Spider

mite

-LRB-

Acari

Tetranychidae

-RRB-

mitochondrial

COI

phylogeny

reviewed

host

plant

relationships

phylogeography

reproductive

parasites

and

barcoding

The

past

years

have

witnessed

a

number

of

molecular

studies

that

aimed

to

resolve

issues

of

species

delineation

and

phylogeny

of

mites

in

the

family

Tetranychidae

The

central

part

of

the

mitochondrial

COI

region

has

frequently

been

used

for

investigating

intra

and

interspecific

variation

All

these

studies

combined

yield

an

extensive

database

of

sequence

information

of

the

family

Tetranychidae

We

assembled

this

information

in

a

single

alignment

and

performed

an

overall

phylogenetic

analysis

The

resulting

phylogeny

shows

that

important

patterns

have

been

overlooked

in

previous

studies

whereas

others

disappear

It

also

reveals

that

mistakes

were

made

in

submitting

the

data

to

GenBank

which

further

disturbed

interpretation

of

the

data

Our

total

analysis

clearly

shows

three

clades

that

most

likely

correspond

to

the

species

T.

urticae

T.

kanzawai

and

T.

truncatus

Intraspecific

variation

is

very

high

possibly

due

to

selective

sweeps

caused

by

reproductive

parasites

We

found

no

evidence

for

host

plant

associations

and

phylogeographic

patterns

in

T.

urticae

are

absent

Finally

we

evaluate

the

application

of

DNA

barcoding

Introduction

Species

identification

is

the

basis

for

understanding

species

diversity

phylogenetic

patterns

and

evolutionary

processes

Only

correct

identifications

allow

for

comparisons

between

studies

and

the

repetition

or

expansion

of

earlier

experiments

In

pest

species

species

identification

is

also

important

in

the

development

of

-LRB-

biological

-RRB-

pest

control

strategies

Identification

and

delineation

of

species

within

the

Tetranychidae

has

been

an

issue

of

debate

for

the

past

few

decades

Within

the

family

about

different

species

are

described

many

of

which

are

of

agronomical

importance

-LRB-

Bolland

et

al.

-RRB-

The

genus

Tetranychus

is

well

studied

and

includes

two

common

major

agricultural

pest

species

with

a

worldwide

distribution

Tetranychus

urticae

Koch

and

Tetranychus

kanzawai

Kishida

Morphological

identification

of

tetranychid

species

is

difficult

The

number

of

potential

diagnostic

characters

is

limited

-LRB-

partly

due

to

the

small

size

of

the

mites

-RRB-

and

key

traits

often

exhibit

large

phenotypic

plasticity

As

a

result

many

species

can

not

be

distinguished

on

the

basis

of

external

morphology

For

example

in

Japan

Tetranychus

species

are

recognized

-LRB-

Ehara

-RRB-

However

morphological

identification

using

adult

females

is

possible

for

only

two

of

the

species

The

remaining

eight

species

can

only

be

identified

by

microscopic

examination

of

the

shape

of

the

aedeagus

-LRB-

part

of

the

male

genitalia

-RRB-

Another

example

that

shows

our

inability

to

identify

species

on

the

basis

of

morphology

is

the

well-studied

two-spotted

spider

mite

species

T.

urticae

This

species

is

considered

a

species

complex

-LRB-

Navajas

et

al.

-RRB-

and

as

many

as

synonymous

names

are

known

-LRB-

Bolland

et

al.

-RRB-

The

question

whether

red

T.

urticae

mites

should

be

considered

a

separate

species

-LRB-

T.

cinnabarinus

-RRB-

has

occupied

taxonomists

for

many

years

-LRB-

Dupont

Gotoh

and

Tokioka

Zhang

and

Jacobson

-RRB-

The

fact

that

there

are

only

few

taxonomists

specialized

in

morphological

identification

of

mites

and

that

their

number

is

decreasing

adds

to

the

problem

of

spider

mite

identification

DNA

sequences

are

currently

an

indispensable

tool

for

delineating

and

identifying

species

In

this

context

it

is

important

to

distinguish

between

DNA

taxonomy

and

DNA

barcoding

DNA

taxonomy

concerns

the

circumscription

and

delineation

of

species

using

evolutionary

species

concepts

-LRB-

Vogler

and

Monaghan

-RRB-

DNA

barcoding

aims

at

the

identification

of

pre-defined

species

and

does

not

address

the

issue

of

species

delineation

per

se

-LRB-

Monaghan

et

al.

-RRB-

In

DNA

barcoding

a

short

standardized

DNA

sequence

usually

the

end

part

of

the

mitochondrial

cytochrome

c

oxidase

subunit

I

-LRB-

COI

-RRB-

gene

is

used

to

identify

species

DNA

barcoding

can

be

used

to

-LRB-

i

-RRB-

identify

and

assign

unknown

specimens

to

species

that

have

been

previously

described

and

-LRB-

ii

-RRB-

enhance

the

discovery

of

new

species

using

a

threshold

of

sequence

divergence

-LRB-

Hebert

et

al.

Moritz

and

Cicero

-RRB-

DNA

taxonomy

may

be

based

on

one

or

several

mitochondrial

as

well

as

nuclear

DNA

regions

and

can

serve

as

a

database

for

DNA

barcoding

DNA

taxonomy

is

an

offshoot

of

phylogenetics

in

which

the

evolutionary

relationships

between

taxa

-LRB-

e.g.

species

-RRB-

are

investigated

Throughout

this

paper

we

use

the

term

species

delineation

when

it

concerns

DNA

taxonomy

and

species

identification

when

it

concerns

DNA

barcoding

The

usefulness

of

the

COI

region

for

delineating

tetranychid

species

has

been

investigated

in

several

studies

-LRB-

Hinomoto

et

al.

Hinomoto

and

Takafuji

Lee

et

al.

Navajas

et

al.

Toda

et

al.

Xie

et

al.

-RRB-

Recently

a

DNA

barcoding

approach

was

used

to

identify

tetranychid

species

-LRB-

Hinomoto

et

al.

-RRB-

Each

of

these

studies

used

a

different

or

sometimes

partially

overlapping

subset

of

tetranychid

sequences

Many

studies

extended

their

dataset

with

one

or

several

tetranychid

sequences

from

the

GenBank

database

serving

as

a

reference

for

phylogeny

reconstruction

or

species

identification

However

sequence

diversity

within

T.

urticae

is

substantial

-LRB-

e.g.

