erithonioides
erithonioides
erithonioides
grosesmithi
44.
45.
46.
47.
Papilio
Papilio
Papilio
Papilio
grosesmithi
grosesmithi
grosesmithi
grosesmithi
AY569114
AY569104
AY569115
May 16, 1989
May 16, 1989
April 2002
April 2002
September 30,
2002
June 1998
May 10, 2001
May 10, 2001
June 10, 2001
CU: Lot No. 1204
UAFS:a48
UAFS:1650
UAFS:1651
SHY:1855
DL1
DL1
DL1
DL1
DL2
AY569049
AY569050
AY569051
AY569058
UAFS:a398
NP:DL01-Q677
NP:DL01-Q676
UAFS:1656
DL3
DL4
DL1
DL1
AY569048
AY569055
AY569056
AY569052
March 11, 2002
UAFS:1871
DL5
AY569053
September 2002
September 2002
September 2002
November 12,
2000
November 12,
2000
March 15, 1989
March 15, 1989
March 11, 2002
March 11, 2002
March 11, 2002
2001
January 16, 2003
January 16, 2003
January 16, 2003
January 16, 2003
February 12, 2002
May 16, 1989
January 19, 2002
February 22, 2002
February 22, 2002
2001
2001
1967
1967
February 6, 2002
January 31, 2001
January 17, 2002
February 4, 2002
March 24, 2002
March 9, 2002
March 19, 2002
Mar 13, 2002
January 20, 2002
SHY:1856
SHY:1857
SHY:1858
NP:RE02-A018
DL1
DL1
DL1
DL6
AY569059
AY569057
AY569060
AY5690926
NP:RE01-H070
DL7
AY569067
UAFS:a146
UAFS:1872
UAFS:1653
UAFS:1654
UAFS:1655
UAFS:1652
UAFS:1927
UAFS:1928
UAFS:1930
UAFS:1929
BMNH(E):697423
UAFS:a58
CASENT:8001338
CAS:8001418
CAS:8001794
UAFS:1801
UAFS:1802
BMNH(E):220150
BMNH(E):220151
BMNH(E):697422
CAS:8001477
CAS:8001225
CAS:8001520
CAS:8002601
CAS:8002221
CAS:8002417
CAS:8002274
BMNH(E):697420
DD1
DD2
DD3
DD4
DD5
DD6
DD7
DD8
DD9
DD10
DD11
DD11
DD12
DD13
DD11
DD14
DD14
AY4575885
AY569070
AY569071
AY569068
AY569069
AY569072
AY569073
AY569074
AY569075
AY5690916
AY569064
AY569063
AY569065
AY569066
AY569067
AY569061
AY569062
AY4576145
AY569112
AY569103
AY569113
E1
E1
E1
E1
E1
E1
E1
E1
G1
AY5690956
AY5690966
AY569076
AY569077
AY569078
AY569079
AY569080
AY569081
AY5690896
AY569099
AY569100
AY569108
AY569109
AY569101
AY569110
BMNH(E):697421
CAS:8001969
CAS:8001315
CAS:8034300
G2
G3
G2
G3
AY5690906
AY569082
AY569083
AY569084
AY569102
AY569111
January 31, 2002
March 1, 2002
January 18, 2002
February 6, 2003
GALLEY 180
demoleus
demoleus
demoleus
demoleus
Wingless
AF0448252,4
DTPro System
Papilio
Papilio
Papilio
Papilio
EF-1a
•
6.
7.
8.
9.
10. Papilio demoleus demoleus
Penang Island
Penang Island
Perak
Perak
AF0440003
Allen Press
Malaysia:
Malaysia:
Malaysia:
Malaysia:
Malaysia
COI, COII
pg 180 # 5
File # 19TQ
malayanus
malayanus
malayanus
malayanus
malayanus
Haplotype
Plate # 0-Composite
demoleus
demoleus
demoleus
demoleus
demoleus
Voucher ID 1
11/12/2004 04:17PM
Papilio
Papilio
Papilio
Papilio
Papilio
Date
EVGUENI V. ZAKHAROV ET AL.
1.
2.
3.
4.
5.
List of species and specimens examined.
L
Species
Name /evol/58_1219
TABLE 1.
Allen Press
Name /evol/58_1219
•
Locality
Madagascar: Ambovonaomby
Madagascar: Androngonibe
Madagascar: Androngonibe
51. Papilio morondavana
52. Papilio morondavana
Outgroup taxa
53. Papilio helenus
54. Papilio paris
55.
56.
57.
58.
59.
60.
Papilio
Papilio
Papilio
Papilio
Papilio
Papilio
constantinus
delalandei
lormieri
oribazus
xuthus
machaon
61. Papilio anactus
62. Papilio canadensis
63. Papilio thoas
Japan (ex pupa)
China: Guandong Province
Kenya (ex pupa)
Madagascar: Talatakely
Central African Republic
Madagascar: Talatakely
Japan: Tokyo
France: Coudoux (ex
pupa)
Australia: Queensland
USA: New York: Richford
French Guiana
Haplotype
COI, COII
EF-1a
Wingless
February 6, 2003
November 30,
2001
November 6, 2002
CAS:8034301
CAS:8000801
G4
M1
AY569085
AY5690936
AY569097
AY569106
CAS:8002996
M1
AY5690946
AY569098
AY569107
November 20,
2002
November 20,
2002
CAS:8033022
M1
AY569086
CAS:8033048
M1
AY569087
March 29, 1990
June 20, 2001
UAFS:a90
UAFS:1572
AY4575755
AY4575745
AY4576195
AY4576055
AY569121
AY569116
1989
April–May 1998
July 22, 2001
April–May, 1998
August 30, 1990
February 18, 1988
CU: Lot No. 1204
UAFS:939
UAFS:1875
UAFS:941
CU: Lot No. 1204
CU: Lot No. 1204
AF0440023
AY4575855
AY5690886
AY4575915
AF0439993
AF0440063
AF0448362, 4
AY4576135
AY569105
AY4576265
AF0448382, 4
AF0448282, 4
AY569118
AY569117
AY569122
AY569119
AY569123
AY569124
1997
June 5, 1997
UAFS:942
CU: Lot No. 1204
AY4575925
AF0440143
AY4576085
AF0448162, 4
AY569120
AY569125
May 30, 1990
UAFS:302
AY4576015
AY4576325
AY569126
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50. Papilio morondavana
Voucher ID 1
Plate # 0-Composite
Madagascar: Zombitse
Madagascar: Kirindy
Date
ORIGIN OF ISLAND ENDEMICS
48. Papilio grosesmithi
49. Papilio morondavana
Continued.
