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Accepted Manuscript

Title: Characterization of two complete Isospora mitochondrial


genomes from passerine birds: Isospora serinuse in a domestic
canary and Isospora manorinae in a yellow-throated miner

Authors: Rongchang Yang, Belinda Brice, Charlotte Oskam,


Yujuan Zhang, Frances Brigg, Dave Berryman, Una Ryan
PII: S0304-4017(17)30037-7
DOI: http://dx.doi.org/doi:10.1016/j.vetpar.2017.01.027
Reference: VETPAR 8248

To appear in: Veterinary Parasitology

Received date: 22-8-2016


Revised date: 23-1-2017
Accepted date: 31-1-2017
Please cite this article as: {http://dx.doi.org/

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Short Communication

Characterization of two complete Isospora mitochondrial genomes from passerine

birds: Isospora serinuse in a domestic Canary and Isospora manorinae in a

yellow-throated miner

Rongchang Yanga*, Belinda Briceb, Charlotte Oskama, Yujuan Zhanga, Frances

Briggc, Dave Berrymanc, Una Ryana

a
School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western

Australia, 6150.

b
Kanyana Wildlife Rehabilitation Centre, 120 Gilchrist Road, Lesmurdie, Western

Australia 6076.

C
WA State Agricultural Biotechnology Centre, Murdoch University, Murdoch,

Western Australia, 6150.

________________________________

*Corresponding author: Rongchang Yang

Mailing address: School of Veterinary and Life Sciences, Murdoch University,

Murdoch, Western Australia, Australia, 6150.

E-mail: R.Yang@murdoch.edu.au

Graphical abstract
1
Arrangement of the mitochondrial genomes for I. serinuse (a) and I. manorinae (b)

a
Isospora serinuse
1 1000 2000 3000 4000 5000 6000 6260

Putative Start

Isospora manorinae
b
1 1000 2000 3000 4000 5000 6000 6223

Putative Start

Highlights
Mitochondrial (mt) genomes were sequenced from two avian Isospora species
First phylogenetic analysis of the Isospora mt with other coccidian mt
sequence.
Further support the monophyletic nature of avian Isospora

Abstract

The genus term Isospora is now applied specifically to parasites of birds, with the term

Cystoisospora preferred for parasites which infect mammals. Isospora is a common

parasitic coccidian in birds worldwide, especially in passerine birds, in which it can

cause systemic coccidiosis. The complete mitochondrial genome sequences from two

recently identified Isospora species; Isospora serinuse in a domestic canary and

Isospora manorinae in a yellow-throated miner, were sequenced and compared with

those of other closely related coccidian species. The complete mitochondrial genome
2
sequence for Isospora serinuse is 6,260 bp in size and 6,223 bp for Isospora manorinae.

The mitochondrial genomes of Isospora serinuse and

Isospora manorinae include three protein-coding genes (COI, COIII and CytB), 19 LSU

and 14 SSU rDNA fragments, including one newly identified putative LSU fragment in

Isospora sp. The arrangement of coding regions in these two Isospora species were

identical to that of available Isospora sp. and Eimeria spp. mitochondrial genomes and

the start codon usage for protein coding genes was conservative. Phylogenetic analysis

of the mt genome of the two Isospora species based on the

three coding regions further support that the monophyletic nature of avian Isospora.

Keywords: Isospora serinuse; Isospora manorinae; mitochondrial genome;

phylogenetic analysis; Sanger sequencing; next generation sequencing.

1. Introduction

Coccidiosis is usually caused by protozoa of the genera Eimeria or Isospora.

Clinical signs include diarrhea, fever, inappetence, weight loss, emaciation, and in

extreme cases, death. Nevertheless, many infections are subclinical (Constable, 2015).

Coccidiosis is an important disease of most species of livestock (e.g. poultry, cattle,

sheep, goats, pigs, rabbits), as well as wildlife (e.g. marsupials, reptiles and birds). The

genus Isospora belongs to the phylum Apicomplexa, which also includes

3
Plasmodium, Babesia, Theileria, Toxoplasma, Eimeria, Cystoisospora and

Cryptosporidium (Frlich et al., 2012).

Mitochondria (mt) are organelles derived from the primitive symbiosis of

archeon ancestors with prokaryotic -proteobacteria species (Margulis, 1975).