Navajas

et

al.

-RRB-

so

that

different

T.

urticae

sequences

are

available

from

GenBank

some

of

which

bear

an

incorrect

species

name

-LRB-

due

to

misidentification

-RRB-

As

a

consequence

different

T.

urticae

reference

sequences

were

used

in

above-mentioned

studies

leading

to

the

emergence

of

variable

taxonomic

groupings

and

phylogenetic

patterns

This

combined

with

the

analyses

of

restricted

subsets

in

each

study

gives

an

incomplete

and

fragmented

view

of

species

delineations

and

phylogenetic

relationships

within

the

family

Tetranychidae

In

this

study

we

create

an

extensive

COI

dataset

of

the

family

Tetranychidae

with

a

wide

coverage

of

the

species

T.

kanzawai

and

T.

urticae

-LRB-

including

T.

cinnabarinus

which

is

currently

considered

synonymous

to

T.

urticae

-RRB-

We

have

collected

all

currently

available

mitochondrial

COI

sequences

from

GenBank

and

added

data

on

mites

collected

from

Europe

and

North

America

We

critically

evaluate

the

assembled

data

and

perform

an

overall

phylogenetic

analysis

This

approach

reveals

novel

patterns

on

species

delineation

and

phylogenetic

relationships

We

discuss

the

use

of

COI

for

DNA

barcoding

purposes

by

considering

the

intra

and

interspecific

variation

In

addition

we

discuss

the

observed

variation

in

COI

in

relation

to

associated

host

plant

phylogeographic

patterns

and

the

presence

of

endosymbionts

-LRB-

e.g.

Wolbachia

Cardinium

-RRB-

Finally

we

provide

guidelines

for

future

phylogenetic

studies

on

-LRB-

tetranychid

-RRB-

mites

Material

and

methods

Additional

tetranychid

samples

Tetranychid

mites

were

collected

in

Europe

-LRB-

six

locations

-RRB-

in

North

America

-LRB-

one

location

-RRB-

and

from

two

cultures

maintained

in

our

lab

for

years

-LRB-

Table

-RRB-

Mites

were

not

identified

morphologically

to

the

species

level

DNA

was

extracted

from

single

individuals

using

a

modified

CTAB

extraction

method

-LRB-

Doyle

-RRB-

A

single

adult

female

was

ground

in

μl

of

proteinase

K

-LRB-

mg/ml

-RRB-

and

μl

CTAB

-LRB-

CTAB

w/v

in

mM

Tris-HCl

-LSB-

-RSB-

mM

EDTA

and

M

NaCl

-RRB-

buffer

was

added

After

vortexing

samples

were

incubated

at

°C

for

h.

Next

μl

chloroform

isoamylalcohol

-LRB-

-RRB-

was

added

and

contents

were

gently

mixed

for

min

Tubes

were

centrifuged

for

min

at

g

After

centrifugation

μl

of

the

supernatant

was

transferred

to

a

clean

tube

and

DNA

was

precipitated

by

adding

μl

ice-cold

ethanol

Tubes

were

incubated

at

°C

for

at

least

h

prior

to

centrifugation

at

g

for

min

at

°C

The

supernatant

was

removed

and

the

DNA

pellet

was

washed

with

ethanol

Next

the

DNA

was

air

dried

for

at

least

min

eluted

in

μl

sterile

water

and

stored

at

°C

Table

of

samples

sequenced

in

this

study

and

the

primer

sequences

used

for

COI

amplificationStrainCountryLocalityHost

plantCollection

date

PrimerbCommon

nameScientific

SpindleEuonymus

HoneysuckleLonicera

-LRB-

greenhouse

-RRB-

RoseaRosa

StatesTucson

-LRB-

AZ

-RRB-

de

MonchiqueCitrusCitrus

del

campOrangeCitrus

in

the

lab

on

bean

-LRB-

Phaseolus

vulgaris

-RRB-

for

over

GGAGGATTTGGAAATTGATTAGTTCC

-LRB-

Navajas

and

Boursot

-RRB-

AAWCCTCTAAAAATRGCRAATACRGC

-LRB-

modified

from

Hinomoto

and

Takafuji

-LRB-

-RRB-

TGATTTTTTGGTCACCCAGAAG

-LRB-

Navajas

et

al.

-RRB-

TACAGCTCCTATAGATAAAAC

-LRB-

Navajas

et

al.

-RRB-

Part

of

the

mitochondrial

COI

gene

was

amplified

using

various

primer

combinations

-LRB-

Table

-RRB-

Depending

on

the

primer

combination

this

yielded

a

fragment

size

of

basepairs

-LRB-

bp

-RRB-

excluding

the

primer

annealing

sites

-LRB-

Fig.

-RRB-

PCR

was

performed

in

a

μl

reaction

mix

containing

μl

Super

Taq

buffer

-LRB-

HT

BioTechnology

Cambridge

U.K.

-RRB-

μl

bovine

serum

albumin

-LRB-

mg/ml

-RRB-

μl

-LRB-

mM

-RRB-

μl

dNTP

mix

-LRB-

mM

of

each

nucleotide

-RRB-

μl

of

each

primer

-LRB-

μM

each

-RRB-

μl

of

super

Taq

-LRB-

u

μl

-RRB-

-LRB-

HT

BioTechnology

-RRB-

μl

water

and

μl

of

DNA

extract

PCR

cycling

conditions

were

min

at

°C

followed

by

cycles

of

min

at

°C

min

at

°C

and

min

at

°C

and

a

final

extension

at

°C

for

min

Products

-LRB-

μl

-RRB-

were

visualized

on

a

agarose

gel

stained

with

ethidium

bromide

in

X

TBE

buffer

-LRB-

mM

Tris

base

mM

boric

acid

and

mM

EDTA

pH

-RRB-

Fig.

of

the

COI

fragments

sequenced

in

different

studies

and

their

relative

position

after

alignment

The

position

of

the

fragment

analyzed

in

this

study

and

of

the

standard

DNA

barcoding

fragment

-LRB-

Folmer

fragment

-RRB-

on

the

total

mitochondrial

COI

gene

-LRB-

position

-RRB-

are

indicated

on

top

Base

pair

numbers

correspond

to

the

Drosophila

melanogaster

mitochondrial

DNA

sequence

-LRB-

GenBank

accession

nr

-RRB-

For

each

study

thick

horizontal

lines

represent

the

fragment

sequenced

in

all

specimens

and

thin

lines

indicate

the

maximum

sequence

length

Number

of

sequences

-LRB-

between

parentheses

-RRB-

and

references

are

listed

at

the

right

For

references

of

unpublished

studies

see

Appendix

PCR

products

were

purified

using

a

DNA

extraction

kit

-LRB-

Fermentas

St.

