The chromosomes and the mitogenome of Ceratitis fasciventris (Diptera: Tephritidae): two genetic approaches towards the Ceratitis FAR species complex resolution
Abstract
Ceratitis fasciventris is a serious agricultural pest of the Tephritidae family that belongs to the African Ceratitis FAR species complex. Species limits within the FAR complex are obscure and multidisciplinary approaches have attempted to resolve phylogenetic relationships among its members. These studies support the existence of at least three additional species in the complex, C. anonnae, C. rosa and C. quilicii, while they indicate the presence of two structured populations (F1 and F2) within the C. fasciventris species. In the present study we present the mitotic karyotype, polytene chromosome maps, in situ hybridization data and the complete mitochondrial genome sequence of an F2 population of C. fasciventris. This is the first polytene chromosome map and complete mitogenome of a member of the FAR complex and only the second reported for the Ceratitis genus. Both polytene chromosomes and mitochondrial sequence could provide valuable information and be used as reference for comparative analysis among the members of the complex towards the clarification of their phylogenetic relationships.
Introduction
The Tephritidae family consists of more than 500 genera and 4600 species being one of the most speciose groups of Diptera1. About 40% of the species of this family are characterized as true fruit flies since they infect a great variety of fruit producing plants and a significant number of them are considered serious agricultural pests. The Anastrepha, Bactrocera, Ceratitis, Dacus and Rhagoletis genera include some of the world’s most destructive insect pests, causing extensive damages in a wide variety of crops with enormous economic impact2.
Ceratitis fasciventris is an African fruit fly, which, together with C. anonae, C. rosa and the recently described C. quilicii comprise the“Ceratitis FAR complex”3, 4. All members of the complex are agricultural pests with C. rosa being the most aggressive one. They attack a great number of wild and cultivated plants belonging to more than 25 different families, and present greatly overlapping host range while each also has unique hosts5, 6. Ceratitis fasciventris is found mainly in eastern and western Africa sympatrically with C. anonae, while C. rosa and C. quilicii are distributed in eastern and southern Africa overlapping with each other and partially with C. fasciventris in Kenya and Tanzania4, 6–8. There is increasing concern about the possibility that these species could expand outside their native range similarly to the spread of C. capitata almost worldwide in the past century2, 3, 6, 9, 10. Populations of C. rosa and C. quilicii have been already introduced in the Indian ocean islands Mauritius and La Réunion4, 11 and their invasive potential is a major consideration taking into account their potential adaptation to a wider temperature range than C. capitata6, 12–14 and their capability to attack fruits grown also in temperate climates (e.g. peaches and apples)4, 15.
Because of the high risk of their expansion, the delimitation and accurate identification of species within the FAR as well as other complexes of economic relevant tephritid pests is of crucial importance for regulatory agencies and agricultural countries. It affects the international trade and quarantine policies implemented on fruit and vegetable hosts with significant economic impact16. Besides, the clarification of genetic relationships among the different entities of species complexes is critical for the effective development and application of environmental friendly methods for their control, such as the Sterile Insect Technique (SIT)17, 18. Within the FAR complex the species boundaries are obscure and have been the object of research and discussion for some time.The members of the complex are almost identical morphologically. Their identification has been based on differences in the setal pattern of the adult male mid legs, while females are almost indistinguishable4, 19, 20. Due to the absence of clear diagnostic morphological features a number of molecular approaches, including mitochondrial markers and the ITS1 sequence, have been used unsuccessfully to clearly resolve the limits among the morphospecies of the complex3, 21–23. A more recent microsatellite analysis revealed the presence of five genotypic clusters within the complex: two (R1, R2) representing populations that were at the time considered as C. rosa, two (F1, F2) for C. fasciventris and one for C. anonae8, thus complicating further the phylogenetic relationships and species discrimination in the FAR complex.
Recent studies have shown that efforts to resolve complex species status require multidisciplinary approaches24, 25, well-characterized material and extended sampling26–28. An example is the study of the members of the Bactrocera dorsalis complex, B. dorsalis s.s., B. papayae, B. philippinensis, B. invadens and B. carambolae, for which morphological/morphometric, behavioral/sexual compatibility, chemoecological, molecular/genetic and cytogenetic data lead to the synonymization of the four members, maintaining B. carambolae as a discrete entity29. Pluralistic approaches have been also followed for species delimitation within species complexes of other Tephritidae genera, such as the Anastrepha fraterculus complex18, 30–35. In this context, the issue of the number and the limits of species within the FAR complex has been recently addressed by an integrative approach on the basis of: (i) adult and larvae morphology36, 37, (ii) wing morphometrics38, (iii) microsatellite analysis8, (iv) cuticular hydrocarbons39, (v) pheromones40, (vi) developmental physiology41 and (vii) geographical and altitudinal distribution42. The above analysis supported the species identity of C. anonae36. For C. rosa, it was proposed that it consists of two separate entities (R1 and R2) that should be considered as different species: C. rosa representing group R1 and the newly described species C. quilicii representing group R24, 36. Although two entities (F1 and F2) were identified for C. fasciventris as well, the available data are insufficient to support their separation in two different species36.
The above analysis, although incorporating a volume of data from several disciplines, lacks chromosome/cytogenetic and mitochondrial evidence. Cytogenetic analyses can be proven valuable in efforts to resolve phylogenetic relationships and species boundaries among closely related species. Many years of cytogenetic studies in Drosophila and mosquitoes have shown that chromosomal rearrangements (CRs), especially inversions, play a causative role in speciation and they can be used as interspecific phylogenetic markers43–48. Modern genomic support the above observations and propose that CRs enhance speciation through the restriction of recombination within and near inverted regions resulting in restriction of gene flow49–58.