However, in the evolutionary process leading to modern eukaryotic cells, mitochondria

lost the ability to synthesize most of the proteins encoded by the primitive bacterial

DNA, and only the small circular polycystronic mtDNA was conserved, with the

bacterial genes being transferred to the nuclear genome (Arciuch et al., 2012). Most

apicomplexan species possess mt genomes of ~6kb in size with considerable variability

in structure and organization (Gray et al., 2004). The genomes of apicomplexan

mitochondria encode three mt protein coding genes (cytochrome c oxidase subunits,

COI and COIII, and cytochrome b, CytB) (Cinar et al., 2015), in addition to highly

fragmented ribosomal RNA (rRNA) genes. Among the

apicomplexan organisms, Eimeria is phylogenetically closest genus to Isospora based

on 18S gene sequences (Yang et al., 2015a). The complete mt genome sequences for 19

Eimeria species have been reported (Hikosaka et al., 2010; Lin et al., 2011; Liu et al.,

2012 and 2015; Heitlinger et al., 2014; Ogedengbe et al., 2014; Hafeez et al., 2016; Tian

et al., 2015), all of which range from 6.1 to 6.4 kb in size with highly conserved content

and structure (Liu et al., 2015). Currently only one Isospora mt genome sequence has

been reported (Ogedengbe et al., 2015). In the present study, we generated the complete

mt DNA sequences of two Isospora species using next generation sequencing (NGS)

with the Illumina MiSeq platform and Sanger clonebased sequencing for I. manorinae

and Sanger clone-based sequencing for I. serinuse We also analysed features of their
4
gene content and genome organization and compared these with closely related

coccidian species.

2. Materials and Methods

2.1 Sampling and DNA extraction

The sample information, DNA extraction and species identification at both

morphological and molecular levels for Isospora serinuse in the domestic canary and

for Isospora manorinae in the yellow-throated miner have been previously described

(Yang et al., 2015b; Yang et al., 2016).

2.2 Amplification and sequencing of two Isospora spp. mtDNA fragments

Due to the low number of I. serinuse oocysts available and hence the low DNA

yield from PCR amplicons, insufficient DNA was available for NGS sequencing.

Therefore long-range PCR combined with clone-based Sanger

sequencing was used for this species. As large numbers of oocysts were available for I.

manorinae, this species was sequenced using the NGS MiSeq (Illumina) platform. For

comparative purposes, I. manorinae was also sequenced using the Sanger sequencing

method described for I. serinuse. Mitochondrial whole genome amplification for I.

serinuse and I. manorinae initially used two sets of specific primers that generated

overlapping PCR fragments: 1) Cocci_MT-WG-F(5TAC ACC TAG CCA ACA CGA

T-3) and Cocci_MT-WG-R (5-GCA GCT GTA GAT GGA TGC TT-3); and

2) Inv_COI_262R(5-AAW GCG GCA TCR TAG AAT TG-3) and

5
Inv_COI_461F(5 CTAG CYA TGG GAT GTA TTA CTG-3) (Ogedengbe et. al.,

2014). PCR reactions were carried out in a 50 l reaction mixture consisting of 25l 2

LA Taq polymerase buffer (CloneTech Laboratories Mountain View, CA USA),

23.0l sterile deionized water, 0.5l each primer (50 pmol/l), and 1.0l DNA template

(50ng/l), with the following amplification conditions: 94C for 5 min, then

94C for 30 s, 50C for 30s and 68C for 6 min for 35 cycles, followed by 68C for 5

min. To complete the mt genome of I. serinuse and I. manorinae, PCR products were

generated using a reverse primer downstream of the original forward primer (i.e.

Cocci_MT-WG-F or Inv_COI_461F) and a forward primer upstream of the original

reverse primer (i.e. Cocci_MT-WG-R or Inv_COI_262R). Each PCR product was

cloned into the pGEMT-Easy vector (Promega, USA) and sequenced in both directions,

using an ABI PRISM 3730 (Applied Biosystems Inc.). For I. serinuse,

PCR products were also sequenced directly with the primers listed in Supplementary

Table 1, to further confirm the complete mt sequence was correct.