Leon-Rot

Germany

-RRB-

The

purified

products

were

directly

sequenced

using

the

ABI

PRISM

BigDye

Terminator

Sequence

Kit

-LRB-

Applied

Biosystems

Nieuwerkerk

a/d

IJssel

The

Netherlands

-RRB-

according

to

the

manufacturer

's

instructions

but

diluted

times

Both

strands

of

the

products

were

sequenced

using

the

same

primers

as

used

in

the

PCR

amplification

Sequences

were

run

on

an

ABI

automated

DNA

sequencer

Obtained

sequences

were

aligned

using

ClustalX

v

-LRB-

Thompson

et

al.

-RRB-

and

compared

to

the

sequences

obtained

from

GenBank

-LRB-

see

below

-RRB-

Database

compilation

Sequence

collection

A

single

database

was

constructed

comprising

all

available

tetranychid

COI

sequences

from

GenBank

and

the

sequences

obtained

in

this

study

Sequences

were

collected

from

GenBank

on

September

-LRB-

keywords

for

search

were

cytochrome

oxidase

subunit

I

AND

Tetranychidae

-RRB-

This

yielded

a

total

of

sequences

-LRB-

from

GenBank

from

this

study

-RRB-

of

which

-LRB-

-RRB-

were

published

in

peer

reviewed

journals

An

overview

of

all

sequences

their

GenBank

accession

numbers

assigned

species

names

references

sample

locations

and

associated

host

plants

-LRB-

if

known

-RRB-

is

given

in

the

Appendix

Sequences

were

aligned

using

ClustalX

Due

to

the

use

of

different

primer

combinations

in

the

various

studies

the

sequences

differed

in

length

and

in

position

on

the

COI

region

-LRB-

Fig.

-RRB-

A

central

part

of

bp

was

chosen

for

subsequent

analysis

-LRB-

highlighted

region

in

Fig.

-RRB-

Considering

this

central

part

sequences

were

found

more

than

once

-LRB-

see

Appendix

-RRB-

Prior

to

phylogenetic

analysis

identical

sequences

-LRB-

except

one

-RRB-

were

removed

from

the

dataset

resulting

in

unique

sequences

In

addition

six

sequences

with

accession

numbers

-LRB-

Lee

et

al.

-RRB-

were

excluded

from

analysis

because

of

too

many

missing

data

in

the

region

of

overlap

-LRB-

bp

of

the

bp

part

are

missing

Fig.

-RRB-

The

dataset

was

further

adjusted

for

wrongly

submitted

sequences

-LRB-

see

next

paragraph

-RRB-

leading

to

the

addition

of

one

corrected

sequence

yielding

a

final

number

of

aligned

COI

sequences

Of

these

were

published

in

peer

reviewed

journals

and

belong

to

the

genus

Tetranychus

according

to

the

GenBank

submission

info

A

Clustal

alignment

of

the

unique

sequences

can

be

obtained

from

the

corresponding

author

upon

request

Data

validation

Incongruencies

in

the

database

When

compiling

the

dataset

two

discrepancies

were

encountered

between

sequence

information

submitted

to

GenBank

and

the

description

in

the

associated

articles

The

first

one

concerns

accession

Its

sequence

was

wrongly

submitted

to

GenBank

In

GenBank

accessions

and

are

listed

as

T.

neocaledonicus

and

T.

gloveri

respectively

According

to

the

associated

article

these

sequences

should

differ

-LRB-

Navajas

et

al.

-RRB-

However

accessions

and

show

identical

sequences

both

concurring

with

the

sequence

T.

neocaledonicus

from

the

article

The

correct

sequence

of

T.

gloveri

was

obtained

from

the

original

article

and

added

to

the

dataset

The

second

discrepancy

concerns

accessions

and

According

to

the

description

in

GenBank

accession

was

obtained

from

the

Amphitetranychus

quercivorus

strain

Sapporo

and

accession

from

the

A.

quercivorus

strain

Tsukuba

Comparing

this

to

sequences

in

Navajas

et

al.

-LRB-

-RRB-

the

GenBank

sequence

concurs

with

A.

quercivorus

strain

Tsukuba

in

the

article

GenBank

sequence

concurs

with

the

sequence

of

A.

viennensis

in

the

article

-LRB-

and

therefore

is

identical

to

the

GenBank

sequence

of

previously

named

T.

viennensis

-RRB-

This

means

that

the

sequences

of

accessions

and

are

different

from

the

article

sequences

We

included

accessions

as

A.

quercivorus

and

as

A.

viennensis

in

the

dataset

Accession

was

excluded

from

the

dataset

Phylogenetic

analysis

PAUP

version

-LRB-

Swofford

-RRB-

and

DAMBE

version

-LRB-

Xia

and

Xie

-RRB-

were

used

to

calculate

numbers

of

variable

sites

uncorrected

pairwise

divergences

nucleotide

composition

and

transition

and

transversion

ratios

PAUP

was

used

to

perform

a

chi-square

test

of

base

frequency

homogeneity

across

all

taxa

Phylogenetic

analyses

were

conducted

in

PAUP

using

Neighbour-Joining

-LRB-

NJ

-RRB-

algorithms

-LRB-

p-distance

-RRB-

and

Maximum

Likelihood

-LRB-

ML

-RRB-

algorithms

-LRB-

TBR

heuristics

random

addition

sequence

with

five

replicates

reconnection

limit

of

-RRB-

Both

PAUP

and

Modeltest

-LRB-

Posada

and

Crandall

-RRB-

were

used

to

select

the

optimal

evolution

model

for

the

ML

analysis

The

selected

model

was

further

optimized

by

critically

evaluating

the

selected

parameters

-LRB-

Swofford

and

Sullivan

-RRB-

using

the

Akaike

Information

Criterion

-LRB-

AIC

Akaike

-RRB-

Because

COI

is

a

protein

coding

gene

we

tested

if

the

likelihood

of

models

with

the

lowest

likelihood

score

could

be

further

improved

by

incorporating

specific

rates

for

each

codon

position

-LRB-

Shapiro

et

al.