In Tephritidae flies, mitotic karyotypes have been used to distinguish between different members of species complexes based on differences of the sex chromosomes33, 35, 59–64. Polytene chromosome maps are available for 11 tephritid species belonging to five genera65–79. Comparative analyses among most of them revealed specific CRs that are diagnostic at the genus, subgenus and species level70, 75–78, 80, supporting the possible involvement of CRs in speciation.
The mitochondrial DNA (mt DNA) is considered a very useful molecular marker for phylogenetic analyses as it can be informative at a variety of taxonomic levels81, 82. Partial mitochondrial gene sequences have been extensively used for inferring phylogeny among species of the Tephritidae family83–94, and within the Ceratitis genus, in particular3, 22, 23. However, the use of complete mitochondrial genome sequences, facilitated by the continuously increasing number of them in the databanks, has become a preferable approach for phylogenetic and molecular systematic studies in several insect groups95–103 including Tephritidae104–110. When the issue is the discrimination of closely related species for quarantine and management applications the availability of complete mitogenome data is particularly valuable, as it can allow identification of the most informative diagnostic markers/sequences through intra and inter-specific comparative analysis106.
In the current study, we present the mitotic karyotype, a detailed salivary gland polytene chromosome analysis, as well as the complete mitochondrial genome sequence of C. fasciventris. This is the first description of the mitotic and polytene chromosomes, as well as the first complete mitogenome sequence from a member of the Ceratitis FAR complex and only the second available for the whole genus, after the model species, C. capitata. Both cytogenetic and mitochondrial information provided can be used as reference for comparative studies towards species delimitation and resolution of phylogenetic relationships within this species complex, but also among other species of the Ceratitis genus.
Results and Discussion
Mitotic chromosomes
The mitotic karyotype of Ceratitis fasciventris consists of six pairs of chromosomes: five pairs of meta- or sub-metacentric autosomes and one pair of sex chromosomes (Fig. 1). According to the labeling system used for the Mediterranean fruit fly (medfly), C. capitata (Wiedemann)67, the sex chromosomes are designated as the first pair of the mitotic karyotype, while the five autosomes are labeled from 2 to 6, in order of descending size. The first autosome pair (2) of C. fasciventris is easily distinguished due to its larger size. The remaining four autosomes are almost equal in size. One of them is clearly submetacentric and has been designated as autosome pair 3 in accordance to C. capitata, while the other three cannot be reliably distinguished (Fig. 1b). The sex chromosomes are identified as the heteromorphic pair (XY) of heavily stained chromosomes in the mitotic complement (Fig. 1b). This karyotype is consistent with the karyotype of most Tephritidae species analyzed so far59–61, 65, 67, 68, 70, 71, 73–77, 79, 111.
The above described mitotic karyotype of C. fasciventris is quite similar to the karyotype of C. capitata66, 67. The most apparent difference between the two species is the size of the sex chromosomes. In C. capitata, the X chromosome is about the same size with the largest autosome, while the Y is the shorter chromosome of the mitotic complement. On the contrary, in C. fasciventris the X chromosome is shorter than any autosome, while the Y chromosome is significantly smaller with its length reaching about ¼ of the length of the X. Size and shape variation of the sex chromosomes is common among tephritids and, based on their highly heterochromatic nature, it could be attributed to the accumulation or loss of heterochromatin30, 31, 33, 35, 59–61, 63–65, 67, 68, 70–79, 111–116.
Polytene chromosomes
The analysis of the salivary gland polytene chromosomes of C. fasciventris showed that the polytene complement consists of five long, well banded chromosomes (10 polytene arms) corresponding to the five autosomes. The sex chromosomes are under-replicated in polytene tissues, do not form discrete polytene elements and are apparent as a heterochromatic network (Fig. 2). No typical chromocenter exists; the two arms of each individual chromosome are loosely connected or can be found separated from each other. The polytene chromosomes of C. fasciventris were numbered from 2 to 6 and divided into sections from 1 to 100 based on their banding pattern similarities to C. capitata. For each polytene chromosome, the longer arm is designated as left (L) and the shorter one as right (R) (Fig. 3).
Detailed comparison of the polytene chromosomes of C. fasciventris to the reference map of C. capitata67, 117 revealed significant similarities. Characteristic structures such as the distal ends (telomeres) and the centromeric region of each chromosome seem identical between the two species. Furthermore, extensive similarity in the banding pattern of eight out of the ten polytene arms, namely 2L, 2R, 3R, 4L, 4R, 5R, 6L and 6R, can be identified (Supplementary Figs S1–S8). However, some differences in the puffing pattern were observed, which could reflect species-specific differences, different developmental stages118 and/or differences at rearing conditions. Regarding chromosome arms 3L and 5L, despite the banding pattern similarity in both ends of each arm, differences can be observed in the inner parts. After thorough analysis of the banding pattern, specific chromosome segments that seem to be rearranged in comparison to C. capitata have been identified (Figs 4 and 5). These changes could have been derived from consecutive overlapping inversions resulting in both inverted and transposed chromosome fragments119. Comparative analyses of polytene chromosomes among several tephritids showed that CRs are restricted to specific chromosome arms, mainly to the 3L and 5L arms70, 76–78, 80.
Chromosome localization of molecular markers
Four gene markers, namely hsp70, w, sxl and st (Table 1), were localized on the polytene chromosomes of C. fasciventris by in situ hybridization. Each heterologous probe gave a unique hybridization signal (Table 1 and Fig. 6): (i) the hsp 70 specific probe on region 27 of the 3L arm (Fig. 6a), (ii) the w probe on region 67 of the 5L polytene arm (Fig. 6b), (iii) the sxl probe on region 79 of the 5R arm (Fig. 6c) and (iv) the st probe on region 83 of the 6L polytene arm (Fig. 6d).