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TABLE 1.
1
File # 19TQ
Voucher locations are indicated as: CU, insect collection (see Caterino and Sperling 1999) of the Department of Entomology at Cornell University, Ithaca, NY; UAFS, DNA and tissue collection
in F.A.H.S. Laboratory at the University of Alberta, Edmonton, AB; BMNH (E), Department of Entomology at Natural History Museum, London, U.K.; CASENT, Department of Entomology at
California Academy of Sciences, Golden Gate Park, San Francisco, CA; SHY, specimens from private collection of Shen-Horn Yen; NP, DNA and tissue collection in Naomi Pierce lab at Harvard
University, Cambridge, MA.
2 A 245-bp fragment was sequenced to extend 39 end of previously published sequence of EF-1a (Reed and Sperling 1999).
3 (Caterino and Sperling 1999).
4 (Reed and Sperling 1999).
5 (Zakharov et al. 2004).
6 Specimens were sequenced for full 2.3 kb of COI 1 COII in addition to 825-bp fragment at 39 end of COI.
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EVGUENI V. ZAKHAROV ET AL.
FIG. 1. Edited digital images showing morphological characters, with circled numbers referring to numbered character states (Appendix).
(A) Upper and undersides of Papilio demoleus demoleus. Ellipses on wings indicate the position of character traits. (B) Male genitalia
of P.demodocus bennetti. (C) Female genitalia of P. erithonioides. All character states shown are coded as (1) unless indicated otherwise.
of a three-dimensional cost matrix derived from a simple biogeographic model. Distributions are described in terms of a
set of unit areas, and speciation is assumed to divide ancestral
distributions allopatrically into mutually exclusive sets of unit
areas. DIVA finds the optimal distributions of ancestral species
by minimizing the number of dispersal and extinction events.
Unlike other methods in historical biogeography, areas are not
required to be hierarchically related, and DIVA does not make
any assumptions about the shape or existence of general patterns of area relationships.
We used the trees obtained from bootstrap analysis of our
combined and partitioned datasets. The distribution of each
species was classified as present/absent in four different areas.
In the optimization we allowed a maximum of two geographical areas for ancestral species, under the assumption that ancestral populations had limited geographical distributions.
RESULTS
Data Description
Among 60 morphological characters scored for the P. demoleus group, 16 characters in one or more outgroup species
were scored missing or inapplicable being dependent on a
character itself scored as absent (0; Table 2). Two characters
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ORIGIN OF ISLAND ENDEMICS
TABLE 2. Morphological data matrix, for Papilio species. Characters that could not be scored with confidence were coded as doubtful
(?), missing or inappropriate (2), or polymorphic (*).
Characters
Species
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
thoas
canadensis
machaon
anactus
xuthus
paris
helenus
constantinus
lormieri
delalandei
d. demodocus
d. bennetti
oribazus
erithonioides
morondavana
grosesmithi
demoleus
123456789111111111122222222223333333333444444444455555555556
012345678901234567890123456789012345678901234567890
0--010110010100001111000310000100001101101100001110110001110
0--0?011?0101011111111002000001000000000011000011111101010-10-01111?0101111111111002001000---0100110110010111010----100
0--01111?010101110-111000-000011010100100110000111010----100
1100111100101011111111003001000---01000101100001110110101100
10-00000000*101100-10-003100000---01001001100001110010011100
110000000000101000-10-003110000---000110011000010-1111101100
10-0111100100-0000-010103111000---01000001100101101110000100
110001010010100001011111311000101010000100100001110110101100
10-000010010101110-011113110000---11000101100001100110101100
1111111110111000010110000-0110100000001101110001110010001100
111111111011100001011000100110100001001101110001110010001100
0--0110100000-1110-011002100000---010000011000010-0010111100
111111111111110001111100100010100000001101110001110010001100
10-111111111101011111100200010100001001111110001111111100110
111111111011110001111100200111110111001101110001111011100100
111111*011*11000*11111000-01101110001000011011110-1010110101
were scored as doubtful, and four characters were polymorphic. Across all species, two characters were nonvariable and
eight variable characters were parsimony uninformative,
leaving 50 parsimony informative characters for the whole
dataset. For the ingroup alone, the number of nonvariable
characters increased to 11 and number of informative characters decreased to 31.
The final alignment of DNA sequences numbered 3929
nucleotides in the combined dataset, including 1532 bp of
COI, 68 bp of tRNA-leu, 685 bp of COII, 404 bp of wg, and
1240 bp of EF-1a. GenBank accession numbers for sequences
are given in Table 1. Statistics for the ingroup and for all
taxa are given in Table 3. Within the ingroup, COI and COII
had the highest proportion of informative characters (10.9%
and 10.3%, respectively), while wg had 5.9% and EF-1a only
3.9% parsimony informative characters. The tRNA-leu sequences had only one variable nucleotide in the ingroup, and
for that reason this fragment was excluded from all further
analyses except the combined dataset with all outgroup taxa.
The number of informative characters varied over genes and
nucleotide positions and, as is common in such data (Reed
and Sperling 1999), most substitutions were observed in third
codon positions and second codon positions were the most
conservative in all genes. Very few or no substitutions were
registered in first and second codon positions in nuclear genes
in the ingroup. Relatively low levels of homoplasy were registered in all genes for the ingroup, while the inclusion of
outgroups increased homoplasy in nucleotide data as seen
from CI and RI values (Table 3). The same was true for
saturation levels, with transition/transversion ratios declining
in the total dataset compared to the ingroup.