The mt genome of I. manorinae was also sequenced using an Illumina MiSeq

(San Diego, CA). DNA was extracted from 50 L of purified oocysts at a concentration

of 1x107 oocysts per L. Briefly, the genomic DNA (2 g) was sheared to an average

size of 500 bp using a M220 ultrasonicator (Covaris, MA, USA) and measured using a

Qubit 2.0 Fluorometer (Invitrogen, Scoresby, Vic. Australia).

Libraries were prepared using the KAPA HyperPlus Library Preparation kit (KAPA

BIOSYSTEMS, South Africa) and barcoded using index 2 CGATGTC(A) from the

TruSeq DNA kit from Illumina. Precapture amplification was conducted using thirteen

6
PCR cycles and was quantified by Qubit 2.0. Paired end sequencing (2 x250bp) was

performed using a MiSeq v2 chemistry kit from Illumina. Data were initially processed

using MiSeq Reporter.

The complete mt genomic sequences of both I. serinuse and I. manorinae

generated by Sanger sequencing, were assembled using CLC genomics workbench

CAP3 plugin (Huang and Madan, 1999) (http://doua.prabi.fr/software/cap3) and

manually annotated by comparison of sequences with other mt Isospora and Eimeria

genomes available in the GenBank (accessed on 08Nov 2016) (Supplementary Table

2). For the I. manorinae mt sequences generated by NGS, the average coverage was

10.7 fold with a minimum of 4.0 and maximum of 166. Overall, 2,031 out of 2.76

million reads matched the mt sequence of I. manorinae while the majority of the

remaining sequences matched I. manorinae genomic sequences.

2.3 Sequence analyses

Protein-coding genes were identified using BLAST searches and ORF Finder

software at NCBI, with the genetic code set for Isospora sp. Transfer RNA (tRNA)

genes were annotated using tRNAscan-SE (Schattner et al. 2005), combined with

recognizing anticodon sequences and potential secondary structures by visual

inspection. The rRNA genes were identified by sequence comparisons with the

published Isospora mt rRNA genes. Tandem repeat elements were detected with

Tandem Repeats Finder program (Benson, 1999). The nucleotide composition (%) of

each gene and non-coding regions were determined using MEGA7 (Kumar et al.

7
2016).

2.4 Phylogenetic Analyses

Three coding regions (COI, COIII and Cytb) from 34 mt genome sequences were used

for analysis (Supplementary Table 2), including 21 Eimeria spp. (8 from chickens, 6

from the domestic turkeys (Ogedengbe et al., 2014), 6 from rabbits (Liu et al., 2015;

Tian et al., 2015) and one from a striped skunk (Mephitis mephitis). Sequences from

seven Isospora sp. were used in the phylogenetic analysis and included four from

passerine birds in Canada (KP658103, KT203397 KR108298 and KT203396) and one

from the central bearded dragon (KR108297) from Canada. Analysis also included one

Caryospora bigenetica sequence from a snake (Ogedengbe and Barta, 2015), two

Cyclospora cayetanensis from human stool samples (Cinar et al., 2015; Tang et al.,

2015) and one Lankesterella sequence from a green frog in Canada (KT369005).

Haemoproteus columbae (Perkins, 2008) was used as the out-group. Phylogenetic

analyses were conducted using Neighbor-joining (NJ),

Maximum Likelihood (ML) and Maximum Parsimony (MP methods) in MEGA7

(Kumar et al., 2016). Evolutionary history was conducted using the Maximum

Likelihood (ML) method based on the General Time Reversible model (Nei, 2000)

which was selected based on the model selection in MEGA 7. The tree with the highest

log likelihood is shown. The percentage of trees in which the associated taxa clustered

together is shown next to the branches. Initial tree(s) for the heuristic search were

obtained automatically by applying NJ and BioNJ algorithms to a matrix of pairwise

distances estimated using the Maximum Composite Likelihood (MCL) approach, and
8
then selecting the topology with the highest log likelihood value. The tree was drawn

to scale, with branch lengths depicting substitutions per site. 3. Results

The complete mt genomes of I. serinuse (Acc. No. KX276860) and I.

manorinae (Acc. No. KX276861) were 6,260 bp and 6,223 bp in length, respectively.

Comparison of the consensus sequences for I. manorinae from NGS and Sanger

sequencing identified some single nucleotide polymorphisms (SNPs) and small

insertions and deletions (INDELS) in the non-coding regions in the NGS sequences.