Gene symbol | Description | Species of origin | DNA type | Localization site C. fasciventris/C. capitata | References | |
---|---|---|---|---|---|---|
hsp70 | The gene for the heat-shock 70 protein | Ceratitis capitata | genomic | 27-3L | 24-3L | 121 |
w | The orthologue of the D. melanogaster white gene | Bactrocera tryoni | genomic | 67-5L | 65-5L | 120, 147 |
sxl | The orthologue of the D. melanogaster sex lethal gene | Bactrocera oleae | cDNA | 79-5R | 79-5R | 117, 148 |
st | The orthologue of the D. melanogaster scarlet gene | Bactrocera tryoni | genomic | 83-6L | 83-6L | 117, 149 |
The four gene markers studied here have been previously mapped in C. capitata at putative corresponding polytene chromosome regions70, 117, 120, 121. The localization of the sxl and st genes supports the conservation of the 5R and 6L polytene chromosome arms between the two species, as it was deduced by banding pattern similarities (Supplementary Figs S6 and S7). On the other hand, the hybridization of the hsp70 gene at region 27 of C. fasciventrisis is in discordance with the localization of the above gene at region 24 of C. capitata (Table 1 and Fig. 4), supporting the proposed rearrangements of the 3L polytene arm between the two species (Fig. 4). Similarly, the different chromosome region of the w locus (67 on 5L for C. fasciventris, 65 on 5L for C. capitata; Table 1 and Fig. 5) is further support for the rearrangements of the 5L chromosome arm between the two species suggested by banding pattern analysis (Fig. 5).
The banding pattern comparison and gene localization data provide evidence for the conservation of chromosome synteny between the closely related species C. capitata and C. fasciventris. However, a more thorough chromosome mapping analysis would be needed in order to confirm the above observation. The idea that the chromosome elements have maintained their basic content and identity was initially proposed for Drosophila species122, 123. Later comparative studies using biochemical and morphological markers showed a remarkable conservation of linked gene clusters among a wider phylogenetic range of higher Diptera124–126. In situ hybridization of well characterized genes on polytene chromosomes further supported the extensive conservation of linkage groups in Tephitidae70, 117, 127–131. On the other hand, the differences identified in the structure of the 3L and 5L polytene arms of C. fasciventris and C. capitata, are congruent with the concept that chromosome rearrangements, mainly paracentric inversions, are implicated in species differentiation in Diptera which is substantiated by comparative cytogenetic and genomic analyses in Drosophila and mosquitoes46–54, 56–58. In accordance with the above notion, comparisons among tephritids, reveal that inversions and/or transpositions on specific chromosomes, including chromosomes 3 and 5, differentiate species and support the potential value of chromosome rearrangements as phylogenetic and diagnostic markers among closely related species70, 75–78, 80, 111.
Mitochondrial genome
The mitochondrial DNA of C. fasciventris was amplified and sequenced in 27 overlapping fragments. The above analysis resulted in the nucleotide sequence of the complete C. fasciventris mitogenome consisting of 16,017 bp with very high A + T (77.17%) and very low G + C (22.83%) contents. The organization of the C. fasciventris mt genome is typical to that of other tephritids studied104, 106–110, 132–136. It comprises 13 protein-coding, two rRNA (12S and 16S rRNA) and 22 tRNA genes, and one major non-coding sequence, the control region (Table 2), all of which are similar in size to their counterparts in other insects.
Gene/Element | Abbreviation | Strand | Position | Size (bp) | Inter-genic spacer | Start codon | Stop codon | Codon recognized | Amino acids |
---|---|---|---|---|---|---|---|---|---|
tRNAIle | I | H | 1–67 | 67 | 62 | ATC | |||
tRNAGln | Q | L | 130–198 | 69 | 16 | CAA | |||
tRNAMet | M | H | 215–283 | 69 | 1 | ATG | |||
NADH dehydrogenase subunit 2 | ND2 | H | 285–1307 | 1023 | 4 | ATT | TAA | 340 | |
tRNATrp | W | H | 1312–1379 | 68 | −8 | TGA | |||
tRNACys | C | L | 1372–1436 | 65 | 20 | TGC | |||
tRNATyr | Y | L | 1457–1523 | 67 | −2 | TAC | |||
Cytochrome c oxidase subunit 1 | COI | H | 1522–3057 | 1536 | 6 | TCG | TAA | 511 | |
tRNALeu (UUR) | L | H | 3064–3129 | 66 | 5 | TTA | |||
Cytochrome c oxidase subunit 2 | COII | H | 3135–3821 | 687 | 8 | ATG | TAA | 228 | |
tRNALys | K | H | 3830–3900 | 71 | 4 | AAG | |||
tRNAAsp | D | H | 3905–3971 | 67 | 0 | GAC | |||
ATP synthase F0 subunit 8 | ATP8 | H | 3972–4133 | 162 | −7 | ATT | TAA | 53 | |
ATP synthase F0 subunit 6 | ATP6 | H | 4127–4804 | 678 | −1 | ATG | TAA | 225 | |
Cytochrome c oxidase subunit 3 | COIII | H | 4804–5592 | 789 | 6 | ATG | TAA | 262 | |
tRNAGly | G | H | 5599–5663 | 65 | 0 | GGA | |||
NADH dehydrogenase subunit 3 | ND3 | H | 5664–6017 | 354 | 2 | ATA | TAA | 117 | |
tRNAAla | A | H | 6020–6083 | 64 | 19 | GCA | |||
tRNAArg | R | H | 6103–6166 | 64 | 3 | CGA | |||
tRNAAsn | N | H | 6170–6234 | 65 | 0 | AAC | |||
tRNASer(AGY) | S | H | 6235–6302 | 68 | 0 | AGC | |||
