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Deng et al. BMC Evolutionary Biology 2013, 13:275 
http://www.biomedcentral.com/1471-2148/13/275 



Evolutionary Biology 



RESEARCH ARTICLE Open Access 



Cophylogenetic relationships between Anicetus 
parasitoids (Hymenoptera: Encyrtidae) and their 
scale insect hosts (Hemiptera: Coccidae) 

Jun Deng 1,2 , Fang Yu 1 , Hai-Bin Li 1,2 , Marco Gebiola 3,4 , Yves Desdevises 5,6 , San-An Wu 2 * and Yan-Zhou Zhang 1 " 



Abstract 

Background: Numerous studies have investigated cospeciation between parasites and their hosts, but there have 
been few studies concerning parasitoids and insect hosts. The high diversity and host specialization observed in 
Anicetus species suggest that speciation and adaptive radiation might take place with species diversification in scale 
insect hosts. Here we examined the evolutionary history of the association between Anicetus species and their scale 
insect hosts via distance-based and tree-based methods. 

Results: A total of 94 Anicetus individuals (nine parasitoid species) and 1 13 scale insect individuals (seven host 
species) from 14 provinces in China were collected in the present study. DNA sequence data from a mitochondrial 
gene (COI) and a nuclear ribosomal gene (28S D2 region) were used to reconstruct the phylogenies of Anicetus 
species and their hosts. The distance-based analysis showed a significant fit between Anicetus species and their 
hosts, but tree-based analyses suggested that this significant signal could be observed only when the cost of 
host-switching was high, indicating the presence of parasite sorting on related host species. 

Conclusions: This study, based on extensive rearing of parasitoids and species identification, provides strong 
evidence for a prevalence of sorting events and high host specificity in the genus Anicetus, offering insights into 
the diversification process of Anicetus species parasitizing scale insects. 

Keywords: Host-parasitoid interactions, Sorting, Speciation, COI, 28S-D2 



Background 

The study of the evolution of host-parasite associations 
has a long history, with the first paper published a cen- 
tury ago [1-6]. Since then, numerous host-symbiont sys- 
tems have been observed and several analytical methods 
proposed. When the host and parasite phylogenetic trees 
are the same, that is when visual inspection show that 
the two trees precisely match, with hosts and corres- 
ponding parasites at the same positions, a cospeciation 
pattern can be directly inferred. In other situations, the 
reconstruction of a hypothetical revolutionary scenario 
is not straightforward, as it can involve different events 
including cospeciation, duplication, lineage sorting and 



* Correspondence: sananwu@bjfu.edu.cn; zhangyz@ioz.ac.cn 

2 Key Laboratory for Silviculture and Conservation of Ministry of Education, 

Beijing Forestry University, Beijing 100083, China 

Vey Laboratory of Zoological Systematics and Evolution, Institute of 

Zoology, Chinese Academy of Sciences, Beijing 100101, China 

Full list of author information is available at the end of the article 



host-switching [7]. In such cases, a rigorous and specific 
method must be used to differentiate cospeciation from 
a number of potential scenarios. 

In the last two decades, several methods were devel- 
oped to assess the level of cospeciation in symbiotic as- 
sociations [8], and the availability of programs such as 
TreeMap [9], TreeFitter [10,11] and ParaFit [12] has led 
to an increased level of accuracy in host-parasite cospe- 
ciation studies [13-15]. These software search for an op- 
timal evolutionary scenario for the association between 
hosts and their symbionts (for example, parasites). Pre- 
vious work has investigated cospeciation between para- 
sites and their hosts, such as lice and mammals [16-21], 
plants and insects [22-25], plants and fungi [26], fish 
and Platyhelminthes [7,27,28], and animals and viruses 
[29,30]. However, cophylogeny between parasitoids and 
their insect hosts has been rarely investigated, with the 
few previous studies focusing on Lepidoptera-parasitoids 
systems [31,32]. 



O© 2013 Deng et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative 
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stated. 