The validity of these SNPs and INDELS in the NGS sequences were confirmed by

repeated direct Sanger sequencing of PCR products. Genome content and organization

of the two Isospora sp. mt genomes consisted of three protein-coding genes (COI, COIII

and Cytb), interspersed with 19 LSU and 14 SSU rDNA fragments (Tables 1 and 2; Fig.

1a and Fig. 1b). Both I. serinuse and I. manorinae had identical sized COI, COIII and

CytB genes at 1,080, 1,443 and 756 bp, respectively. A pseudogene tRNA (tRNA F)

was identified in I. serinuse from position 3,120 to 3,179 bp, while three pseudogene

tRNA fragments (tRNA F, tRNA E and tRNA P) were detected in I. manorinae at

positions 3,120-3,179, 3,205-3,263 and 3,365-3,416 bp, respectively.

The initiation codon, ATG was found in CytB and COI genes, whilst TTA was

detected in the COIII gene. All the three genes were terminated by the TAA codon. The

nucleotide compositions of the mtDNA sequences for I. serinuse and I. manorinae are

biased toward A and T, with T being the most common nucleotide and G the least

common. The A+T content is 65.5% for I. serinuse (30.0% A, 35.5% T, 17.8% C and

16.6% G), 65.4% for I. manorinae (30.3% A, 35.5% T, 17.6% C and

9
16.6% G).

To determine the phylogenetic relationships of the two Isospora species with other

related Apicomplexa parasites, a concatenated analysis of the DNA from the three

coding regions (Cytb, COI and COIII) were analyzed using NJ, ML and MP methods.

Topologies of all trees inferred by three methods were identical or similar (Fig. 2 ML

tree shown). Phylogenetic relationships among species were well resolved with very

high nodal support throughout. Phylogenetic analysis showed that I. serinuse and I.

manorinae were in the same clade as the other four Isospora sp.from the passerine birds

(I. sp. JRB-2015, I. sp. JRB-2016, I. superbusi and I. greineri). Isospora serinuse and

I. sp. JRB-2015 were both identified in faecal samples from the domestic

canary (Serinus canaria forma domestica) and grouped closely in the phylogenetic

tree (Fig. 2).

Multiple sequence alignments among the six Passerine bird Isospora sp mt DNA

sequences were conducted. Overall, I. serinuse exhibited 96.9%, 96.0%, 96.0%, 95.7%

and 95.1%, similarity with I. sp. JRB-2015 from a domestic canary, I. greineri from a

superb glossy starling (Lamprotornis superbus), I. sp. JRB-2016 from a blackthroated

laughingthrush (Garrulax chinensis), I. superbusi from a superb glossy starling

(Lamprotornis superbus) and I. manorinae from a yellow-throated miner (Manorina

flavigula), respectively at the nucleotide level. Isospora manorinae exhibited the

highest similarity of 95.6% with I. sp. JRB-2015 and I. sp. JRB-2016 at

95.4%. Both I. superbusi and I. greineri shared 95.1% similarity with I. manorinae.

Individual mt rDNA regions annotated in the Isospora sp. genomes and the

10
corresponding mt rDNA fragments of Isospora sp. JRB-2015 (KP658103), I. sp. JRB-

2016, I. superbusi and I. greineri demonstrated that the sequence identities ranged from

85.7% to 100% from I. manorinae and 89.3 to 100% for I. serinuse. For the three

protein-coding genes (COI, COIII and Cytb), similarities between I. manorinae and I.

sp. JRB-2015 ranged from 94.3% to 95.6% for Cytb, from 94.3 to 95.2% for COI and

from 92.3 to 93.1% for COIII. Similarly, similarities for I. serinuse and I. sp. JRB-2015

accross the three regions ranged from 95.5 to 96.2% for

Cytb, 94.8 to 95.7% for COI and 93.3 to 93.9% in COIII .

4. Discussion

The mt genomes of I. serinuse and I. manorinae are similar with respect to

genome size, organisation, start codon positions and overall base composition.