tRNAGlu | E | H | 6303–6370 | 68 | 18 | GAA | |||
tRNAPhe | F | L | 6389–6455 | 67 | 0 | TTC | |||
NADH dehydrogenase subunit 5 | ND5 | L | 6456–8175 | 1720 | 18 | ATT | T* | 573 | |
tRNAHis | H | L | 8194–8258 | 65 | 12 | CAC | |||
NADH dehydrogenase subunit 4 | ND4 | L | 8271–9611 | 1341 | −1 | ATG | TAA | 446 | |
NADH dehydrogenase subunit 4L | ND4L | L | 9611–9901 | 291 | 2 | ATG | TAA | 96 | |
tRNAThr | T | H | 9904–9968 | 65 | 0 | ACA | |||
tRNAPro | P | L | 9969–10034 | 66 | 2 | CCA | |||
NADH dehydrogenase subunit 6 | ND6 | H | 10037–10561 | 525 | −1 | ATT | TAA | 174 | |
Cytochrome b | CYTB | H | 10561–11697 | 1137 | −2 | ATG | TAG | 378 | |
tRNASer(UCN) | S | H | 11696–11762 | 67 | 15 | TCA | |||
NADH dehydrogenase subunit 1 | ND1 | L | 11778–12717 | 940 | 9 | ATT | T* | 313 | |
tRNALeu(CUA) | L | L | 12727–12792 | 66 | 0 | CTA | |||
16S rRNA | 16S | L | 12793–14133 | 1341 | 0 | ||||
tRNAVal | V | L | 14134–14205 | 72 | 0 | GTA | |||
12S rRNA | 12S | L | 14206–14993 | 788 | 0 | ||||
Control region | CR | 14994–16017 | 1024 | 0 |
Protein coding genes
The location of the 13 protein coding genes (PCGs) was determined by the identification of initiation and termination signals as well as by sequence comparisons to the respective genes of C. capitata. Nine of them are encoded by the H strand, while ND1, ND4, ND4L and ND5 are encoded by the L strand (Table 2). Only six protein coding genes (COII, ATP6, COIII, ND4, ND4L, CYTB) begin with the canonical initiation codon ATG, five of them (ND2, ATP8, ND5, ND6 and ND1) initiate with ATT, while for ND3 the first codon is ATA and for COI it is TCG (Table 2), similarly to other Tephritidae species104, 106–108, 110, 132, 133. In C. fasciventris, two of the protein coding genes (ND5 and ND1) possess an incomplete termination codon (T) (Table 2). This is common in animal mitochondrial DNA; the stop codon is likely completed by post-transcriptional polyadenylation137.
The overlapping of seven nucleotides between ATP8 and ATP6 genes is the longest observed between protein coding genes of C. fasciventris and is also common (presenting variable size) among tephritids104, 106, 132, 133. Overlapping restricted to one or two nucleotides can also be observed between ATP6 and COIII, ND4 and ND4L, ND6 and CYTB and CYTB and tRNASer (Table 2). However, in these cases one could assume an incomplete stop codon for the preceding gene, which could mean lack of overlap with the following one.
RNA genes
The C. fasciventris 16S rRNA and 12S rRNA genes consist of 1,341 (positions: 12,793–14,133) and 788 (positions: 14,206–14,993) nucleotides, respectively (Table 2). As in other insects, these genes are located in the L strand at the end of the mtDNA molecule between the gene for tRNALeu (CUA) and the control region, and are separated by the tRNAVal gene. Dispersed among the protein-coding and the rRNA genes there are 22 tRNA genes; 14 on the H and 8 on the L strand. Their sizes range from 64–72 nucleotides (Table 2) and are predicted to fold into the expected cloverleaf secondary structures.
Non-coding regions
Following the rule for animal mitochondrial DNA, the mt genome of C. fasciventris contains only one long non-coding region with very high A + T content (90.24%) that regulates replication and transcription, the control region. It is located between the 12SrRNA and the tRNAIle genes and its length is 1024 bp (positions 14,994–16,017; Table 2), within the range of the respective region in other Tephritidae species. At the 5′ end of the control region (positions 15,947–15,973) a poly(T) followed by a [TA(A)n]-like stretch can be observed. This feature is conserved among insect species and has been proposed to play a role in the control of transcription and/or replication107, 110, 133, 138. Two motifs, TTAAATTAATAATTAT and TATTTTTATTTTTAAATT, were found to be tandemly repeated three (positions 15,263–15,315) and two times (positions 15,871–15,909), respectively. Tandem repeats have been identified in the control regions of other Tephritidae species, as well107, 133.
The C. fasciventris mitogenome contains 20 intergenic spacers (IGS) of 232 bp total length (Table 2). The longest one (62 bp) is located between tRNAIle and tRNAGln, as is the case in B. zonata107. In C. capitata, an IGS is present at the respective position but it is not the longer one. In the latter species, the longer IGS (46 bp) is found between tRNAGln and tRNAMet132 similarly to several Bactrocera species106, 108, 109. Quite long IGS in species of the Tephritidae family are also present between tRNACys and tRNATyr, which is able to form a secondary structure and between tRNAArg and tRNAAsn104, 106, 108–110, 132–134. The second longest IGS in C. fasciventris lies between tRNACys and tRNATyr; however it is only 20 bp long and cannot form a stem loop. On the other hand, IGS separating tRNAArg and tRNAAsn is only 3 bp long. It has been observed thatspecific IGS (the ones between tRNAGlu and tRNAPhe, ND5 and tRNAHis, tRNASer and ND1 and ND1 and tRNALeu) are conserved in size and sequence among several tephritids and have their counterparts in the control region107, 133. This is also valid for C. fasciventris.