-RRB-

Under

the

selected

model

parameters

and

tree

topology

were

optimized

using

the

successive

approximations

approach

-LRB-

Sullivan

et

al.

-RRB-

For

the

NJ

analyses

robustness

of

nodes

was

assessed

with

NJ-bootstrap

replicates

For

the

ML

analyses

bootstrap

support

was

assessed

by

performing

a

NJ

bootstrap

-LRB-

replicates

-RRB-

with

distances

calculated

according

to

the

selected

ML

model

-LRB-

because

of

computational

constraints

-RRB-

Phylogenetic

analyses

were

performed

for

i

-RRB-

the

family

Tetranychidae

and

ii

-RRB-

the

genus

Tetranychus

separately

The

analysis

of

the

family

Tetranychidae

included

all

species

As

the

species

T.

kanzawai

T.

urticae

and

T.

truncatus

are

over-represented

eight

strains

were

selected

representing

these

three

species

-LRB-

Fig.

-RRB-

The

dataset

for

this

analysis

included

sequences

The

final

tree

was

rooted

using

the

species

Petrobia

harti

and

Bryobia

kissophila

These

two

species

belong

to

a

separate

subfamily

-LRB-

Bryobiinae

-RRB-

from

all

other

species

-LRB-

subfamily

Tetranychinae

-RRB-

The

analysis

of

the

genus

Tetranychus

included

sequences

For

this

analysis

two

Panonychus

sequences

and

one

Petrobia

and

one

Bryobia

sequence

were

used

as

an

outgroup

Because

saturation

of

the

third

codon

position

is

frequently

observed

for

COI

-LRB-

Gleeson

et

al.

Söller

et

al.

-RRB-

an

analysis

excluding

this

position

was

performed

to

see

if

this

improved

the

resolution

of

the

phylogeny

Results

Data

acquisition

New

sequencing

Each

COI

sequence

that

we

obtained

in

this

study

was

identical

to

several

sequences

already

present

in

the

GenBank

dataset

Two

samples

from

the

USA

-LRB-

Arizona

-RRB-

and

Portugal

were

identical

to

Eutetranychus

banksi

The

sample

from

citrus

in

Spain

was

identical

to

Panonychus

citri

from

Japan

and

the

sample

from

Poland

-LRB-

from

Prunus

spec

-RRB-

to

P.

ulmi

from

Japan

The

remaining

samples

-LRB-

the

two

lab

strains

and

a

sample

from

France

-RRB-

were

identical

to

an

Asian

T.

urticae

strain

-LRB-

sampled

from

Japan

Thailand

and

Taiwan

-RRB-

-LRB-

see

Appendix

-RRB-

Alignment

and

analysis

of

patterns

of

molecular

evolution

All

sequences

could

be

unambiguously

aligned

no

insertions

or

deletions

were

found

Translation

of

all

sequences

into

amino

acids

revealed

no

stop

codons

The

total

alignment

of

the

tetranychid

sequences

was

bp

-LRB-

minimum

sequence

length

was

bp

Fig.

-RRB-

sites

were

phylogenetically

informative

sites

were

variable

but

uninformative

and

sites

were

constant

On

average

across

all

taxa

the

AT

content

was

-LRB-

A

T

C

and

G

-RRB-

This

high

AT

content

is

a

general

feature

of

the

COI

region

in

arthropods

and

is

comparable

to

other

studies

on

insect

and

mite

taxa

-LRB-

Lunt

et

al.

Navajas

et

al.

-RRB-

However

the

distribution

of

bias

in

base

composition

was

not

uniform

with

respect

to

the

three

codon

positions

-LRB-

Fig.

-RRB-

First

second

and

third

codon

positions

showed

AT

biases

of

and

respectively

In

some

haplotypes

no

C

or

G

base

was

found

at

the

third

codon

position

Nevertheless

a

chi-square

test

of

base

frequency

homogeneity

revealed

no

significant

differences

across

taxa

for

the

overall

data

set

or

for

the

three

base

positions

separately

-LRB-

Fig.

-RRB-

Note

that

this

test

ignores

correlation

due

to

phylogeny

and

therefore

tends

to

reject

the

hypothesis

too

easily

so

that

failure

to

reject

can

safely

be

taken

as

evidence

of

homogeneity

-LRB-

Frati

et

al.

-RRB-

Fig.

compositions

for

each

codon

position

of

the

aligned

COI

region

averaged

over

all

tetranychid

samples

Error

bars

depict

minimum

to

maximum

range

Results

of

the

homogeneity

test

are

given

for

each

codon

position

The

extent

of

saturation

was

assessed

by

plotting

the

transition

and

transversion

rates

against

uncorrected

p-distance

divergences

-LRB-

Fig.

-RRB-

At

the

third

codon

position

transversions

outnumber

transitions

and

the

number

of

transversions

begins

to

plateau

-LRB-

Fig.

-RRB-

indicating

saturation

and

making

this

position

unsuitable

for

resolving

more

basal

branching

patterns

However

removing

the

third

codon

position

from

the

analysis

did

not

result

in

a

more

resolved

phylogeny

-LRB-

results

not

shown

-RRB-

This

is

probably

due

to

a

conserved

amino

acid

sequence

-LRB-

limiting

the

amount

of

variation

in

first

and

second

base

pair

positions

as

changes

in

these

positions

in

most

cases

change

the

amino

acid

sequence

-RRB-

Fig.

plots

of

transversion

and

transition

rates

against

uncorrected

p-distance

at

each

codon

position

The

model

selected

by

Modeltest

for

the

tetranychid

dataset

was

the

General

Time

Reversible

Model

with

invariable

sites

and

a

gamma

distribution

of

rate

heterogeneity

-LRB-

GTR+I+G

-RRB-

However

implementing

the

rate

class

a

b

a

b

e

f

significantly

improved

the

likelihood

-LRB-

AIC

-RRB-

and

was

therefore

used

for

parameter

and

tree

topology

estimation

For

the

Tetranychus

dataset

the

General

Time

Reversible

Model

-LRB-

GTR

-RRB-

with

site-specific

rates

for

the

three

coding

positions

was

further

optimized

by

incorporating

the

following

rate

class

a

b

c

d

e

a

This

slightly

simpler

model

significantly

improved

the

likelihood

-LRB-

AIC

-RRB-

and

was

used

for

parameter

and

tree

topology

estimation

Phylogenetic

relationships

between

tetranychid

genera

The

ML

tree

of

the

overall

analysis

is

shown

in

Fig.