Plotting p-distances versus maximum-likelihood corrected
distances under GTR 1 G 1 I revealed higher levels of
saturation in COII and COI and lower amounts in nuclear
datasets (data not shown). The average percent sequence divergence (p-distance) for the ingroup ranged from 1.7% in
EF-1a to 2.4% in wg and 5.1% both in COI and COII, while
adding outgroups increased the amount of divergence to 4.4%
in EF-1a, 7.7% in wg, 8.0% in COII, and 8.4% in COI.
Phylogenetic Analyses
A branch-and-bound search in PAUP retained six most
parsimonious trees for morphological data (140 steps, CI 5
0.429, RI 5 0.529). Alternative topologies for both ingroup
and outgroup relationships were recovered and no resolution
was obtained in the strict consensus tree for any outgroups
except paris 1 helenus and machaon 1 xuthus (Fig. 2). The
strict consensus also had an unresolved trichotomy between
P. erithonioides, P. demodocus and ((P. demoleus, P. grosesmithi), P. morondavana). Levels of support on the morphological tree were very low, and did not allow confidence about
phylogenetic relationships between species in the P. demoleus
species-group. However, the bootstrap consensus tree gave
a basal position for P. morondavana. Also, when the most
distant outgroups (based on highest pairwise sequence divergences from the ingroup), P. thoas and P. canadensis, were
removed from the maximum parsimony analysis and P. anactus was used as an outgroup as the next most distant relative
of lime swallowtails (Zakharov et al. 2004), the strict consensus of four most parsimonious trees also showed a basal
position for P. morondavana. NONA heuristic searches of
the morphological data recovered the same six trees that were
found by branch-and-bound algorithm in PAUP.
Heuristic searches of molecular data with the full set of
outgroup species resulted in a single tree (1157 steps) for
COI data, five trees for COII (469 steps), one tree for COI
1 COII (1641 steps), one tree for wg (267 steps), one tree
for EF-1a (476), four trees for wg 1 EF-1a (757 steps), and
one tree (2415 steps) for all genes combined (Fig. 3, outgroup
taxa are not shown). Two most parsimonious trees (2571
steps) were found for all data including molecular and morphological characters. When more than one most parsimonious tree was found for a particular partition, the ingroup
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EVGUENI V. ZAKHAROV ET AL.
TABLE 3.
Morphological
No. characters
60
Morphological and nucleotide variability.
tRNAleu
COI
All
1532
1st
511
2nd
510
3rd
511
All
68
COII
All
685
1st
229
2nd
228
3rd
228
Ingroup only (Papilio demoleus group)
No. variable
53
196
No. informative
36
158
Autapomorphies
17
38
CI
0.679
0.831
RI
0.591
0.840
Ti/Tv
1.960
Overall average p
0.051
29
24
5
0.882
0.900
7.810
0.022
4
3
1
1.000
1.000
0.706
0.003
163
131
32
0.824
0.831
1.636
0.129
1
1
0
1.000
1.000
—
0.005
88
75
13
0.872
0.884
1.844
0.051
16
13
3
0.800
0.833
6.000
0.027
4
1
3
1.000
1.000
0.657
0.005
68
61
7
0.902
0.917
1.328
0.119
Ingroup 1 outgroup
No. variable
No. informative
Autapomorphies
CI
RI
Ti/Tv
Overall average p
92
56
36
0.587
0.594
5.587
0.040
15
5
10
0.882
0.875
1.244
0.005
355
254
101
0.510
0.489
0.877
0.205
5
2
3
1.000
1.000
1.450
0.013
205
132
73
0.561
0.569
1.311
0.080
48
25
19
0.610
0.709
6.562
0.051
9
3
6
0.923
0.750
0.620
0.006
148
100
48
0.552
0.352
0.923
0.184
58
50
8
0.529
0.429
462
315
147
0.561
0.569
1.130
0.084
topologies were identical among trees found for all data except for the wg 1 EF-1a dataset. Each gene partition produced alternative groupings for the P. demoleus group. However, only COI, COI 1 COII, and all combined data produced
relatively robust trees.
MitochondrialDNA data suggest that P. morondavana is the
most basal species in the group, while nuclear genes indicate
P. grosesmithi. All partitions confirm the monophyly of the P.
demoleus species-group with 91–100% bootstrap proportions
and decay indices of 6–12, except for morphological data that
had a 76% bootstrap proportion and decay index of 4. Combining data increased support both for monophyly of the P.
demoleus species-group and intragroup relationships.
Bayesian analyses of mitochondrialDNA genes and partitioned wg and EF-1a revealed the same patterns of relationship among ingroup taxa that were obtained in corresponding maximum parsimony heuristic searches. Bayesian
analysis of combined nuclear genes converged on the tree
topology recovered for the wg dataset. As in maximum parsimony searches of molecular data, the same tree topology
was found for COI, COI 1 COII and all four genes combined.
Combining all four genes and morphological data in Markov
chain Monte Carlo analyses with all substitution model parameters being estimated individually for gene partitions resulted in the highest levels of ingroup node support.
The trees shown in Figure 4 illustrate the cladograms obtained for all data combined, for both the P. demoleus group
and representative Papilio outgroups. MP and Bayesian analyses recovered trees with an almost identical pattern of relationships, with the only difference in the position of P.
anactus. Both maximum parsimony and Bayesian analyses
showed P. helenus as the sister taxon to the P. demoleus
group; however, support for this relationship was rather low.
Partitioned Bremer support (Baker and DeSalle 1997) was
calculated for each node of the combined maximum parsimony tree to provide a measure of how individual gene data
contribute to the total decay indices. Most relationships
among outgroups had low bootstrap proportions and conflicting phylogenetic signals from independent gene partitions.
However, our main concern is the relationship within the
ingroup, where all genes gave high support to monophyly of
the P. demoleus group. The gene wg demonstrates the lowest
support for a sister relationship between P. demodocus and
P. erithonioides and is in greatest conflict with grouping P.
demoleus with these two species. This partition also has negative support for allying P. grosesmithi with ((P. demodocus,
P. erithonioides), P. demoleus). Data from COII also demonstrate visible conflict with the grouping of P. demoleus
with P. demodocus 1 P. erithonioides.