Sequence comparisons with C. capitata and other Tephritidae species
The C. fasciventris mitogenome is the second complete mt DNA sequence of the Ceratitis genus analyzed, with the first one being that of C. capitata132. The mitochondrial genomes of these two species are highly similar both in terms of organization and structure as well as of sequence similarity. The overall sequence identity is 92.11%, while identity of the PCGs is 92.25% (Table 3). In comparison to other tephritid mitogenomes, the C. fasciventris mtDNA presents the lowest sequence identity with B. minax (79.18% for complete mitogenome; 79.31% for PCGs). The latter species presents the lowest identity percentages in comparison with any other tephritid, even the ones of the same genus, both in the complete mitosequence, as well as in the PCGs sequence (Table 3).
fas | cap | are | cor | car | dor | try | zon | mel | lat | umb | ole | min | cau | cuc | dia | scu | tau | lon | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
fas | — | 92.11 | 84.05 | 84.40 | 84.92 | 84.94 | 84.21 | 84.57 | 84.83 | 82.91 | 82.53 | 84.19 | 79.18 | 84.87 | 84.30 | 85.53 | 85.04 | 85.63 | 82.69 |
cap | 92.25 | — | 84.03 | 84.73 | 84.89 | 84.89 | 84.27 | 84.67 | 85.04 | 83.07 | 82.54 | 84.36 | 78.90 | 84.59 | 84.13 | 85.59 | 84.67 | 85.56 | 82.97 |
are | 83.77 | 83.68 | — | 90.12 | 91.26 | 91.15 | 90.76 | 90.86 | 90.52 | 88.08 | 87.92 | 86.47 | 81.29 | 85.55 | 84.57 | 86.08 | 86.09 | 85.96 | 83.47 |
cor | 84.65 | 84.97 | 90.09 | — | 91.62 | 91.64 | 90.53 | 94.09 | 90.58 | 87.53 | 87.24 | 86.65 | 81.06 | 85.75 | 84.98 | 86.37 | 85.81 | 86.07 | 83.57 |
car | 84.70 | 84.64 | 90.13 | 91.59 | — | 98.83 | 92.22 | 92.30 | 91.61 | 88.34 | 88.41 | 87.01 | 81.93 | 85.97 | 85.67 | 87.11 | 86.70 | 86.77 | 84.04 |
dor | 84.72 | 84.62 | 89.98 | 91.57 | 98.60 | — | 92.12 | 92.29 | 91.68 | 88.35 | 88.33 | 87.05 | 82.06 | 85.99 | 85.70 | 87.14 | 86.59 | 86.81 | 84.00 |
try | 83.87 | 83.88 | 89.73 | 90.38 | 91.12 | 90.99 | — | 91.32 | 90.98 | 88.21 | 88.03 | 86.50 | 81.18 | 85.60 | 85.10 | 86.36 | 86.17 | 86.20 | 83.74 |
zon | 84.62 | 84.68 | 90.14 | 93.71 | 91.46 | 91.42 | 90.52 | — | 91.34 | 87.98 | 87.69 | 86.88 | 81.10 | 85.65 | 85.16 | 86.71 | 86.10 | 86.19 | 83.70 |
mel | 84.57 | 84.77 | 89.42 | 90.50 | 90.76 | 90.86 | 89.84 | 90.69 | — | 87.80 | 87.71 | 86.64 | 81.14 | 85.79 | 84.99 | 86.74 | 86.22 | 86.14 | 83.63 |
lat | 82.19 | 82.36 | 86.77 | 87.26 | 86.93 | 86.97 | 87.04 | 87.06 | 86.66 | — | 86.07 | 85.38 | 80.40 | 84.46 | 83.60 | 85.03 | 84.71 | 84.86 | 82.68 |
umb | 81.91 | 81.81 | 86.59 | 86.73 | 87.15 | 87.08 | 86.75 | 86.65 | 86.36 | 84.58 | — | 84.75 | 80.35 | 83.48 | 82.89 | 83.88 | 83.86 | 83.75 | 82.24 |
ole | 83.89 | 83.97 | 85.63 | 86.31 | 85.90 | 85.95 | 85.39 | 86.26 | 85.54 | 84.33 | 83.41 | — | 81.19 | 85.93 | 84.74 | 86.38 | 86.12 | 86.08 | 83.49 |
min | 79.31 | 79.04 | 80.97 | 81.27 | 81.84 | 81.90 | 80.82 | 81.13 | 80.72 | 79.86 | 79.86 | 80.70 | — | 81.17 | 80.69 | 81.26 | 81.29 | 81.24 | 80.03 |
cau | 84.57 | 84.15 | 84.66 | 85.42 | 84.91 | 84.91 | 84.59 | 85.06 | 84.99 | 83.50 | 82.25 | 84.97 | 81.05 | — | 87.60 | 92.52 | 92.52 | 89.23 | 85.07 |
cuc | 84.28 | 83.89 | 83.79 | 84.72 | 84.89 | 84.91 | 84.30 | 84.77 | 84.16 | 82.75 | 81.87 | 83.96 | 81.86 | 86.87 | — | 88.63 | 88.43 | 94.58 | 84.06 |
dia | 85.28 | 85.34 | 85.21 | 86.16 | 86.10 | 86.15 | 85.44 | 86.32 | 85.92 | 84.05 | 82.49 | 85.43 | 81.12 | 91.48 | 87.77 | — | 94.40 | 90.66 | 85.51 |
scu | 84.74 | 84.17 | 85.06 | 85.44 | 85.60 | 85.41 | 85.13 | 85.48 | 85.12 | 83.64 | 82.44 | 85.17 | 81.32 | 91.35 | 87.49 | 93.36 | — | 90.07 | 85.25 |
tau | 85.13 | 84.84 | 84.92 | 85.52 | 85.67 | 85.67 | 85.06 | 85.50 | 85.02 | 83.67 | 82.34 | 84.88 | 81.72 | 88.01 | 94.32 | 89.37 | 88.54 | — | 85.38 |
lon | 81.95 | 82.14 | 82.14 | 82.86 | 82.66 | 82.63 | 82.44 | 82.81 | 82.39 | 81.32 | 80.78 | 82.23 | 79.65 | 83.63 | 83.14 | 83.98 | 83.71 | 83.97 | — |
In the upper part the identities of complete mitochondrial DNA sequences are given. In the lower part the identities of the concatenated protein coding gene sequences are given. Species abbreviations and GenBank accession numbers of the mtDNA sequences used are given in Supplementary Τable S1.