Arrangement of coding regions was identical to that of available Isospora sp. mt

genomes and start codon usage for protein-coding genes was conventional. Comparison

of the consensus sequences for I. manorinae from NGS and Sanger sequencing

identified some INDELs in the non-coding regions and 15 SNPs in the coding regions

identified by NGS but not Sanger. Repeated direct Sanger sequencing of PCR products

confirmed these sequence variations. SNP and INDEL errors in NGS-generated

genomes can arise from a number of mechanisms but increasing coverage due to the

decreasing cost of NGS and improved INDEL calling algorithms continue to reduce

these errors (Fang et al., 2014). In the present study, within population variation may

account for the disparity between NGS and Sanger, particularly as the DNA was

11
extracted from a mixed population of oocysts and not single oocysts. This is because

Sanger sequencing of PCR products presents the average or most abundant sequence in

the population whereas sequencing by NGS presents "all" sequences individually.

Therefore the deeper coverage afforded by NGS was able to detect the additional SNPs

and indels in the I. manorinae mt genome that were not initially detected by Sanger

sequencing.

The genus Isospora was first described by Schneider in 1881 and its taxonomy

has been a source of controversy since. In 2005, Barta et al. assigned all tetrasporozoic,

diplosporocystic oocysts from mammals without Stieda bodies in their sporocysts, to

the genus Cystoisospora (Sarcocystidade), and all such oocysts from birds with Stieda

bodies in their sporocysts to the genus Isospora (Barta et al., 2005). In the present study,

phylogenetic analysis of concatenated analysis of the DNA from the three coding

regions (Cytb, COI and COIII) confirmed the monophyletic nature of avian Isospora.

Previous phylogenetic analysis of chromosomal 18S rDNA sequences from both these

species also confirmed this (Yang et al., 2015a; Yang et al., 2016).

Interestingly, in the present study, the Isopsora sp. from the central bearded

dragon (I. amphiboluri ) grouped in a clade with Lankesterella sp.from a green frog in

Canada. As this was the only mtDNA sequence available from a lizard-derived Isopsora

sp. in GenBank, it further analysis requiring additional mtDNA sequences from lizard-

derived Isospora spp. is required to better understand the phylogenetic relationships

between Lankesterella and lizard-derived Isospora sp. Recently, MegaPalma et al.

2016 genetically characterized 9 Isospora species from Squamata at 18S locus, it was

shown that the lizard-derived Isospora sp was also monophyletic, they set in a separate
12
clade in the 18S phylogenetic tree, which was far distanced from the clade for avian

Isospora species. This result also support the outcome from this study that the avian and

lizard Isospora species were genetically different even they have the similar

oocyst/sporocyst structures. In addition, the single Choleoeimeria sp. isolate grouped

in a separate clade from all the other coccidian species in the mt phylogenetic three

(Fig.2), further supporting its status as a separate genus (Yang et al., 2015).

In conclusion, the mtDNA of I. serinuse in a domestic Canary and I.

manorinae in a yellow-throated miner characterised in the present study will provide

novel mt genetic markers for further studies on the taxonomy, epidemiology and

population genetics of avian Isospora sp.

Acknowledgements

The authors are grateful to Dr. Maoyun She for assistance with drawing the

mitochondrial annotation maps.

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Fig. 1. Arrangement of the mitochondrial genomes for I. serinuse (1a) and I. manorinae

(1b). Transcriptional direction and order of the three coding regions for Cytb, COI and

COIII (direction indicated by arrowed end). Drawings were generated from Geneious

software and modified by Microsoft Office PowerPoint.

Arrangement of the mitochondrial genomes for I. serinuse (a) and I. manorinae (b)

a
Isospora serinuse
1 1000 2000 3000 4000 5000 6000 6260

Putative Start

Isospora manorinae
b
1 1000 2000 3000 4000 5000 6000 6223

Putative Start

Fig. 2. Evolutionary relationships of I. serinuse and I. manorinae inferred by Maximum

Likelihood analysis of concatenated DNA sequences from 3 mt coding regions (Cytb,

COI and COIII). Percentage support (>50%) from 1000

pseudoreplicates from ML analysis, is indicated at the left of the support node.

18
Table 1. Protein-coding genes and fragmented rDNAs found in the complete
mitochondrial genome of an Isospora serinuse (Eimeriidae, Apicomplexa).