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Almost every plant-feeding insect species is attacked 
by at least one parasitoid species [33] and even without 
strict host specificity, there are at least as many (and 
possibly more) parasites than free-living species. Among 
Hymenopteran parasitoids, Encyrtidae (Hymenoptera: 
Chalcidoidea) is an economically important group of 
nearly 4000 species of natural enemies of Lepidoptera, 
scale insects and other insect orders [34]. The genus 
Anicetus Howard is well known for its important eco- 
nomic significance. Several Anicetus species, such as A. 
beneficus Ishii & Yasumatsu, are frequently used as bio- 
logical control agents of wax and soft scales of the genus 
Ceroplastes Gray (Homoptera: Coccidae), which are sig- 
nificant pests of important agricultural crops [35-37]. 
However, due to their small size and frequent lack of 
distinct morphological characters, the accurate identi- 
fication of wax scales and parasitoids is still a great 
challenge for taxonomists. The study of cophylogenetic 
patterns between species of Anicetus and Ceroplastes is 
therefore difficult, however, it is also crucial for a better 
understanding of speciation and diversification processes 
in this parasitoid genus. Two recent DNA barcoding 
studies of Anicetus and their wax scale hosts were used 
as a taxonomic reference for the present study [38,39]. 

Several recent DNA-based studies strongly suggest 
that morphologically similar lineages traditionally con- 
sidered as single species are instead genetically isolated, 
and in many cases host-specific [40-43]. Koinobiont pa- 
rasitic Hymenoptera, in particular, display an intricate 
physiological relationship with their hosts and conse- 
quently tend to have relatively narrow host ranges [44]. 
The degree of host specificity of Encyrtidae is variable. 
For example, Anagyrus sp. nov. nr. sinope and Leptomas- 
tix dactylopii Howard are two parasitoids of mealybug 
species; the former is highly host specific, whereas the 
latter displays a wider host range, having been recorded 
from more than 20 host species [45]. Some Encyrtidae 
species such as Copidosoma floridanum (Ashmead) [46] 
exclusively parasitize a given host family or subfamily, 
while other Copidosoma species have a wider host range 
and attack different families of Lepidoptera [47]. High 
host specificity has been reported in Comperia merceti 
(Compere) [48], Gyranusoidea tebygi Noyes [49,50], and 
more recently in Encyrtus sasakii [51]. Zhang et al. [38] 
recently showed that host specificity tends to be strict in 
the Anicetus group, where species are usually restricted 
to one host species. Furthermore, Anicetus species have 
a low mobility and individuals that leave the host die 
within a few hours or days, hence they are totally reliant 
upon their hosts for survival. This makes the genus Ani- 
cetus a good candidate for evolution via cospeciation 
with their insect hosts. 

The nine Anicetus species used for this study exhibit 
narrow host ranges and only parasitize wax scales. A 



large number of Ceroplastes individuals were collected 
throughout China (see Materials and methods). The 
aims of this study were to reconstruct molecular phylo- 
genies for wax scale insects and their Anicetus parasi- 
toids, and to assess the degree of cospeciation in this 
host-parasitoid association in order to better understand 
the drivers of species diversification in this group of 
parasitoids. 

Results 

Phylogenetic analyses 

The partition homogeneity test indicated that the COI 
and 28S datasets did not display any significant signal of 
heterogeneity (P = 0.35 for host dataset and P = 0.66 for 
parasitoids dataset). This test compared the summed 
lengths of most-parsimonious trees computed from each 
dataset (i.e. gene) to the lengths of trees generated from 
random partitions of the combined sequences of both 
genes [52], and calculated the probability of obtaining a 
random tree similar or shorter to the length of observed 
summed tree. The two datasets were then combined 
for subsequent phylogenetic analysis. In the host tree, 
Parasaissetia sp. was strongly supported as basal clade 
and Pulvinaria aurantii was sister group to the clade of 
all Ceroplastes species, which was strongly supported 
(Figure 1). For parasitoids, most Anicetus species were 
strongly supported except for two groups of A. benifi- 
cus and A. rubensi individuals (PP = 0.58) (Figure 2). 
These two species are morphologically very similar, 
reflecting the taxonomic uncertainty at this level. 

The parasite and host phylogenies built from consen- 
sus sequences were used to assess their phylogenetic 
congruence (Figure 3). These trees, using consensus se- 
quences, were identical to previous phylogenies (Figure 1, 
Figure 2). Furthermore, not all parasitoids from the same 
host clustered in the same clade, for example, A. dodo- 
nia Ferriere and A. aligarhensis Hayat, Alam & Agarval 
clustered together even though they use different hosts. 