The phylogenetic analysis based on the concatenated sequences of the 13 PCGs of 19 Tephritidae species places the currently analyzed C. fasciventris sequence in the same clade with the sequence of C. capitata (Fig. 7) confirming the closer relationship of the two species22, 139. Furthermore, species of the Zeugodacus subgenus are grouped separately from all other Bactrocera subgenera in a sister clade to Dacus (Fig. 7), supporting the recent suggestion of raising Zeugodacus to the genus level, as well its closer phylogenetic relationship to Dacus in comparison to Bactrocera88, 92, 94, 110, 140.
Conclusions
In the crucial issue of species boundaries within complexes of destructive insect pests, a multidisciplinary approach is considered the best way for reaching solid results and reliable species discrimination24, 25, 29. To this direction, cytogenetic and complete mitogenome analysis can provide important information and potentially reveal species diagnostic characters. The mitotic karyotype and polytene chromosome maps of C. fasciventris are the first presented for the FAR complex and the second for the genus Ceratitis. The comparison and the differences identified with the mitotic and polytene chromosomes of C. capitata proves the cytogenetic information on C. fasciventris is valuable for comparative analysis possibly providing important insight in the genetic relationships of the species of the FAR complex, as well as among other species of the Ceratitis genus. Likewise, the first complete mitochondrial sequence of a member of the FAR complex can be used as reference for future sequence comparisons among FAR species, aiming to reveal polymorphic mitochondrial regions suitable for the development of molecular diagnostic markers. The fact that the insects used originate from a well-characterized established colony eliminates possible problems resulting from sampling and species misidentification80 and supports the use of both cytogenetic and mitochondrial present results as reference material.
Methods
Specimens and rearing conditions
Insects used in the present study came from a C. fasciventris colony maintained at the FAO-IAEA Insect Pest Control Laboratory (IPCL), Seibersdorf Austria. The colony was established in the summer of 2013 from pupae found in field infested Citrus sp. fruits from Kenya. Male adults were identified as belonging to the F2 group of C. fasciventris, based on the coloration and setal ornamentation of the mid tibia36. Identified adult voucher specimens of the colony are preserved at the entomology collections of the Royal Museum for Central Africa (Tervuren, Belgium).
Rearing was accomplished by keeping adults in groups of 300–500 individuals, in 2-side fine-mesh covered, rectangular cages (50 × 30 × 20 cm). Adults had ad libitum access to water and adult diet consisting of yeast hydrolysate and sugar at 1:3 ratio. For oviposition, females were provided with bananas, which were pin punctured with 50–70 holes to serve egg deposit. The oviposited bananas were placed over a thin sawdust layer, in plastic trays. The fully developed 3rd instar larvae that exited the bananas and pupated in the sawdust were collected by sieving. The colony was reared under controlled temperature, humidity and light conditions (22 °C, 65 ± 2% RH, 14 h L: 10 h D).
Mitotic chromosome preparations
Spread chromosome preparations were made from nerve ganglia of third instar larvae67, 111. Brain tissue was dissected in 0.7% NaCl solution. The material was transferred to 1% sodium citrate on a well slide for at least 15 min and in fresh fixation solution (methanol/acetic acid 3:1) for 3 min. Samples were transferred to a small drop of 60% acetic acid and dispersed using a micropipette. The cell suspension was dried by laying it on a clean slide placed on a hotplate (40–45 °C). Chromosomes were stained with Giemsa solution (5% Giemsa in 10 mM phosphate buffer, pH 6.8). Chromosome slides were analyzed at 100× magnification, using a phase contrast microscope (Leica DMR), and photographs were taken using a CD camera (ProgResCFcool; Jenoptik Jena Optical Systems, Jena, Germany). More than 20 chromosome preparations representing 20 individual larvae and at least 10 well spread nuclei per preparation were analyzed.
Polytene chromosome preparations
Polytene chromosome preparations were made from well fed third-instar larvae or 1–2 days old pupae. Larvae were dissected in 45% acetic acid and salivary glands were carefully transferred to 3 N HCL on a depression slide for 1 min. Glands were fixed in glacial acetic acid:water:lactic acid (3:2:1) for about 5 min before stained in lacto-acetic-orcein for 5–7 min67, 111. Early pupae were dissected in Ringer’s solution and the salivary glands were transferred to 45% acetic acid for 2–3 min and post-fixed in 1 N HCL for 2 min. The material was passed through lacto- acetic acid (80% lactic acid:60% acetic acid, 1:1) and stained in lacto-acetic-orcein for 10–20 min. Excess stain was removed by washing the glands in lacto-acetic acid69, 111. For best chromosome preparations, each salivary gland was cut in two pieces, and each piece was squashed for one preparation. More than 200 chromosome slides (representing at least 150 single larvae or pupae) were prepared and observed at 63× and 100× objectives in a phase contrast microscope (Leica DMR). At least 200 well spread nuclei and/or isolated chromosomes were selected for analysis and photographed using a digital camera (see above). Selected photographs for each chromosome arm showing the best morphology were used for the construction of the C. fasciventris polytene chromosome maps, as well as for the comparison with the available maps of C. capitata117.