The COI gene appears to have the most stable and reliable
phylogenetic signal among closely related species and so the
39 half of COI was selected for investigation of intraspecific
variability within the P. demoleus species-group. A total of
27 unique haplotypes were identified from 50 ingroup individuals (Fig. 5). We found seven haplotypes for P. demoleus,
with the most common haplotype (DL1) shared between populations of P. d. malayanus and P. d. libanius. None of these
populations contained haplotypes of P. d. demoleus (DL5) or
P. d. sthenelus (DL6 and DL7). Papilio demodocus revealed
14 haplotypes with no haplotypes shared between different
localities in Africa. Also, none of the haplotypes of P. demodocus from Africa were found in Madagascar. Papilio grosesmithi revealed only four haplotypes, two of those (G2 and
G3) were found in more than one collecting site. All four
specimens of P. morondavana, collected in different years
from three distant localities, had the same COI haplotype.
No intraspecific variation was revealed within P. erithonioides, with all eight analyzed specimens from a wide range of
sampled sites in southern and western Madagascar producing
identical sequence for the 39 half of COI.
Bayesian estimation of phylogenetic relationships of the
27 haplotypes identified for the five species of the P. demoleus
group and 11 outgroup species of Papilio is shown in Figure
6. Very little nucleotide variability was found in P. grosesmithi with zero to two substitutions (p 5 0–0.242%) in the
825-bp fragment. The degree of sequence divergence between
two subspecies of P. demoleus (ssp. malayanus and ssp. libanius) was also low, with p ranging from 0% to 0.242%.
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ORIGIN OF ISLAND ENDEMICS
TABLE 3.
Extended.
EF-1a
Wg
All
404
1st
135
2nd
134
3rd
135
All
1240
1st
2nd
413
413
3rd
414
32
24
8
0.943
0.946
2.930
0.024
1
1
0
1.000
1.000
—
0.003
0
0
0
—
—
—
0.000
31
23
8
0.941
0.944
2.730
0.071
56
48
8
0.851
0.872
3.109
0.017
2
2
0
1.000
1.000
—
0.002
0
0
0
—
—
—
0.000
54
54
8
0.846
0.865
2.958
0.050
125
75
50
0.665
0.716
2.282
0.077
16
2
14
1.000
1.000
0.878
0.014
3
1
2
1.000
1.000
—
0.005
106
72
34
0.643
0.709
2.147
0.228
242
132
110
0.625
0.609
3.477
0.044
11
4
7
0.846
0.846
1.538
0.005
4
2
2
1.000
1.000
0.671
0.002
227
126
101
0.632
0.623
3.437
0.126
Average p between these two subspecies and P. demoleus
demoleus was only about 1%, whereas the Australian subspecies P. demoleus sthenelus had a 3.8% sequence divergence from other subspecies of P. demoleus. Papillio demodocus samples from Africa did not form a monophyletic
lineage. All Malagasy specimens of P. demodocus differed
from each other by zero to two substitutions (p 5 0–0.242%)
and grouped into a monophyletic assemblage (88% posterior
probability) characterized by a single synapomorphic substitution. Number of substitutions among African P. demodocus varied from one to 10 (p 5 0.121–1.212%). The average distance between P. demodocus from Africa and from
Madagascar was 0.78%, ranging from 0.36% to 1.212%. All
species of the P. demoleus group are well diverged, as indicated by the amount of sequence divergence ranging from
4.9% to 6.5%.
Biogeography and Ancestral Areas
Dispersal-vicariance analysis (Ronquist 1996) was applied
to the inferred alternative cladograms (Fig. 7) with the distribution of each species classified as present/absent in four
different areas. There were several alternative optimizations
but in all cases the most optimal reconstruction required three
dispersal events (four if P. demodocus arrived in Madagascar
naturally) with different sequences of vicariance events.
Based on this form of analysis, the ancestral area of the group
was inferred to be Madagascar.
DISCUSSION
Conflicting Phylogenetic Signals in MitochondrialDNA,
Nuclear Genes, and Morphology
FIG. 2. Phylogeny for the Papilio demoleus species group and
Papilio outgroups computed by PAUP branch-and-bound search
from 60 morphological characters, shown as a strict consensus of
six most parsimonious trees (TL 5 140, CI 5 0.429, RI 5 0.529)
rooted with P. thoas. Refer to Figure 3 for ingroup bootstrap and
Bremer support values.
Except for the most basal relationships within the P. demoleus species-group, as well as outgroups, we obtained
largely congruent evidence from the multiple sources of data,
including nucleotide sequences from three independent gene
regions (COI 1 COII, wg, EF-1a) and morphology. We observed more discordances between gene partitions and morphological data but here, as for mitochondrial versus nuclear
genes, the conflict stemmed primarily from disagreement
about basal relationships. Tests for incongruence between
partitions revealed that COI and COII were the most congruent genes in our data, as should be expected because
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FIG. 3. Phylogenetic relationships for species of the Papilio demoleus group inferred from partitioned and combined molecular and
morphological data using maximum parsimony (MP) and Bayesian analyses. Trees represent ingroup relationships from MP bootstrap
consensus trees that were identical to ingroup topologies of MP trees in each analysis that included full set of outgroups (except F and
H) and were identical to all Bayesian reconstructions (except H). For F (wg 1 EF-1a), two MP trees had the given ingroup topology,
two other trees showed sister relationships for P. grosesmithi with P. demoleus 1 P. morondavana. For H (morphological data), the MP
bootstrap consensus is on the left, and Bayesian reconstruction is on the right. Numbers above nodes show bootstrap proportions and
Bremer support; numbers under nodes indicate Bayesian posterior probabilities.
mitochondrialDNA genes are generally not considered to recombine due to almost exclusively maternal inheritance of
mitochondrialDNA (with few exceptions, e.g., Kondo et al.
1990; Kaneda et al. 1995) and therefore to represent a single
locus with a single history. The observed disagreement between the position of P. grosesmithi in COI and COII trees
is thus likely to be due to nucleotide sampling error and the
lower number of parsimony-informative characters in the second gene. Although some incongruence was observed between mitochondrialDNA genes and nuclear genes, the alternative phylogenetic hypotheses inferred from nuclear
genes were not very robust. Phylogeny estimation based on
wg data (the partition with the lowest number of informative
characters) had the lowest average bootstrap support for the
ingroup. In fact, there was strong correlation between number
of informative characters and average bootstrap support on
the tree for each gene partition (R2 5 0.75, data not shown).