Topology-based analyses: Treemap 3.0P and Jane 4 

The tanglegram built from the phylogenetic trees and in- 
dividual associations between Anicetus species and their 
scale insect hosts (Figure 3) suggested that the trees did 
not perfectly match. We then used Treemap 3.0p that 
generated 64 optimal solutions to reconcile the two trees 
with the lowest number of revolutionary events consid- 
ering their costs (Figure 4), none of which indicated sig- 
nificant congruence. We used different cost sets for each 
of these coevolutionary events to produce different re- 
sults in Jane 4 (Table 1). In both methods, each event is 
given a cost inversely related to the likelihood of that 
event [53], and a global cost is computed by summing 
the costs of all events needed to fit the parasitoid tree 
onto the host tree (i.e. tree reconciliation). A significant 



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S3_523a 
S3_523b 
S3_523c 
S3 523d 



Acanthococcus sp. 



| Parasaissetia sp. 



4 l2"i6ib | Pulvinaria a in until 



0.99 



S2 161b 
C0002a 
C0002b 
C0002c 
C0002d 
C0003a 
C0003b 
C0003c 
C0003d 
S2011_070a 
S2011_070b 
S2011_128a 
S2011_128b 
S2011_129a 
S2011_129b 
S2011_142a 
S2011_142b 
S2011_142c 
S2011_030a 
S2011_030b 
S2011_030c 
S2011_030d 
S2011_030e 
S2011_036a 
S2011_036b 
S2011_003a 
S2011_003b 
S2011_003c 
S2011_046a 
S2011_046b 
S2011_058a 
S2011_058b 
S2011_076a 
S2011_076b 
S2011_076c 
WHa 
WHb 
WHc 
WHd 

S2011_129c 
S2011_129d 
S2011_046c 
S2011_046d 
S2011_001a 
S2011_001b 
S2011_141a 
S2011_141b 
S2011_141c 
S2011_146a 
S2011_146b 
S2011_146c 
S2011_022a 
S2011_022b 
S2011_022c 
S2011_028a 
S2011_028b 
S2011_010a1 

1S2011_010a2 
S2011_010b1 
S2011_010b2 
S2011_053a 
S2011_053b 
S2011_001c 
S2011_135a 
S2011_135b 
S2011_135c 
S2011_135d 
S2011_135e 
C0020B 
S2011_137a 
S2011_137b 
S2011_137c 
S2011_139a 
S2011_139b 
S2011_138a 
S2011_138b 
S2011_138c 
S2011_014a 
S2011_014b 
S2011_014c 
S2011_005a 
S2011_005b 
S2011_006a 
S2011_006b 
S2011_006c 
S2011_016a 
S2011_016b 
S2011_016c 
S2011_139c 
S2011_051a 
S2011_051b 
S2011_085a 
S2011_085b 
S2011_085c 
S2011_139d 
S2011_139e 
S2011_04a 
S2011_04b 
S2011_074a 
S2011_074b 
S2011_074c 
S2011_082a 
S2011_082b 
S2011_082c 
S2011_002a 
S2011_002b 



C. rubens var 



C. rubens 



C. ceriferus 



C. japonicus 



0.4 

Figure 1 Bayesian trees of scale insect species based on combined COI and 28S data. Support values (posterior probabilities) are provided 
for each node. 



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IsS | Eusemion sp. 



, ■ 08-07a 
1 I 08-07b 
08-07C 
08-07d 



rl 



A. sp2 



0.5X 



07-96a 
07-96b 
07-96C 

07- 96d 

08- 28a 
08-28b 
07-961a 
07-961 b 
07-962a 

07- 962b 

08- 53a 
08-53b 
08-23a 
08-23b 

08-03-1 
08-03-2 
08-03-3 
08-03-4 
08-03-5 
08-03-6 
08-03a 
08-03b 
08-03c 
08-03d 
08-03e 
08-03f 
08-04a 
08-04b 
08-04c 
08-04d 
10-084a 
10-084b 
10-084c 
10-084d 
10-084e 

10- 084f 

11 - 025a 
,11 -025b 



07-97a 

07- 97b 

08- 30a 
08-30b 
08-32a 
08-32b 
10-066a 
10-066b 
08-29a 
08-29b 
10-1 15a 
10-115b 
10-1 14a 
10-1 14b 
07-971 a 
07-971 b 
07-971 c 

07- 971 d 

08- 24a 
08-24b 

10-111a 
10-111b 
10-1 12a 
10-1 12b 
08-35a 
08-35b 



H 



A. benificus & A. beniflcus var 



A. rubensi 



A. ceroplastis 



A. dodina 



0.98 



I | 08-27a 
1 | 08-27b 
08-54a 
08-54b 



10-078a 
10-078b 
10-078C 
10-078d 
10-078e 
10-078f 



07-01 a 
07-01 b 
07-01 c 
07-01 d 
07-01 e 

07- 01 f 

08- 31 a 
08-31 b 
10-1 13a 

10- 1 13b 

11 - 030a 
11 -030b 
11 -030c 
11-030d 



A. aligarhensis 



A.ohgushii 



A. spl 



0.3 



Figure 2 Bayesian trees of Anicetus species based on combined COI and 28S data. Support values (posterior probabilities) are provided for 
each node. 