The

phylogenetic

relationship

among

the

taxa

is

not

well

resolved

This

is

probably

due

to

the

strongly

biased

nucleotide

composition

and

the

saturation

at

the

third

codon

position

It

shows

that

this

portion

of

the

COI

gene

is

not

suitable

for

resolving

the

branching

order

of

the

genera

and

the

more

distantly

related

species

P.

harti

and

B.

kissophila

both

belonging

to

the

subfamily

Bryobiinae

cluster

together

and

group

outside

the

subfamily

Tetranychinae

There

is

however

no

high

support

for

monophyly

of

any

of

the

genera

The

NJ

tree

shows

a

similar

non-resolved

phylogeny

-LRB-

not

shown

-RRB-

with

the

only

difference

that

the

genus

Eotetranychus

appears

as

a

monophyletic

group

with

high

bootstrap

support

-LRB-

-RRB-

Differences

between

genera

range

from

to

and

between

species

within

genera

from

to

Fig.

likelihood

tree

of

the

tetranychid

dataset

based

upon

COI

sequences

GenBank

accession

numbers

and

associated

species

names

are

given

If

a

haplotype

is

found

more

than

once

the

accession

number

is

followed

by

the

haplotype

number

-LRB-

see

Appendix

-RRB-

and

the

number

of

times

the

haplotype

is

found

between

parentheses

Numbers

on

the

branches

indicate

the

percentage

bootstrap

values

-LRB-

-RRB-

based

on

NJ

bootstrapping

with

ML

settings

-LRB-

replicates

-RRB-

Bar

at

the

lower

left

corner

depicts

the

branch

length

corresponding

to

maximum

likelihood

distance

The

genus

Tetranychus

Just

over

of

the

sequences

in

GenBank

are

unpublished

Hong

Xie

and

colleagues

have

submitted

sequences

-LRB-

accessions

through

submitted

March

-RRB-

as

T.

cinnabarinus

It

is

unclear

why

all

these

accessions

were

named

T.

cinnabarinus

The

fact

that

these

T.

cinnabarinus

accessions

are

scattered

all

over

the

phylogeny

shows

that

these

sequences

do

not

concern

a

single

species

Besides

the

species

name

T.

cinnabarinus

is

not

generally

accepted

-LRB-

Dupont

Gotoh

and

Tokioka

-RRB-

and

is

also

not

mentioned

in

the

World

Catalogue

of

the

spider

mite

family

-LRB-

only

as

synonym

of

T.

urticae

-RRB-

-LRB-

Bolland

et

al.

-RRB-

In

the

remainder

of

this

paper

we

will

not

use

the

name

T.

cinnabarinus

For

the

genus

Tetranychus

the

ML

tree

is

shown

in

Fig.

The

NJ

tree

shows

a

similar

topology

as

the

ML

tree

bootstrap

support

values

are

slightly

lower

in

the

ML

tree

-LRB-

NJ

tree

not

shown

-RRB-

Several

clades

emerge

although

the

exact

branching

order

remains

unresolved

-LRB-

Fig.

-RRB-

The

species

T.

kanzawai

T.

urticae

and

presumably

T.

truncatus

-LRB-

see

below

-RRB-

have

been

widely

sampled

and

intraspecific

variation

is

substantial

These

species

form

a

monophyletic

group

-LRB-

bootstrap

support

-RRB-

The

relationship

between

all

other

Tetranychus

species

remains

unresolved

except

that

T.

pacificus

and

T.

mcdanieli

cluster

together

-LRB-

bootstrap

support

-RRB-

Two

potentially

new

species

are

found

-LRB-

and

-RRB-

Sequence

divergence

between

these

two

accessions

is

which

is

of

the

same

order

as

found

between

other

species

Although

these

accessions

are

described

in

GenBank

as

T.

cinnabarinus

this

seems

incorrect

-LRB-

see

above

-RRB-

Fig.

likelihood

tree

of

the

genus

Tetranychus

based

upon

COI

sequences

GenBank

accession

numbers

and

associated

species

name

are

given

-LRB-

except

for

T.

cinnabarinus

see

text

-RRB-

If

a

haplotype

is

found

more

than

once

the

accession

number

is

followed

by

the

haplotype

number

-LRB-

see

Appendix

-RRB-

and

the

number

of

times

the

haplotype

is

found

between

parentheses

Accessions

deposited

on

GenBank

as

T.

turkestani

are

marked

in

grey

Accessions

followed

by

the

letter

A

or

B

indicate

samples

belonging

to

clade

A

and

B

respectively

deduced

from

Navajas

-LRB-

-RRB-

and

Navajas

et

al.

-LRB-

-RRB-

Numbers

on

the

branches

indicate

the

percentage

bootstrap

values

-LRB-

-RRB-

based

on

NJ

bootstrapping

with

ML

settings

-LRB-

replicates

-RRB-

Bar

at

the

lower

left

corner

depicts

the

branch

length

corresponding

to

maximum

likelihood

distance

Tetranychus

urticae

Tetranychus

kanzawai

and

Tetranychus

truncatus

The

species

T.

urticae

and

T.

kanzawai

have

been

investigated

in

several

different

studies

and

were

sampled

from

all

over

the

world

-LRB-

see

Appendix

-RRB-

The

analysis

of

COI

variation

reveals

the

existence

of

very

divergent

lineages

-LRB-

Fig.

-RRB-

Clade

contains

all

T.

kanzawai

specimens

-LRB-

bootstrap

value

-RRB-

This

clade

contains

two

subclades

that

were

previously

described

by

Hinomoto

and

Takafuji

-LRB-

-RRB-

On

the

other

hand

T.

urticae

specimens

form

a

highly

diverse

group

in

which

several

well-supported

clades

are

recognized

One

clade

-LRB-

clade

in

Fig.