In situ hybridization
Polytene chromosome preparations for in situ hybridization were made from salivary glands of late third instar larvae or young pupae (1–2 day old)128. Four heterologous gene sequences, namely hsp70, w, sxl and st were used as probes (Table 1). Labeling was performed using the DIG-High Prime and detection using the Anti-Digoxigenin-AP, Fab fragments and the NBT/BCIP Stock Solution, all purchased by ROCHE, Mannheim, Germany. Hybridization was performed at 60 °C for the hsp70, sxl and st probes and at 52 °C for the w probe. Hybridization and detection procedures were performed as previously described111, 128. Four to five preparations were hybridized with each probe, and at least ten well spread nuclei per preparation were observed at 63x or 100x magnification with a Nikon Eclipse 80i or a Leica DMR phase contrast microscope, respectively. Photographs were captured using a Nikon DS-5 M-U1 (63x) or a Jenoptik ProgRes (100x) CCD camera.
DNA isolation and mitochondrial genome amplification and sequencing
Total genomic DNA was extracted from single flies, using the CTAB protocol141.
The complete mtDNA sequence was obtained from a single specimen by standard PCR amplifications using: (i) heterologous primers that were designed based on the mitochondrial sequences of Ceratitis capitata and Bactrocera dorsalis (Supplementary Table S1) and (ii) homologous primers that were designed based on the sequences determined by the analysis in this study. In total 27 pairs of primers amplifying overlapping fragments were designed by the Oligoexplorer and Oligoanalyzer programs (Supplementary Table S2). The reaction mixture (25 μl) contained 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 μM of the appropriate primers and 1 U Taq polymerase (BIOTAQ, BIOLINE). Approximately 30 ng of template DNA was used in each reaction. PCR reactions comprised an initial denaturation at 94 °C for 3 min, followed by 40 cycles of 45 min denaturation at 94 °C, 30 sec primer annealing at 49–56 °C and 1 min DNA chain extension at 72 °C, for standard PCR. This was followed by a final extension at 72 °C for 7 min. The resulting PCR products were analyzed in a 1.0% agarose gel stained with ethidium bromide. PCR products were purified by the Nucleospin Gel and PCR Clean up kit (Macherey Nagel, Germany).
Sequencing reactions were performed by Macrogen Europe (Amsterdam, The Nederlands). The full-length mtDNA sequence was assembled using EMBOSS Merger142.
Sequence analysis
Nucleotide sequence analysis was performed using the programs BLASTn (www.ncbi.nlm.nih.gov) and ClustalOmega (www.ebi.ac.uk). The regions of the protein-coding, rRNA, and tRNA genes were initially identified by comparison with the corresponding known sequences of C. capitatα (Supplementary Table S1). Furthermore, the 13 mitochondrial protein-coding genes were defined by the presence of initiation and stop codons whereas the 22 tRNAs were checked for their capability to fold into cloverleaf secondary structures and the presence of specific anticodons by tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/)143 and MITOS (http://mitos.bioinf.uni-leipzig.de/index.py)144. Repeats in the control region were found by the “Tandem Repeat Finder” program (http://tandem.bu.edu/trf/trf.html)145.
Phylogenetic analysis based on the concatenated protein coding gene sequences from available Tephritidae mitogenomes (one for each species) (Supplementary Table S1) was performed using MEGA 7.0146. Multiple sequence alignment (11,242 positions total length) was constructed by ClustalW using default parameters. Phylogenetic trees were inferred by the Maximum Likelihood (ML) method based on the General Time Reversible (GTR) model with 1000 bootstrap replicates.
Data Availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files).
Electronic supplementary material
Footnotes
Electronic supplementary material
Supplementary information accompanies this paper at doi:10.1038/s41598-017-05132-3
Accession Codes:Ceratitis fasciventris mitochondrial genome is available in GenBank database under the accession number KY436396.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Drs Sunday Ekesi and Fathiya Khamis for providing the biological material from Kenya for the establishment of the colony. We also thank Dr. Marc de Meyer for the identification of the specimen and his valuable suggestions on the manuscript. The present study has been funded by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture through CRP and SSA projects.
Author Contributions
E.D., A.A., K.B., P.M.-T., and A.Z. designed the study; C.P., T.C. and G.K. performed the experiments, E.D., A.A., A.G.-P., P.M.-T. and A.Z. analyzed the data; A.G.-P. prepared the figures; E.D., A.A., K.B., P.M.-T., and A.Z. wrote the manuscript. All authors reviewed the manuscript.
Competing Interests
The authors declare that they have no competing interests.
References
- 1. Pape, T., Bickel, D. J. & Meier, R. Diptera diversity: status, challenges and tools (Brill, 2009).
- 2. White, I. M., Elson-Harris, M. M. & others. Fruit flies of economic significance: their identification and bionomics (CAB International, 1992).
- 3. De. Molecular evaluation of nominal species in the Ceratitis fasciventris, C. anonae, C. rosa complex (Diptera: Tephritidae)Mol. Phylogenet. Evol.200848270280[PubMed][Google Scholar]
- 4. Description of new Ceratitis species (Diptera: Tephritidae) from Africa, or how morphological and DNA data are complementary in discovering unknown species and matching sexesEur. J. Taxon.2016233123[Google Scholar]
- 5. De Meyer, M. et al. Annotated check list of host plants for Afrotropical fruit flies (Diptera: Tephritidae) of the genus Ceratitis (Vol. 27). (R. Museum Cent. Africa, Belgium, 2002).
- 6. Geographic distribution, host fruit, and parasitoids of African fruit fly pests Ceratitis anonae, Ceratitis cosyra, Ceratitis fasciventris, and Ceratitis rosa (Diptera: Tephritidae) in KenyaAnn. Entomol. Soc. Am.200699261278[PubMed][Google Scholar]
- 7. Distribution patterns and host-plant relationships within the genus Ceratitis MacLeay (Diptera: Tephritidae) in AfricaCimbebasia200117219228[Google Scholar]
- 8. Cryptic diversity and gene flow among three African agricultural pests: Ceratitis rosa, Ceratitis fasciventris and Ceratitis anonae (Diptera, Tephritidae)Mol. Ecol.20132225262539[PubMed][Google Scholar]
- 9. A review of relationships between interspecific competition and invasions in fruit flies (Diptera: Tephritidae)Ecol. Entomol.200429511520[PubMed][Google Scholar]
- 10. Globalization and fruitfly invasion and expansion: the medfly paradigmGenetica200713119[PubMed][Google Scholar]
- 11. White, I. M. et al. A review of native and introduced fruit flies (Diptera, Tephritidae) in the Indian Ocean islands of Mauritius, Réunion, Rodrigues and Seychelles. in Proceedings of the Indian Ocean Commission, Regional Fruit Fly Symposium, Flic en Flac, Mauritius, 5th-9th June, 2000. 15–21 (2000).