Increased numbers of characters have been shown to contribute to both accuracy (e.g., Cummings et al. 1995; Hillis
1998) and support (e.g., Felsenstein 1985; Sanderson 1995;
Bremer et al. 1999) of phylogenetic trees. Combined data
provided robust phylogenetic reconstructions that we considered the departure point for further examination of lime
swallowtail evolution.
Phylogenentic Relationships
Hancock (1983) considered lime swallowtails to belong to
the subgenus Princeps (Princeps), with the Papilio menestheus species-group as their sister-group. Based on experimental hybridization of P. demoleus with 13 other Papilio
species, Ae (1979) considered P. dardanus and P. phorcas
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FIG. 4. One of two equally parsimonious trees (TL 5 2533, CI 5 0.548, RI 5 0.554) based on combined dataset. Most parsimonious
trees were different only in some outgroup rearrangements. Bayesian analysis produced tree with almost identical topology (-lnL 5
17024.14) with different position of Papilio anactus shown by a circled star. Histograms above nodes show partitioned Bremer supports,
numbers under nodes indicate bootstrap proportions, total Bremer support, and clade posterior probabilities from Bayesian analyses.
to be part of the P. demoleus group. However, there is little
correlation between hybridization success and genetic similarity of swallowtail species (Zakharov et al. 2004), and hybridization data can be misleading. A similar situation was
reported for water-striders in the genus Limnoporus, where
one species that was clearly closely related to others, as was
shown by mitochondrialDNA, allozymes, and morphology,
demonstrated much higher hybrid incompatibility (Sperling
et al. 1997). Häuser et al. (2002) place the lime swallowtails
within the subgenus Papilio (Princeps), a group of 19 species
that, with the single exception of P. demoleus, is restricted
to Africa and Madagascar.
The most recent evidence from a more comprehensive molecular phylogeny of Papilio (Zakharov et al. 2004) indicates
that Princeps itself is not monophyletic and lime swallowtails
occupy a phylogenetic position between two Papilio subdi-
visions, Menelaides and Achillides, which are recognized by
Häuser et al. (2002) as subgenera, with good support for
Menelaides as the sister taxon to the P. demoleus group. Our
data continue to support this relationship, as P. (Menelaides)
helenus was found to be the closest outgroup to the P. demoleus species-group. Papilio lormieri, from the P. menestheus group, is one outgroup species that was added to the
current study to evaluate Hancock’s (1983) hypothesis that
this species is closely related to the P. demoleus group. However, this species was shown to have a stronger relationship
with P. delalandei and P. constantinus, which are relatively
distantly related to the P. demoleus group.
Our results leave no doubt as to the monophyly of the P.
demoleus species-group, and give strong resolution of intragroup relationships in the combined analyses. Our data do not
agree with traditional assumptions about phylogenetic rela-
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EVGUENI V. ZAKHAROV ET AL.
FIG. 5. Sampled localities and distribution of 27 unique haplotypes identified from 50 ingroup individuals of Papilio demoleus species
group from 825-bp fragment of the COI gene. Haplotypes shared between different localities are shown in regular font. Haplotypes
restricted to a single locality are shown as bold. For haplotype definitions and complete list of specimens, refer to Table 1. Arrows
indicate prevailing wind and currents in the Indian Ocean that are influenced by the Asiatic monsoon and trade winds (Walter 1970),
including North Equatorial Current (I), Mozambique Current (II), Alguhas Stream (III), West Wind Drift Current (IV), West Australian
Current (V), and South Equatorial Current (VI).
tionships within the P. demoleus group, in which P. demodocus
or P. demoleus are considered the most basal species (Carcasson 1964). None of the analyses supports a basal position
for P. demodocus and this species instead appears to be relatively derived. The strongest evidence indicates a clear sister
relationship between P. demodocus and P. erithonioides, with
P. demoleus likely being their sister taxon. There is good support for P. grosesmithi as the sister taxon of these three species,
and a basal position for P. morondavana. Thus, the endemic
species whose conservation status is of most concern, P. grosesmithi and P. morondavana, appear to represent the oldest
lineages among lime swallowtails. These relic species speak
for additional protection being given to the dry deciduous
forests of Madagascar as a conservation priority (Vane-Wright
et al. 1991; Stiassny and de Pinna 1994).
Another interesting finding is the degree of genetic variation among and within species of the P. demoleus group.
Although percent sequence divergence is quite variable
among closely related Lepidoptera and is not necessarily a
reliable indicator of species status, there is an obvious trend
with overall sequence divergence increasing at higher taxonomic levels. Almost 98% of species recognized through prior morphological studies are reported to fit a species level
threshold value of 3% divergence in COI proposed by Hebert
et al. (2003). However, the amount of sequence divergence
in COI at the interspecific level within lepidopteran species
complexes ranges from 0.1–0.6% (0.4% average) in ermine
moths Yponomeuta (Sperling et al. 1995), 0–3.6% (2.1% average) in Choristoneura (Sperling and Hickey 1994), and 0.1–
3.7% (2.2% average) in Feltia (Sperling et al. 1996). Divergences of less than 1% and up to to 2.5% divergence in COI
was reported for sister species of torticid moths of the genus
Archips (Kruse and Sperling 2001). Higher levels of variation
in interspecific mitochondrialDNA divergences was demonstrated for the one of the most well studied species groups
of the genus Papilio, the P. machaon complex, with values
ranging from ,1% to 8% (Sperling and Harrison 1994).
In lime swallowtails, intraspecific mitochondrialDNA divergences ranged from nothing within P. morondavana and
P. erithonioides to less than 0.5% between closely related
subspecies of P. demoleus, but up to almost 4% sequence
divergence between more distant subspecies of P. demoleus.
At the same time, the species themselves were separated by
nucleotide differences of 5–6%, which leaves no doubt that
all lime swallowtail species are very distinct by any comparison to other Lepidoptera species.