global cost (P = 0.004) was only observed in Jane with 
the TreeFitter default cost model, that is 5 for cospecia- 
tion, 4 for duplication, 0 for host-switch, 7 for loss and 0 
for failure to diverge. Setting the costs of host-switch to 
high values in the TreeFitter default model caused the 
overall fit to become significant, suggesting that host- 
switch is rare in this host-parasitoid system. Meanwhile, 
a large number of sorting events (7) were found with 
the TreeFitter default model, in contrast to 0-1 sorting 
events with the other models. In addition, we compared 
the patristic distances (phylogenetic divergence) between 



parasitoid and hosts in copaths using TreeMap (Figure 5), 
to assess whether branch lengths are correlated in cospe- 
ciating hosts and parasitoids (corresponding branches in 
the two trees are called "copaths"). A strong positive cor- 
relation would support cospeciation, and in this case the 
slope of the linear relationship indicated the relative evo- 
lutionary rates in hosts and parasitoids because the same 
genes were used to build the phylogenies. The branch 
length randomization test suggested a strong significant 
correlation between copaths (r = 0.8145), supporting the 
hypothesis that cospeciation has occurred in this host- 



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Table 1 Results of cophylogenetic analyses with Jane for Anicetus and their hosts 



Model 


Event 
costs 


i otai 
cost 


Cospeciation 


Duplication 


nosi 
switch 


Sorting 
event 


Failure 
to diverge 


P-value 


Jane default model 


01211 


10 


4 


1 


4 


1 


0 


0.22 


TreeMap default model 


01111 


6 


3 


1 


5 


0 


0 


0.53 


TreeFitter default model 


00211 


7 


5 


4 


0 


7 


0 


0.004* 


Host switch-adjusted TreeFitter model 


00111 


5 


2 


2 


5 


0 


0 


0.13 


Codivergence adjusted TreeFitter model 


10111 


7 


0 


2 


7 


0 


0 


0.56 


Equalweights 


11111 


9 


0 


0 


9 


0 


0 


1 



Asterisks indicate significance at the 1% level. Columns indicate the number of each event type necessary to reconcile host and parasite trees under different 
event cost schemes. Event costs are for cospeciation, duplication, host switching, sorting event, and failure to diverge, respectively. P-values were computed from 
999 random reconstructions. 



parasitoid association. The slope of the linear relationship 
using the reduced major axis method was 3.6, suggesting 
that Anicetus species have evolved more rapidly than their 
scale insect hosts. This result is consistent with previ- 
ous results obtained for Achrysocharoides (Hymenoptera: 
Eulophidae) [32]. 

Distance-based analysis: ParaFit 

We used ParaFit to compare patristic distance between 
hosts and their corresponding parasitoids, to test the 
global fit between the two trees. In addition the method 
assesses if each individual host-parasitoid association 
(link) significantly contributes to the global fit, to evalu- 
ate which ones have a structuring effect. The global test 
indicated a significant congruence between Anicetus and 
scale insect trees (P = 0.01602). However, the test of indi- 
vidual links showed that not all host-parasite associa- 
tions significantly contributed to this global fit: 4 out of 
10 individual links were significant (Eusemion sp.-Acan- 
thococcus sp., A. ohgushii-C. japonicus, A dodina-C. ceri- 
ferus and A. aligarhensis-C. japonicus), suggesting their 
structuring role in the global congruence. 

c ^ 

R=0.8145 





0.7 








0.6 








0.5 






itoids 


0.4 






Paras 


0.3 
0.2 




♦ 




0.1 
n 




♦ 

♦ 

♦ 



♦ 



♦ 

X 



0 0.05 0.1 0.15 0.2 0.25 

Hosts 

Figure 5 Relationship between patristic distances of copaths 

for Anicetus species and their scale insect hosts. 

\ J 



Discussion 

A cophylogenetic signal is weak or absent in most host- 
parasite associations that have been studied to date 
[54-56]. However, significant cospeciation has been in- 
ferred in systems where host-switching is prevented by 
the asocial lifestyle of the host and the low mobility of 
the parasite. Examples include rodent-lice associations 
[6,18] and insect- symbiont systems where bacteria, nee- 
ded for reproduction, are transmitted maternally [57,58]. 
The present study can be added to these few examples of 
extensive cospeciation, supported using various methods. 