-RRB-

comprises

T.

urticae

specimens

all

originating

from

China

-LRB-

bootstrap

value

-RRB-

Hinomoto

et

al.

-LRB-

-RRB-

renamed

this

clade

T.

truncatus

All

other

T.

urticae

specimens

form

a

group

of

highly

divergent

lineages

-LRB-

clade

-RRB-

which

fall

into

several

more

or

less

supported

subclades

Moreover

within

this

group

several

specimens

have

been

identified

as

T.

turkestani

but

these

do

not

form

a

monophyletic

group

Discussion

The

phylogenetic

analysis

of

all

COI

sequences

available

in

GenBank

revealed

novel

patterns

which

alter

current

views

on

species

delineation

and

phylogeographic

patterns

in

spider

mites

In

addition

we

found

that

a

number

of

accessions

are

probably

registered

under

a

wrong

species

name

This

may

in

the

past

have

led

to

erroneous

interpretations

of

phylogenetic

patterns

that

included

these

GenBank

accessions

One

application

of

phylogenetic

analysis

is

the

identification

of

natural

groupings

in

phylogenetic

trees

that

represent

biological

species

-LRB-

DNA

taxonomy

-RRB-

Our

most

inclusive

assemblage

of

data

shows

new

and

previously

unnoticed

groups

that

most

likely

concern

different

species

-LRB-

Fig.

-RRB-

In

particular

the

phylogenetic

patterns

within

T.

urticae

differ

from

these

of

previous

studies

and

provide

new

insights

in

the

evolutionary

history

of

this

group

Up

to

now

two

clades

within

T.

urticae

were

recognized

named

clade

A

and

B

by

Navajas

et

al.

-LRB-

-RRB-

and

Hinomoto

et

al.

-LRB-

-RRB-

and

lineage

I

and

II

by

Xie

et

al.

-LRB-

-RRB-

The

latter

concluded

that

lineage

I

and

II

were

consistent

with

the

two

clades

A

and

B.

However

this

conclusion

is

not

supported

by

our

analysis

Our

clade

-LRB-

Fig.

-RRB-

contains

specimens

of

lineage

II

-LRB-

Xie

et

al.

-RRB-

and

clade

A

and

B

-LRB-

Navajas

et

al.

-RRB-

Moreover

clade

A

and

B

disappear

in

our

total

analysis

and

new

groupings

emerge

Clade

contains

specimens

of

lineage

I

In

fact

clade

is

a

well

supported

clade

restricted

to

China

that

clusters

outside

the

other

T.

urticae

samples

It

is

unclear

from

the

study

of

Xie

et

al.

-LRB-

-RRB-

whether

mites

from

clade

were

morphologically

different

from

other

T.

urticae

samples

Clade

presumably

represents

T.

truncatus

as

suggested

by

Hinomoto

et

al.

-LRB-

-RRB-

based

on

morphological

identification

of

newly

sampled

Japanese

mites

with

highly

similar

COI

sequences

Tetranychus

urticae

and

Tetranychus

turkestani

GenBank

specimens

listed

as

T.

urticae

and

T.

turkestani

do

not

form

separate

monophyletic

clades

-LRB-

Fig.

-RRB-

This

is

in

agreement

with

a

study

by

Navajas

and

Boursot

-LRB-

-RRB-

that

was

based

on

a

smaller

dataset

Although

Navajas

and

Boursot

-LRB-

-RRB-

were

able

to

separate

the

two

species

based

on

sequence

this

distinction

was

based

on

three

diagnostic

sites

only

Moreover

intraspecific

and

intra-individual

variation

within

was

found

which

further

questions

the

recognition

of

two

different

species

In

addition

there

are

no

discrete

morphological

differences

between

the

two

species

Taxonomic

identification

is

based

on

continuous

traits

-LRB-

e.g.

the

shape

of

the

aedeagus

of

males

-RRB-

and

there

is

no

thorough

study

describing

variation

of

these

traits

within

and

between

these

species

The

current

data

do

not

support

the

maintenance

of

T.

turkestani

as

a

separate

species

Host

plant

relationships

Host

race

formation

is

another

evolutionary

process

studied

in

spider

mites

that

may

explain

the

diversity

in

this

group

of

mites

Phylogenetics

is

one

approach

to

assess

spider

mite

host

plant

associations

Most

Tetranychus

species

are

reported

from

many

different

host

plant

species

For

example

Bolland

et

al.

-LRB-

-RRB-

described

different

host

plant

species

for

T.

urticae

belonging

to

plant

families

We

found

no

correlation

between

COI

divergence

and

associated

host

plant

species

-LRB-

Fig.

and

Appendix

-RRB-

similar

to

what

was

found

by

Navajas

-LRB-

-RRB-

Even

strains

with

identical

COI

haplotypes

can

be

found

on

very

different

host

plant

species

Also

the

other

two

relatively

well

sampled

species

T.

kanzawai

and

T.

truncatus

do

not

show

host

plant

associations

Phylogeographic

patterns

Phylogenetic

analysis

is

also

used

for

determining

phylogeographic

distribution

patterns

-LRB-

Avise

-RRB-

Phylogeographic

information

is

important

for

assessing

historic

migration

and

colonization

routes

and

can

also

be

used

for

tracing

the

origin

of

accidental

introductions

For

T.

kanzawai

two

main

clades

are

distinguished

originating

from

eastern

Asia

-LRB-

Japan

Taiwan

and

China

-RRB-

except

one

sample

that

originates

from

Congo

This

suggests

that

T.

kanzawai

has

a

mainly

eastern

Asian

distribution

However

Bolland

et

al.

-LRB-

-RRB-

reported

T.

kanzawai

from

all

over

the

world

but

it

is

not

known

if

these

samples

fall

within

the

clades

found

so

far

Clade

-LRB-

T.

truncatus

-RRB-

appears

restricted

to

China

-LRB-

Fig.

-RRB-

Within

T.

urticae

no

phylogeographic

pattern

is

apparent

with

respect

to

COI

variation

Samples

form

Europe

Asia

and

North

and

South

America

are

scattered

over

the

tree

The

phylogeographic

patterns

previously

described

by

Navajas

et

al.