Lack of variation in the COI gene in P. erithonioides and
P. morondavana, sampled in different localities and time se-
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ORIGIN OF ISLAND ENDEMICS
FIG. 6. Bayesian estimation of the phylogeny of 27 unique haplotypes (from 50 ingroup samples) and 11 outgroups inferred from 825
bp from the 39 end fragment of COI (106 cycles, each 100th sample, contype 5 halfcompat, burnin 5 5000). Numbers above nodes
indicate clade posterior probabilities, and under nodes indicate bootstrap proportions from maximum parsimony analyses. For haplotype
definitions and species list, refer to Table 1. For geographic distribution of haplotypes, see Figure 5.
ries, requires further investigation. While P. erithonioides
may still be too evolutionarily young to have accumulated
much variation, the latter species is probably a relic. Lack
of mitochondrialDNA variation in P. morondavana might indicate that the ancestral population has gone through one or
more recent bottlenecks.
Biogeography
The approximate age of divergence of the P. demoleus
species-group from other Papilio is estimated to be about
16.8 6 6.7 million years (Zakharov et al. 2004). This date
excludes any possibility for older vicariance via Cretaceous
plate tectonics. Recent reports of invasion of P. demoleus
into islands in Southeast Asia where the species was originally absent indicate the ability of lime swallowtails to expand their range, whether through active flight or with the
aid of man and Citrus cultivation (Hiura 1973; Corbet and
Pendlebury 1978, 1992; Vane-Wright and de Jong 1993).
Phylogenetic reconstruction clearly indicates a basal position for P. morondavana and P. grosesmithi within the P.
demoleus species-group. Based on estimated substitution
rates for COI and COII genes (Zakharov et al. 2004), the
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FIG. 7. Phylogenies of the Papilio demoleus group inferred from (a) the morphology bootstrap consensus (this topology was also
recovered from COII data partition); (b) COI-COII; (c) wg; and (d) EF-1a, with areas optimized by dispersal-vicariance analysis (Ronquist
1996). Each species is coded as present in a particular area, disregarding human introductions. Alternative equally parsimonious optimizations are separated by a semicolon at each node. Composite areas (e.g., AB) indicate that the program has calculated the combined
area as the optimal solution. Maps illustrate the present-day distribution of lime swallowtails.
ancestors for P. morondavana and the rest of lime swallowtails diverged between approximately 14.1 and 10.6 million
years ago, and the split of the ancestor for P. grosesmithi
from its sister group is dated at between 13.5 and 10.3 million
years ago Dispersal-vicariance analysis based on these relationships suggests their origin and speciation within Madagascar (Fig. 7), followed by dispersal of lime swallowtails
into Australia and Asia (becoming P. demoleus) around 10.1
to 7.8 million years ago, and Africa (becoming P. demodocus)
around 7.3 to 5.6 million years ago.
Due to weakly supported outgroup relationships, our data
are insufficient to resolve with confidence how the ancestor
of lime swallowtails came to Madagascar. Based on a currently available molecular phylogeny for the genus Papilio
(Zakharov et al. 2004), the P. demoleus species group had a
common ancestor with an Oriental lineage of Papilio, the
subgenus Menelaides. Together they are the sister group to
Papilio (Achillides), another Oriental subgenus. This suggests
that an Asian ancestor of the P. demoleus species-group may
have first reached Madagascar in a manner similar to the
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hypothesized origin of the Madagascan mycalesine butterflies
(Lees 1997; Torres et al. 2001). This scenario is also supported by a close approach to Madagascar of two lineages
of an Indo-Australian genus Euploea (Lepidoptera: Nymphalidae) to the Seychelles and Mascarene Islands (Ackery
and Vane-Wright 1984).
Colonization of Madagascar would have been followed by
speciation of Malagasy endemics, succeeded by dispersal of
the ancestors of each of P. demodocus and P. demoleus to
Africa and back to the Oriental region and Australia. Under
the best-supported parsimony reconstructions, P. morondavana, P. grosesmithi, and P. erithonioides would have diverged from each other on Madagascar itself, whether on
different parts of the island or by a sympatric process where
they were in contact with each other.
The probability of dispersal across the Indian Ocean (c.
4000 km) may seem relatively low compared to a scenario
involving multiple colonizations of Madagascar from Africa
(separated by just c. 400 km). Nonetheless, a similar hypothesis has also been proposed for the origin of other representatives of the Malagasy fauna. Jansa et al. (1999) suggested a single invasion of native rodents (Muridae: Nesomyinae) to Madagascar from Asia followed by secondary
invasion from Madagascar into Africa (but see Jansa and
Weksler 2004). Lees (1997) and Torres et al. (2001) also
suggested that the Malagasy mycalesine butterfly radiation
may have originated from India rather than Africa, and that
a dispersal event from India was followed by subsequent
colonization of Africa by founders from Madagascar.
Long-distance dispersal has been proposed to explain similar patterns of relationships in chameleons, where the oldest
lineages are distributed in Madagascar, and more recently
derived forms that are found in Africa, the Seychelles, the
Comoros, and India are believed to be the result of several
dispersal events across the Indian Ocean (Raxworthy et al.
2002). Both the oldest fossil records that are available for
chameleons and geological age of the volcanic islands of
Comoros are substantially younger than the time of Cretaceous vicariance (see Raxworthy et al. 2002). The Comoros
archipelago has never been in contact with larger land masses
since its formation within the last 5.4 million years (Emerick
and Duncan 1982); thus, the only possible way the ancestor
of the endemic chameleons endemic to these islands could
have arrived there is by transmarine dispersal.
Oceanic dispersal of terrestrial animals to and from Madagascar and other islands in the western Indian Ocean has
been favored by many other studies that hypothesized postGondwanan transmarine migration by rafting on tangles of
vegetation (e.g., Fisher 1996; Yoder et al. 1996; Nagy et al.
2003; Vences et al. 2003). Dispersal across an uninhabitable
space is even more plausible when a taxon possesses a significant degree of vagility, as in actively flying Lepidoptera
and lime swallowtails in particular.
It also appears that the Malagasy biota has provided sources for multiple radiations that have contributed to the biodiversity of the surrounding continents and islands. For example, reconstruction of island radiations in extinct and extant geckos using ancient and recent DNA by Austin et al.