This study is the first to thoroughly investigate the 
cophylogenetic interactions between Anicetus and their 
scale insect hosts, and suggests the ubiquity of sorting 
events coupled with strong host specificity in the genus 
Anicetus. Nine genetically distinct species were clearly 
delineated in the phylogenic tree based on combined 
molecular data (28S-D2 and COI). Anicetus benificus, A. 
benificus_var and A. rubensU all parasitoids of C. rubens, 
were found grouped in the phylogeny, which is congru- 
ent with the current taxonomy (Figure 2). Furthermore, 
morphological data confirmed this pattern, for example, 
the antennal clava and ovipositor of these three species 
are similar to each other [59]. However, not all Anicetus 
species from the same host were found to cluster in the 
phylogenetic tree: A. aligarhensis and A. dodonia, from 
two different hosts, appeared to cluster together as sister 
species with a high posterior probability value. The pres- 
ence of host-switching (one daughter parasitoid lineage 
shifting to a distant host) or sorting events (when the 
parasitoid is absent, for example, has become extinct, 
in one of the daughter host lineages) may explain this 
result. 

The distance-based analysis showed a strong cophylo- 
genetic signal between Anicetus species and their scale 
insect hosts. However, tree-based analyses suggested that 
this signal is significant only when the cost of host- 
switching is high. In addition, a sharp increase in the 
number of sorting events was found using the TreeFitter 
cost model, suggesting that sorting has been an important 



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component of Anicetus diversification. Paterson et al [14] 
have suggested that three processes can lead to the ab- 
sence of parasites from their hosts: sampling error, pa- 
rasite extinction and the patchy distribution of parasites 
(resulting in the so-called "missing the boat" process). We 
believe that our sampling was dense enough to strongly 
reduce, if not eliminate, sampling error. Our observations 
suggest that parasitism rates even within one species are 
not stable and low rates are often found in some locations. 
Chantos et al. [60] observed that the encyrtid wasp Neo- 
dusmetia sangwani (Subba Rao) exhibits a patchy geo- 
graphic distribution. Our investigations showed that most 
Ceroplastes species only carried up to three Anicetus indi- 
viduals and that a patchy distribution of Anicetus species 
may be very common in wax scales. Therefore, Anicetus 
species may have been absent from the host founder 
population because of a patchy distribution and the small 
size of the host population when speciation took place, 
leading to a sorting event via a "missing the boat" process. 
In addition, host specific parasites are likely to possess 
fewer populations than multi-host parasites [56]. These 
observations support the conclusion that some parasites 
in this study may have gone extinct from a host lineage 
after a host speciation event. 

In the present study, we observed that Anicetus species 
only attacked and parasitized single host species. This is 
coherent with the hypothesis that the evolution of ob- 
ligate parasites (or parasitoids) with limited ability to 
transfer between different host species is tightly linked 
to the evolution of their own host species [61]. However, 
the congruence of host-parasite phylogenies is not per- 
fect, which can be explained by a mix of revolutionary 
events such as host switching, parasite speciation with- 
out host speciation (duplication), parasite extinction, 
and non-colonization of all host lineages [62]. A previ- 
ous study suggested that Anicetus species is adapted to 
narrow niches or restricted to particular hosts. Speci- 
fically, A. ceroplastis, A. beneficus, A. rubensi and A. ali- 
garhensis develop on the same host {Ceroplastes spp.), 
and thus far they have not been reared from other hosts 
across China [38]. After investigating a high number of 
samples from different provinces, we found that these 
species and others display strict host specificity (Table 2). 
For example, A. spl and A. sp2 were observed to only 
attack Pulvinaria aurantii and Parasaissetia sp., respect- 
ively. This host specificity is not congruent with former 
multi-host records of the genus Anicetus observed in 
previous studies [63-65], which could be explained by 
the extensive examination carried out in the present 
study, coupled with the use of molecular data. 

Many studies have supported the hypothesis that koi- 
nobionts are more host-specific than idiobionts [66-68], 
and a high degree of host specificity is relatively com- 
mon among parasitic Hymenoptera [43,51,69]. Traditional 



Table 2 A detailed description of host specificity of each 
Anicetus species 



Anicetus species 


Location 


Host 


Date 


Eusemion sp. 