-LRB-

-RRB-

completely

disappeared

They

found

an

entirely

Mediterranean

clade

-LRB-

clade

A

-RRB-

and

a

clade

of

mixed

origin

-LRB-

clade

B

-RRB-

Because

the

Mediterranean

clade

had

the

highest

diversity

they

argued

that

this

region

served

as

a

source

from

which

other

non-Mediterranean

regions

of

the

northern

hemisphere

were

recently

colonized

by

a

subset

of

the

Mediterranean

clades

-LRB-

Navajas

et

al.

Hinomoto

et

al.

-RRB-

However

inclusion

of

all

currently

available

sequences

does

not

support

their

conclusion

Clade

A

and

B

fall

apart

and

moreover

many

more

clades

are

found

There

are

several

possible

explanations

for

the

absence

of

clear

phylogeographic

patterns

First

such

patterns

may

simply

not

exist

Second

T.

urticae

is

a

pest

species

on

many

crops

and

ornamentals

and

it

is

likely

that

the

international

trade

in

crops

has

influenced

the

distribution

of

the

mites

around

the

world

This

will

obscure

any

correlation

between

geographical

location

and

phylogeny

Finally

selective

sweeps

can

greatly

influence

phylogenetic

patterns

-LRB-

Ballard

and

Rand

-RRB-

Evidence

is

accumulating

that

selective

sweeps

are

often

associated

with

the

presence

of

reproductive

parasites

such

as

Wolbachia

and

Cardinium

-LRB-

Hurst

and

Jiggins

-RRB-

Phylogenetic

inferences

and

DNA

barcoding

The

analyzed

COI

region

shows

considerable

variation

among

the

tetranychids

examined

The

diversity

within

species

is

especially

high

with

a

maximum

of

This

is

a

mixed

blessing

it

makes

COI

suitable

for

investigating

intraspecific

variation

but

its

usefulness

for

resolving

phylogenetic

species

relationships

remains

limited

The

latter

is

due

to

a

strongly

biased

nucleotide

composition

at

the

third

codon

position

and

consequently

saturation

at

this

position

Variation

at

first

and

second

codon

positions

is

very

low

As

a

result

relationships

between

taxa

are

difficult

to

resolve

especially

at

the

deeper

nodes

An

extremely

high

AT

content

and

saturation

at

the

third

codon

position

was

also

encountered

in

other

studies

for

the

same

COI

fragment

as

used

in

this

study

in

parasitengona

mites

and

for

the

adjacent

COI

region

in

velvet

worms

-LRB-

Onychophora

-RRB-

-LRB-

Gleeson

et

al.

Söller

et

al.

-RRB-

The

COI

fragment

analyzed

in

this

study

is

different

from

the

usual

DNA

barcoding

fragment

which

is

located

at

the

end

side

of

our

fragment

-LRB-

Fig.

-RRB-

Because

this

barcoding

fragment

is

amplified

by

primers

developed

by

Folmer

et

al.

-LRB-

-RRB-

it

is

referred

to

as

the

Folmer

fragment

-LRB-

Erpenbeck

et

al.

-RRB-

Substitution

patterns

may

differ

between

gene

partitions

and

may

result

in

different

phylogenetic

signals

for

these

partitions

-LRB-

Erpenbeck

et

al.

-RRB-

To

determine

whether

our

fragment

had

the

same

phylogenetic

signal

as

the

commonly

used

Folmer

fragment

we

compared

the

substitution

patterns

of

both

fragments

We

did

not

find

different

substitution

patterns

when

investigating

bp

of

the

Folmer

fragment

for

samples

for

which

this

fragment

was

available

-LRB-

results

not

shown

-RRB-

A

dataset

of

unique

sequences

-LRB-

representing

samples

-RRB-

revealed

a

highly

similar

substitution

pattern

with

transversions

outnumbering

transitions

resembling

the

patterns

in

Fig.

We

therefore

assume

that

analyzing

the

Folmer

fragment

for

tetranychid

mites

will

reveal

similar

patterns

as

found

in

this

study

Currently

the

Folmer

fragment

is

widely

used

as

a

gene

partition

for

barcoding

species

-LRB-

e.g.

Gómez

et

al.

Hebert

et

al.

-RRB-

although

other

fragments

have

also

been

proposed

-LRB-

e.g.

in

plants

Kress

et

al.

-RRB-

DNA

barcoding

assumes

that

genetic

distances

between

species

are

greater

than

within

species

In

that

way

clusters

of

similar

sequences

represent

species

clearly

separated

from

other

clusters

-LRB-

species

-RRB-

Hebert

et

al.

-LRB-

-RRB-

proposed

the

use

of

a

standard

threshold

-LRB-

divergence

value

-RRB-

to

identify

species

Up

to

now

several

studies

have

reported

successful

barcoding

of

species

-LRB-

e.g.

Barret

and

Hebert

Gómez

et

al.

Hebert

et

al.

-RRB-

However

often

intraspecific

variation

was

not

at

all

or

not

thoroughly

investigated

because

only

one

or

two

individuals

per

species

were

analyzed

or

geographic

sampling

was

restricted

-LRB-

Dasmahapatra

and

Mallet

Prendini

-RRB-

This

may

result

in

significant

underestimation

of

the

amount

of

intraspecific

variation

Additionally

interspecific

variation

might

be

overestimated

if

closely

related

sister

taxa

are

not

included

in

the

analysis

Therefore

it

is

necessary

to

analyze

samples

from

more

than

one

geographic

region

and

to

include

closely

related

sister

species

Our

study

comprises

an

analysis

of

three

closely

related

species

that

were

widely

sampled

and

showed

extensive

amounts

of

intraspecific

variation

Moreover

in

several

cases

intraspecific

variation

exceeded

interspecific

variation

between

the

species

as

is

illustrated

by

overlapping

frequency

distributions

of

intra

and

interspecific

pairwise

p-distances

-LRB-

Fig.

-RRB-

For

example

differences

within

T.

urticae

reach

up

to

exceeding

the

minimum

difference

between

T.

urticae

and

T.

kanzawai

Thus

the

general

barcoding

assumption

that

intraspecific

variation

is

smaller

than

interspecific

variation

is

violated

in

tetranychids

indicating

that

simply

relying

on

genetic

distances

is

not

sufficient

for

species

identification

This

clearly

illustrates

the

importance

of

including

samples

of

various

geographically

different

populations

for

each

species

and

to

include

comparisons

with

sister

species

when

investigating

the

efficacy

of

barcoding

It

also

shows

the

need

to

include

phylogenetic

information

to

delineate

species

groupings

instead

of

simply

relying

on

sequence

divergences

-LRB-

Prendini

Rubinoff

et

al.