(2004) suggested that at least four different archipelagos in
the Indian Ocean have been colonized independently by dis-
persals from Madagascar. According to Warren et al. (2003),
Madagascar has given rise to two independent sunbird lineages in the Aldabra archipelago.
Complex seasonal systems of oceanic currents and winds
in the Indian Ocean provide conditions for long-distance
transmarine dispersals (see Fig. 5). The currents of the northern Indian Ocean are influenced by the Asiatic monsoon while
the currents of the southern Indian Ocean are influenced by
anticyclonic circulation in the atmosphere (Walter 1970). The
northwest monsoon stimulates the North Equatorial Current,
which flows from east to west and upon reaching the east
coast of Africa the largest portion turns southward and eventually becomes the Mozambique Current. The Mozambique
Current flows south along the east coast of Africa and at
about 358S merges with the Alguhas Stream, which flows
westward along the southern coast of Madagascar and the
east African coast toward the Cape of Good Hope, where it
partly joins the West Wind Drift Current, which then carries
its waters toward southwest Australia. During the southwest
monsoon in August and September, the North Equatorial Current reverses direction and flows west to east as the Monsoon
Current.
Thus, a westward dispersal of lime swallowtails across the
northern part of the Mozambique Channel is more probable
based on prevailing winds and has been inferred to occur on
several occasions in Lepidoptera (Pierre 1992; Torres et al.
2001), though on more tenuous phylogenetic grounds. Prevailing easterly trade winds and ocean currents in the Indian
Ocean also increase the probability of long-distance dispersal
from Asia to the western Indian Ocean (Donque 1972; Renvoise 1979). At the same time, dispersal from southern Madagascar and southern Africa can be facilitated by West Wind
Drifts, as has been hypothesized for some sea stars (Waters
and Roy 2004).
Alternatively, the assumptions of dispersal-vicariance
analysis may simply be wrong, in that the distribution of
these butterflies may not be parsimonious. For example, extinction of intermediate or ancestral taxa would complicate
and obscure reconstruction of dispersal, refuting an indigenous origin of the group within Madagascar. Under a scenario
of dispersal from Africa, there could have been a number of
successive arrivals to Madagascar from an anagenetically
evolving stem lineage, as suggested by Carcasson (1964).
Additional uncertainty is associated with the origin of P.
demodocus in Madagascar. It is reported that the presence of
P. demodocus on the island is the result of introduction by
man (Paulian 1951; Paulian and Viette 1968; Turlin 1994).
The monophyly of mitochondrialDNA haplotypes found in
Madagascan samples of P. demodocus relative to African
individuals may be a consequence of founder effect from a
single introduction. However, the fact that the mitochondrial
lineage already displays moderate genetic variability (four
haplotypes of seven samples) suggests that colonization of
Madagascar by this lineage may have occurred thousands of
years ago or even longer, prior to island colonization by man
about 2000 years ago (Dewar 1997).
The origin of P. demoleus in Saudi Arabia and Iran is also
believed to be the result of introduction along with the first
importation of Citrus in the 10th century (Wiltshire 1945;
Larsen 1977). The 1% sequence divergence between our sam-
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ple of P. demoleus from Iran and our samples from Southeast
Asia and eastern Asia indicates that this introduction would
probably have come from closer sources, such as the Indian
subcontinent (from which we were unable to obtain samples),
which already had a moderate amount of divergence from
populations farther to the east. Interestingly, all Asian individuals of P. demoleus had very similar or identical haplotypes,
supporting the hypothesis of recent range expansion in this
region. In contrast, there was a striking difference in
mitochondrialDNA between Oriental subspecies and the Australian subspecies P. d. sthenelus. Taking into account host
use differences between this and other subspecies of P. demoleus, these haplotypes appear to be well-diverged lineages
that might be considered separate species (Hebert et al. 2003).
At minimum, it appears likely that ancestral populations of P.
demoleusin the Oriental and the Australian regions have been
isolated for a long time, from 3.24 to 4.24 million years ago
based on substitution rates estimated for mitochondrialDNA
in Papilio (Zakharov et al. 2004). Thus P. d. sthenelus may
represent the oldest lineage in P. demoleus. More detailed
sampling across its range is required to reconstruct the evolutionary history of this species on both sides of Wallace’s
line.
Conclusion
Biogeographic scenarios are strongly dependent on the selection of phylogenetic hypotheses for a particular group of
organisms, while phylogenetic reconstructions themselves
can be subject to different biases in data. Thus, selection of
informative loci for a given taxonomic level remains key to
understanding the evolution and biogeography of any group
of organisms. In our reconstructions of the phylogeny of
species in the P. demoleus group, nucleotide sequences have
provided a useful source of data in addition to traditional
morphology. Comparison of four genes representing three
independent loci supports the utility of the COI gene in reconstructing the phylogeny of closely related species, such
as in the P. demoleus group.
Our results provide strong evidence for the basal relationships of Malagasy endemic species in the P. demoleus group.
Relationships among lime swallowtails on Madagascar include elements similar to those reported in nymphalid butterflies and even chameleons, indicating not only that postCretaceous dispersal across large oceanic barriers has contributed to the diversity of Madagascar, but that the Malagasy
biota has speciated actively on the island, serving as an incubator for radiations that have contributed to the diversity
of surrounding continents on all sides of the Indian Ocean.
Our study may be the best documented case of this pattern
to date, despite the fact that morphology does not yet seem
to provide enough resolution both for ingroup lime swallowtails and outrgoup species of Papilio, as it is supported by
multiple independent genes.
Of more immediate importance, our data support a recommendation that further protection should be given to the
dry forests of Madagascar (generally underrepresented within
the protected area system; Dupuy and Moat 1996) to ensure
survival of the endemic species of lime swallowtails in Madagascar. These large and charismatic butterflies give new in-
sight into the complex history of the unique and ancient biodiversity of this island, as well as add weight to the urgency
of arresting the rapid clearance of dry deciduous forests on
the island via creation of new protected areas, for the mutual
benefit of other species restricted to these ecosystems.
ACKNOWLEDGMENTS
We are grateful to the following people for help in providing specimens for this study: N. Pierce, D. Lohman, and
M. Cornwall from Harvard University (Cambridge, MA,
USA); N. Tatarnik and V. Nazari from the University of
Alberta (Edmonton, AB, Canada); H. F. Wong from Deco
Enterprise (Taiping, Malaysia); S. Schoeman from ARC, Institute for Tropical and Subtropical Crops (South Africa); M.