Guangxi, baise 


Aconthococcus sp. 


2.vi.2013 


A sp2 


Fujian, Nanjing 


Porosoissetio sp. 


23.ix.2008 


A spl 


Shanghai 


pulvinorio ourontii 


19.V.2008 


A oligorhensis 


Shanxi, Taiyuan 


C. joponicus 


3.vi.2007 


A. oligorhensis 


Hubei, Jingzhou 


C. joponicus 


10.V.2011 


A. oligorhensis 


Hubei, Xiangyang 


C. joponicus 


15.viii.2011 


A. ohgushii 


Zhejiang, Yuyao 


C. joponicus 


29.xii.2010 


A. dodonio 


Anhui, Wuhu 


C. ceriferus 


8.vi.2010 


A. ceroplostis 


Beijing 


C. ceriferus 


15.ix.2008 


A. rubensi 


Shanghai 


C. rubens 


1 1 .v.2008 


A. rubensi 


Jiangxi, Yichun 


C. rubens 


13.V.2009 


A. rubensi 


Jiangxi, Xinyu 


C. rubens 


15.xi.2008 


A. rubensi 


Hunan, Changsha 


C. rubens 


11.xi.2006 


A. beneficus 


Shanghai 


C. rubens 


1 1 .v.2008 


A. beneficus 


Jiangxi, Yichun 


C. rubens 


13.xi.2008 


A. beneficus 


Hangzhou 


C. rubens 


24.ix.2009 


A. beneficus 


Sichuan, Chengdu 


C. rubens 


16.V.2009 


A. beneficus 


Austrailia 


C. rubens 


15.xi.2010 


A. beneficus 


Hangzhou 


C. rubens 


24.xi.2008 


A. beneficus 


Anhui, HeFei 


C. rubens 


20.V.201 1 


A. beneficus 


Jiangxi, Xinyu 


C. rubens 


20.xi.2009 


A. beneficus 


Jiangsu, Nanjing 


C. rubens 


9.X.2009 


A. beneficus vor 


Yunnan, Kunming 


C. rubens 


26.iv.2011 



species of parasitoid wasps that use many different hosts 
for their larvae can be complexes of cryptic taxa, each of 
them adapted to use only a few hosts [69]. An increasing 
number of studies using molecular data suggest that spe- 
cies traditionally considered generalists are in fact com- 
plexes of cryptic taxa, each of them adapted to narrow 
niches [38,40,42,70]. To avoid such problematic species 
identification leading to biased patterns of host specificity, 
taxonomic issues such as careful species discrimination 
and recognition of cryptic taxa must be carefully ad- 
dressed before conducting cophylogenetic studies. 

Conclusions 

In this study, we carefully assessed the identity of Anicetus 
species parasitizing wax scales and verified the taxonomic 
status of their hosts using laboratory rearing. Through the 
distance-based analysis (ParaFit) and the topology-based 
analyses (TreeMap 3.0|3 And Jane 4), we presented strong 
evidence for a prevalence of sorting events and high host 
specificity in the genus Anicetus, offering insights into the 
diversification process of Anicetus species parasitizing 
scale insects. Our study emphasizes that extensive rearing 
of parasitoids and accurate identification are important for 



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Page 8 of 1 1 



investigating coevolutionary relationships in host-parasi- 
toid associations. 

Methods 

Sampling 

All species of Anicetus were reared from adults or late- 
stage nymphs of wax scale insects (Ceroplastes spp.) col- 
lected in the field from 14 provinces in China. Different 
Ceroplastes species present on the same twig or leaf 
were isolated and kept individually in glass vials for at 
least 2 months to allow parasitoids to emerge. The col- 
lected parasitoids were stored in 95% ethanol for taxo- 
nomic identification and molecular study. Parasitoids 
were identified by author ZYZ and Ceroplastes hosts by 
author SAW. In total, we collected seven out of twelve 
Anicetus species known from China [34] and two other 
species tentatively named as Anicetus spl (reared form 
Pulvinaria aurantii) and Anicetus sp2 (reared from 
Parasaissetia sp.). Although we have collected six out of 
ten Ceroplastes species known in China [39], Anicetus 
species were reared from three of them (see Additional 
file 1 and Additional file 2). Voucher specimens were de- 
posited at the Institute of Zoology, Chinese Academy of 
Sciences, Beijing. 