-RRB-

A

careful

analysis

of

the

DNA

phylogeny

preferably

in

a

multi

disciplinary

approach

-LRB-

including

multiple

gene

data

morphological

ecological

or

other

relevant

data

-RRB-

can

assist

in

defining

or

delimiting

species

but

the

use

of

single

sequences

in

combination

with

a

threshold

seems

insufficient

to

simply

identify

species

Fig.

of

pairwise

differences

-LRB-

p-distance

-RRB-

between

COI

sequences

within

the

family

Tetranychidae

Pairwise

differences

are

separated

into

three

categories

between

individuals

in

the

same

species

between

individuals

in

the

same

genus

-LRB-

excluding

intraspecific

differences

-RRB-

between

individuals

in

the

same

family

-LRB-

excluding

intraspecific

and

intrageneric

differences

-RRB-

n

number

of

pairwise

comparisons

Reproductive

parasites

and

selective

sweeps

There

are

a

number

of

additional

problems

associated

with

the

use

of

a

single

mitochondrial

gene

for

barcoding

Hybridization

can

result

in

reticulate

evolutionary

relationships

between

species

and

disturb

groupings

into

species

based

on

mtDNA

Selective

sweeps

of

mtDNA

can

both

homogenize

or

increase

mtDNA

diversity

The

widespread

occurrence

of

reproductive

parasites

in

arthropods

can

both

influence

the

frequency

of

hybridization

between

host

species

and

indirectly

cause

selective

sweeps

of

mtDNA

-LRB-

Hurst

and

Jiggins

-RRB-

These

parasites

can

cause

homogenization

of

biological

species

after

hybridization

followed

by

spreading

of

the

intracellular

reproductive

parasite

The

mitochondrial

haplotype

is

dragged

along

with

these

parasites

resulting

in

replacement

of

the

original

mitochondrial

haplotype

and

reducing

mitochondrial

diversity

In

a

recent

study

Whitworth

et

al.

-LRB-

-RRB-

found

a

lack

of

species

monophyly

in

the

blowfly

genus

Protocalliphora

due

to

introgressive

hybridization

associated

with

Wolbachia

infection

On

the

other

hand

the

presence

of

different

reproductive

parasites

co-infecting

the

same

host

species

may

increase

the

levels

of

mitochondrial

diversity

within

that

host

species

if

each

parasite

is

tightly

linked

to

a

different

haplotype

-LRB-

Schulenburg

et

al.

-RRB-

Infection

with

reproductive

parasites

may

thus

increase

or

decrease

mitochondrial

diversity

and

severely

influence

the

patterns

of

mitochondrial

DNA

variation

Intracellular

reproductive

parasites

such

as

Wolbachia

Cardinium

and

Rickettsia

are

widespread

in

tetranychid

mites

-LRB-

Breeuwer

and

Jacobs

Gotoh

et

al.

Hoy

and

Jeyaprakash

Xie

et

al.

-RRB-

They

can

cause

cytoplasmic

incompatibility

-LRB-

CI

-RRB-

and

hybrid

breakdown

in

spider

mites

-LRB-

Breeuwer

Gotoh

et

al.

Vala

et

al.

-RRB-

It

is

possible

that

the

COI

variation

found

within

and

between

closely

related

mite

species

is

a

result

of

selective

sweeps

caused

by

infection

with

reproductive

parasites

Variation

within

species

is

relatively

high

It

is

not

linked

to

geographical

location

nor

associated

with

the

host

plant

An

interesting

next

step

would

be

to

investigate

the

link

between

haplotype

variation

within

COI

and

variation

in

reproductive

parasites

We

should

be

especially

cautious

with

the

use

of

mitochondrial

genes

for

delineating

and

barcoding

biological

species

in

light

of

the

presence

of

reproductive

parasites

Conclusions

and

recommendations

Accurate

species

delineation

and

identification

is

important

for

our

ability

to

understand

and

interpret

evolutionary

processes

and

ecological

diversity

in

mites

It

is

also

clear

that

mites

are

a

difficult

group

to

identify

morphologically

as

many

key

traits

exhibit

large

phenotypic

plasticity

and

lack

suitable

characters

for

identification

Many

ecological

behavioral

genetic

and

pest-control

studies

have

been

conducted

on

various

tetranychid

species

or

strains

without

the

concurrent

storage

of

voucher

specimens

leading

to

subsequent

uncertainty

about

the

identity

of

the

investigated

specimens

In

such

cases

DNA

barcoding

can

be

an

important

and

powerful

tool

to

assist

in

species

identification

-LRB-

Will

et

al.

-RRB-

However

the

use

of

a

single

-LRB-

mitochondrial

-RRB-

gene

for

DNA

barcoding

or

DNA

taxonomy

seems

inappropriate

An

integrative

approach

is

needed

combining

nuclear

and

mitochondrial

genes

morphological

characters

and

ecological

information

-LRB-

and

if

possible

crossing

experiments

-RRB-

A

combined

analysis

of

mitochondrial

and

nuclear

markers

is

commonly

used

to

avoid

the

problem

that

gene

trees

are

not

necessarily

congruent

with

species

trees

and

for

the

detection

of

hybridization

The

challenge

is

to

find

suitable

nuclear

markers

and

robust

geographic

sampling

designs

that

allow

for

the

assessment

of

intra

and

interspecific

variation

Navajas

and

Fenton

-LRB-

-RRB-

and

Cruickshank

-LRB-

-RRB-

have

investigated

the

suitability

of

various

molecular

markers

but

there

is

still

a

need

for

nuclear

markers

suitable

for

distinguishing

closely

related

species

Recently

Sonnenberg

et

al.

-LRB-

-RRB-

suggested

the

region

of

the

nuclear

rDNA

gene

as

a

taxonomic

marker

It

could

complement

DNA

barcoding

studies

based

on

mitochondrial

DNA

sequences

In

addition

molecular

testing

for

reproductive

parasites

and

crossing

experiments

using

isofemale

lines

should

be

standard

procedure

to

delineate

biological

species

O O O O O B O O B O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O

tetranychidae
tetranychus urticae
coi
barcoding
reproductive parasites
spider mites