Gene Strand Strand Location Size (bp) Start Codon Stop Codon
Putative Start (Stem-loop structure) None 1_27 27

RNA9; SSU/8 + 35-81 47

SSUA; SSU/4 + 123-199 77

RNA23t; SSU (Tentative) + 200-234 35

CytB CDS, + 245-1324 1080 ATG TAA

COI CDS + 1357-2799 1443 ATG TAA

RNA20 (Partial); LSU (Tentative) + 2805-2824 20

LSUF; LSU/11 + 2850-2961 112

rrnL + 2887-3047 161

LSUG; LSU/12 + 2962-3067 106

LSU (Tentative) - 3076-3100 25

trnF(aaa) + 3120-3179 60

RNA14; SSU/1 + 3141-3180 40

LSUC; LSU/4 - 3210-3226 17

RNA22; SSU (Tentative) + 3248-3278 31

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SSUF; SSU/12 + 3361-3421 61

rrnS + 3375-3412 38

RNA10; LSU/13 (Partial) + 3436-3495 60

RNA11; LSU/5 + 3519-3567 49

SSUD; SSU/10 + 3583-3647 65

RNA17; SSU/3 + 3687-3726 40

RNA15; SSU (Tentative) + 37333762 30

RNA13; LSU/10 + 37703799 30

RNA6; LSU/15 + 38063863 58

LSUD; LSU/8 - 38873967 81

RNA16 (Partial); LSU (Tentative) - 39763998 23

RNA8; SSU/5 - 40134104 92

rrnS + 4040-4059 20

RNA2; LSU/2 + 41224187 66

LSUA; LSU/1 - 42024382 181

COIII CDS + 43975152 756 TTA TAA

RNA19; SSU/7 - 51625190 29

RNA1; LSU/6 + 52245311 88

LSUB; LSU/3 - 53875413 27

RNA3; LSU/7 - 54495521 73

RNA18; LSU/14 - 55355554 20

SSUB; SSU/6 - 55885706 119

RNA7; LSU (Tentative) - 57115790 80

LSUE; LSU/9 - 58005991 192

SSUE; SSU/11 - 61536185 33

RNA5; SSU/9 - 61096211 103

Imperfect Mirror Repeat None 62486260 13

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Table 2. Protein-coding genes and fragmented rDNAs found in the complete
mitochondrial genome of an Isospora manorinae (Eimeriidae, Apicomplexa).
Gene Strand Strand Location Size (bp) Start Codon Stop Codon
Putative Start (Stem-loop structure) + 1_27 27

RNA9; SSU/8 + 35-81 47

SSUA; SSU/4 + 123-199 77

RNA23t; SSU (Tentative) + 200-234 35

CytB CDS + 245-1324 1080 ATG TAA


COI CDS + 1357-2709 1443 ATG TAA
RNA20 (Partial); LSU (Tentative) + 2805-2824 20

LSUF; LSU/11 + 2850-2961 112

rrnL + 2887-3047 161

LSUG; LSU/12 + 2962-3067 106

LSU (Tentative) - 3076-3100 25

trnF(aaa) + 3120-3179 60

RNA14; SSU/1 + 3141-3180 40

LSUC; LSU/4 - 3190-3205 16

trnE(ttc) + 3204-3262 59

RNA22; SSU (Tentative) + 3231-3261 31

SSUF; SSU/12 + 3344-3404 61

trnP(tgg) + 3365-3416 52

RNA10; LSU/13 (Partial) + 3419-3478 60

RNA11; LSU/5 + 3502-3550 49

SSUD; SSU/10 + 3559-3623 65

RNA17; SSU/3 + 3658-3697 40

RNA15; SSU (Tentative) + 37043733 30

RNA13; LSU/10 + 37413770 30

RNA6; LSU/15 + 37773834 58

LSUD; LSU/8 - 38503930 81

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RNA16 (Partial); LSU (Tentative) - 39393961 23

RNA8; SSU/5 - 39744065 92

RNA2; LSU/2 + 40834148 66

LSUA; LSU/1 - 41774357 181

COIII CDS + 43725127 756 TTA TAA


RNA19; SSU/7 - 51375165 29

RNA1; LSU/6 + 52025286 88

LSUB; LSU/3 - 53625388 27

RNA3; LSU/7 - 54245496 73

RNA18; LSU/14 - 55105529 20

SSUB; SSU/6 - 55585676 119

RNA7; LSU (Tentative) - 56815760 80

LSUE; LSU/9 - 57705961 192

SSUE; SSU/11 - 60656097 33

RNA5; SSU/9 - 60796173 95

Imperfect Mirror Repeat None 62116223 13

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