DNA extraction, amplification and sequencing 

Total DNA was extracted from individuals preserved in 
95% ethanol using DNeasy Blood & Tissue Kit (Qiagen), 
following the manufacturer s protocol. Protocols for PCR 
amplification of COI and 28S followed Zhang et al. [38] 
for parasitoids and Deng et al. [39] for scale insects. 
Products were visualized on 1% agarose and the most 
intense products were sequenced bidirectionally using 
BigDye v3.1 on an ABI3730xl DNA Analyzer (Applied 
Biosystems). GenBank accession numbers are given in 
Additional file 1 and Additional file 2. 

Phylogenetic reconstruction 

Sequences of COI and 28S were aligned using Clustal W 
1.8.3 [71] as implemented in BioEdit 7.0.5 [72]. Some se- 
quences of hosts and parasitoids were retrieved from 
previous studies [38,39]. Several samples collected from 
other cities in China (see electronic supplementary ma- 
terial, Additional file 1 and Additional file 2) were added 
to our data. A total of 94 Anicetus individuals (nine pa- 
rasitoid species) and 113 scale insect individuals (seven 
host species) were used in the present study. To confirm 
that sequence data could be concatenated, the homogen- 
eity of the COI and 28S data sets was assessed using a 
partition homogeneity test (100 replicates) [73] as im- 
plemented in the program PAUP* 4.0bl0 [74]. We esti- 
mated the DNA sequence evolution model that best fit 
the data using jModelTest 0.1.1. [75], applying the Akaike 
Information Criterion (AIC). For the COI data, we used 



a codon model (nucmodel = codon, code = metmt in 
MrBayes, see below). For the 28S data, the selected 
models for hosts and parasitoids were HKY + G and 
GTR + G, respectively. Bayesian analyses (BA) of com- 
bined data sets were performed with MrBayes 3.2 [76] 
with these evolutionary models assigned separately to the 
respective partitions. A Markov chain Monte Carlo search 
was run with four chains of 10,000,000 generations sam- 
pled once every 100 generations. A plot of number of gen- 
erations versus the log probability was used to check for 
stationarity, and posterior probability values (PP) were cal- 
culated after the first 25% of trees were discarded. To test 
the convergence of chains and assess stationarity of BA 
parameter values, the effective sample sizes (ESS) of all 
parameters were calculated using Tracer 1.5 [77]. Analyses 
of these parameters in Tracer 1.5 shown that most ESS 
values were exceeding 500, indicating strong equilibrium 
after discarding burn-in. Eusemion sp. (Hymenoptera: 
Encyrtidae) was chosen as an outgroup of Anicetus para- 
stoids and Acanthococcus sp. (Hemiptera: Eriococcidae) as 
an outgroup of coccids. 

Cophylogenetic analyses 

Seven host species and nine Anicetus species were used 
for cophylogenetic analyses. Consensus sequences of COI 
and 28S were created by collapsing all sequences from the 
same species using BioEdit 7.0.5, and used in the analysis 
of the congruence of parasite and host phylogenies. Sev- 
eral methods using TreeMap [9,78], TreeFitter [11], Jane 4 
[79] and ParaFit [12], are available to study the congru- 
ence between symbiont and host phylogenies. In the 
present study, three methods were used: a distance-based 
method called ParaFit implemented in CopyCat [80] and 
topology (or tree) -based methods implemented in Jane 4 
and TreeMap 3 (developed by Mike Charleston and avail- 
able at http://sites.google.com/site/cophylogeny). 

TreeMap is a popular topology-based program that 
reconciles two trees using four types of events (cospecia- 
tion (C), host-switching (H), duplication (D), and sorting 
(S)) to graphically depict the differences between the phy- 
logenies [9,81]. In our study, TreeMap 3.0|3 was used to 
reconstruct the tanglegram and assess the congruence 
between parasite and host phylogenies (including out- 
groups). We also computed the correlation between evo- 
lutionary divergences in previously identified cospeciating 
pairs ("copaths") in TreeMap to test whether parasitoids 
evolve faster than their hosts [9]. As the same genes were 
used to build host and parasite trees, the slope of the lin- 
ear relationship between corresponding divergences re- 
flect their relative evolutionary rates. 