G. Wright from the University of Hawaii (Honolulu, HI,
USA); D. Goh from the Penang Butterfly Farm (Penang, Malaysia); S. A. Ae (Gifu, Japan); S. H. Yen from The Natural
History Museum (London, United Kingdom); and J. Demay
from SNC Ornithoptera (Agny, France). This study was funded by an NSERC grant to FAHS. Collections from Madagascar were partially supported by grant DEB-0072713 from
the National Science Foundation to B. L. Fisher and C. E.
Griswold. Fieldwork that provided the basis for this work
could not have been completed without the gracious support
of the Malagasy people. We are grateful for comments on an
earlier version of this manuscript by A. Yoder, N. Wahlberg,
and an anonymous reviewer.
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ORIGIN OF ISLAND ENDEMICS
APPENDIX
Morphological characters and states used in the cladistic analysis of five species of the Papilio demoleus species group and eleven outgroup
species of Papilio. Characters are illustrated in Figure 1 and will be fully discussed in C. R. Smith and R. I. Vane-Wright (unpub. ms.).
Wing pattern
Forewing upperside
1. Discal cell with pale scales: absent 5 0; present 5 1.
2. Discal cell scales (patterning): random 5 0; organised 5 1.
3. Discal cell scale pattern: longitudinal stripes 5 0; transverse bands 5 1.
4. Discal cell with an array of three distal marks: absent 5 0; present 5 1.
5. Cell R2 with postdiscal mark: absent 5 0; present 5 1.
6. Cell R4 with discal mark: absent 5 0; present 5 1.
7. Cell R4 postdiscal mark: absent 5 0; present 5 1.
8. Cell M1 with postdiscal mark (in the male): absent 5 0; present 5 1.
9. Cell 1A with both a postdiscal and a submarginal mark: absent 5 0; present 5 1.
Forewing underside
10. Cell R2 with apical mark: absent 5 0; present 5 1.
11. Cell M1 with postdiscal mark: absent 5 0; present 5 1.
Hindwing upperside
12. Cell R1 with an eyespot: absent 5 0; present 5 1.
13. Cell CuA2 with an eyespot: absent 5 0; present 5 1.
14. Cell CuA2 with eyespot with a transverse distal border: absent 5 0; present 5 1.
15. Cell M1 with postdiscal spot: absent 5 0; present 5 1.
16. Cell M2 with postdiscal spot: absent 5 0; present 5 1.
17. Cell M3 postdiscal spot: absent 5 0; present 5 1.
Hindwing underside
18. Cells R1 and discal cell with pale basal band: absent 5 0; present 5 1.
19. Basal band in cells R1 and discal cell: narrow 5 0; broad 5 1.
20. Cells R5 to CuA1 with a pattern of blue and orange bands proximal to the submarginal marks: absent 5 0; present 5 1.
21. Cell R5 with discal mark: absent 5 0; present 5 1.
22. Cell R5 with discal mark reaching root of M1: absent 5 0; present 5 1.
23. Cell M2 with marginal mark subdivided into two large marks: absent 5 0; present 5 1.
24. Cell M3 with marginal mark subdivided into two large marks: absent 5 0; present 5 1.
25. Hindwing tail (size): absent or rudimentary 5 0; short 5 1; medium 5 2; long 5 3.
26. Hindwing tail with club: absent 5 0; present 5 1.
27. Upperside surface of male forewing with androconial scales: absent 5 0; present 5 1.
Antennae
28. Antennal color: unicolorous 5 0; with pale mark on one surface near tip 5 1.
Male genitalia
29. Valve rim with postero-ventral expansion: absent 5 0; present 5 1.
30. Valve rim with terminal notch: absent 5 0; present 5 1.
31. Harpe with vertical projection: absent 5 0; present 5 1.
32. Vertical projection of harpe: free 5 0; fused 5 1.
33. Vertical projection of harpe: simple 5 0; double 5 1.
34. Vertical projection of harpe: contiguous 5 0; detached 5 1.
35. Harpe reaching or surpassing valve rim: absent 5 0; present 5 1.
36. Harpe with ventral flange serrate: absent 5 0; present 5 1.
37. Pseuduncus width: narrow 5 0; broad 5 1.
38. Pseuduncus strongly declivous: absent 5 0; present 5 1.
39. Uncus with prominent, terminal ridge or projection: absent 5 0; present 5 1.
40. Uncus with prominent sub-terminal swelling: absent 5 0; present 5 1.
41. Uncus with prominent serrations: absent 5 0; present 5 1.
42. Saccus: absent 5 0; present 5 1.
Female genitalia
43. Vestibulum with pocket: absent 5 0; present 5 1.
44. External genitalic pocket: shallow 5 0; deep 5 1.
45. Pocket with ventral lining of pocket: rugose 5 0; lanose 5 1.
46. Ostium bursae position: anterior 5 0; central/posterior 5 1.
47. Ostium bursae opening on sagittal ridge: absent 5 0; present 5 1.
48. Peripheral vestibular plates: absent 5 0; present 5 1.
49. Peripheral vestibular plates extending around ostium: absent 5 0; present 5 1.
50. Peripheral vestibular plates joining anteriorly: absent 5 0; present 5 1.
51. Peripheral vestibular plates with posterior expansion: absent 5 0; present 5 1.
52. Peripheral vestibular plates with processes: absent 5 0; present 5 1.
53. Lateral ostial plates: absent 5 0; present 5 1.
54. Lateral ostial plates extended posteriorly: absent 5 0; present 5 1.
55. Lateral ostial plates with lateral ridges: absent 5 0; present 5 1.
56. Lateral ostial plates with peripheral lamella: absent 5 0; present 5 1.
57. Lateral ostial plates with joining anteriorly: absent 5 0; present 5 1.
58. Tonguelike process: absent 5 0; present 5 1.
59. Tonguelike process with posterior transverse ridge: absent 5 0; present 5 1.
60. Posterior central cup: absent 5 0; present 5 1.