Jane 4 uses a polynomial time dynamic programming 
algorithm in conjunction with a genetic algorithm to 
compare the two tree topologies by optimally mapping 
the parasite tree onto the host tree using different event 



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Page 9 of 1 1 



costs to find very good, and often optimal, solutions to 
reconcile the two phylogenetic trees [79,82]. We used 
Jane 4 with 100 generations and a population size of 200 
as parameters of the genetic algorithm. Six different cost 
models were used to find the minimum total cost (see 
Table 1). All models were tested using random tip 
mappings with 100 randomizations. Jane 4 can handle 
polytomies, considered as soft polytomies, which are 
resolved in order to minimize the global cost. We se- 
lected the option "Prevent mid-polytomy" to ensure 
that no revolutionary event was involved in the (very 
short) branches created to resolve polytomies. 

ParaFit is not dependent on fully resolved phylogenies 
and uses matrices of phylogenetic distances for both 
hosts and parasites [12]. Three types of information are 
used to describe the situation in matrix form: a matrix 
of phylogenetic distances among parasites, a matrix of 
phylogenetic distances among hosts, and a matrix of the 
observed host-parasite associations. All of the combined 
consensus data of parasitoids and hosts were used to sta- 
tistically assess the global fit between trees and the sig- 
nificance of the contribution of each individual link 
between taxa to this global congruence. Tests of signifi- 
cance were performed using 999 permutations. 

Availability of supporting data 

GenBank accession numbers are provided in Additional 
file 1: Table SI and Additional file 2: Table S2). The se- 
quence alignments for tree construction have been de- 
posited in the TreeBASE with accession URL (http:// 
purl.org/phylo/treebase/phylows/study/TB2:S15010). 

Additional files 



Competing interests 

The authors declare that they have no competing interests. 
Authors' contributions 

JD, FY and YZZ assembled all of the sequences. JD, FY, HBL, MG, YD and YZZ 
performed data analyses. JD, MG, YD, SAW and YZZ wrote the manuscript. 
All of the authors read and approved the final manuscript. 

Acknowledgments 

We are grateful to two anonymous reviewers for helpful comments, to Bodil 
Cass for kindly revising the English language, and to the following people 
who helped us to collect Ceroplastes samples: Guo-Hua Huang (Hunan 



Agricultural University, Changsha), Shao-Bin Huang (Guangdong Forestry Vo- 
cational Technology College, Guangzhou), Jian-qin Wu (The Administrative 
Bureau of Tianbaoyan National Nature 

Reserve of Yong'an, Yong'an), Kai-Ju Wei (Youxi No.1 Middle School of Fujian 
Province, Youxi), Hong-Liang Li (Institute for Nutritional Sciences, SIBS, 
Chinese Academy of Sciences, Shanghai), Hu Li (Guizhou University, Guiyang), 
Xian Li (Forestry Protection Station of Chengdu, Sichuan), Qiang Shen (Forestry 
Protection Station of Yuyao, Ningbo), Xiu-Hao Yang (Forestry Protection Station 
of Guangxi, Nanning), Ying-Jie Zhang (Yunnan Agricultural University, Kunming), 
Nan Nan and Xu-Bo Wang (Beijing Forestry University, Beijing), Ying Wang 
(Northeast Forestry University, Harbin). The project was supported by the 
Natural Science Foundation of China (NSFC grant no. 31272350, 31372151), the 
Fundamental Research Funds for the Central Universities (BLYJ201 305), 
the Chinese Academy of Sciences (KSCX2-YW-NF-02) and in part by the 
Department of Science and Technology of China (2012FY1 1 1 100). 

Author details 

] Key Laboratory of Zoological Systematics and Evolution, Institute of 
Zoology, Chinese Academy of Sciences, Beijing 100101, China. 2 Key 
Laboratory for Silviculture and Conservation of Ministry of Education, Beijing 
Forestry University, Beijing 100083, China. 3 CNR - Istituto per la Protezione 
delle Piante, UOS di Portici, Via Universita 133, 80055 Portici (NA), Italy, 
department of Entomology, The University of Arizona, 410 Forbes Building, 
Tucson, AZ 85721, USA. 5 Sorbonne Universites, UPMC Univ Paris 06, UMR 
7232, Integrative Biology of Marine Organisms, Observatoire Oceanologique, 
F-66650 Banyuls/Mer, France. 6 CNRS, UMR 7232, Integrative Biology of Marine 
Organisms, Observatoire Oceanologique, F-66650 Banyuls/Mer, France. 

Received: 25 September 2013 Accepted: 18 December 2013 
Published: 23 December 2013 



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Cite this article as: Deng et al.: Cophylogenetic relationships between 
Anicetus parasitoids (Hymenoptera: Encyrtidae) and their scale insect 
hosts (Hemiptera: Coccidae). BMC Evolutionary Biology 2013 13:275. 



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