- 12. Survival and development of different life stages of three Ceratitis spp.(Diptera: Tephritidae) reared at five constant temperaturesBull. Entomol. Res.200292461469[PubMed][Google Scholar]
- 13. Indigenous and invasive fruit fly diversity along an altitudinal transect in Eastern Central TanzaniaJ. Insect Sci.20121212[PubMed][Google Scholar]
- 14. Combining field phenological observations with distribution data to model the potential distribution of the fruit fly Ceratitis rosa Karsch (Diptera: Tephritidae)Bull. Entomol. Res.20131036073[PubMed][Google Scholar]
- 15. Host range and distribution of fruit-infesting pestiferous fruit flies (Diptera, Tephritidae) in selected areas of Central TanzaniaBull. Entomol. Res.200999629641[PubMed][Google Scholar]
- 16. Dyck, V. A., Hendrichs, J. & Robinson, A. S. Sterile insect technique. (Springer, 2005).
- 17. Krafsur, E. S. Role of population genetics in the sterile insect technique in Sterile Insect Technique 389–406 (Springer, 2005).
- 18. Resolving cryptic species complexes of major tephritid pestsZookeys2015540539[PubMed][Google Scholar]
- 19. On the identity of the Natal fruit fly Ceratitis rosa Karsch (Diptera, Tephritidae)Bull. l’Institut R. des Sci. Nat. Belgique, Entomol.2001715562[Google Scholar]
- 20. Revision of the subgenus Ceratitis (Pterandrus) Bezzi (Diptera: Tephritidae)Isr. J. Entomol.200535197315[Google Scholar]
- 21. Ribosomal ITS1 polymorphisms in Ceratitis capitata and Ceratitis rosa (Diptera: Tephritidae)Ann. Entomol. Soc. Am.200194726731[PubMed][Google Scholar]
- 22. Molecular phylogenetics of the genus Ceratitis (Diptera: Tephritidae)Mol. Phylogenet. Evol.200638216230[PubMed][Google Scholar]
- 23. Molecular diagnostics of economically important Ceratitis fruit fly species (Diptera: Tephritidae) in Africa using PCR and RFLP analysesBull. Entomol. Res.20069650521[PubMed][Google Scholar]
- 24. Integrative taxonomy: a multisource approach to exploring biodiversityAnnu. Rev. Entomol.201055421438[PubMed][Google Scholar]
- 25. Species concepts and species delimitationSyst. Biol.200756879886[PubMed][Google Scholar]
- 26. Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wing-shape dataBMC Evol. Biol.201212130[PubMed][Google Scholar]
- 27. Piecing together an integrative taxonomic puzzle: microsatellite, wing shape and aedeagus length analyses of Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay PeninsulaSyst. Entomol.201338213[PubMed][Google Scholar]
- 28. Multi-gene phylogenetic analysis of south-east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomyJ. Appl. Entomol.2014138235253[PubMed][Google Scholar]
- 29. Synonymization of key pest species within the Bactrocera dorsalis species complex (Diptera: Tephritidae): Taxonomic changes based on a review of 20 years of integrative morphological, molecular, cytogenetic, behavioural and chemoecological dataSyst. Entomol.201540456471[PubMed][Google Scholar]
- 30. A genetic–morphological characterization of two cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae)Ann. Entomol. Soc. Am.200598367381[PubMed][Google Scholar]
- 31. Karyotype characterization of Anastrepha fruit flies (Diptera: Tephritidae)Neotrop. Entomol.200534273279[PubMed][Google Scholar]
- 32. Mating incompatibility among populations of the South American fruit fly Anastrepha fraterculus (Diptera: Tephritidae)Ann. Entomol. Soc. Am.200699387397[PubMed][Google Scholar]
- 33. Incipient speciation revealed in Anastrepha fraterculus (Diptera; Tephritidae) by studies on mating compatibility, sex pheromones, hybridization, and cytologyBiol. J. Linn. Soc.200997152165[PubMed][Google Scholar]
- 34. An integrative multidisciplinary approach to understanding cryptic divergence in Brazilian species of the Anastrepha fraterculus complex (Diptera: Tephritidae)Biol. J. Linn. Soc.2016117725746[PubMed][Google Scholar]
- 35. Cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae): a multivariate approach for the recognition of South American morphotypesAnn. Entomol. Soc. Am.2012105305318[PubMed][Google Scholar]
- 36. An integrative approach to unravel the Ceratitis FAR (Diptera, Tephritidae) cryptic species complex: A reviewZookeys2015540405427[PubMed][Google Scholar]
- 37. Description of third instar larvae of Ceratitis fasciventris, C. anonae, C. rosa (FAR complex) and C. capitata (Diptera,Tephritidae)Zookeys2015540443465[PubMed][Google Scholar]
- 38. Wing morphometrics as a possible tool for the diagnosis of the Ceratitis fasciventris, C. anonae, C. rosa complex (Diptera, Tephritidae)Zookeys2015540489506[PubMed][Google Scholar]
- 39. Cuticular hydrocarbons corroborate the distinction between lowland and highland Natal fruit fly (Tephritidae, Ceratitis rosa) populationsZookeys2015540507524[PubMed][Google Scholar]
- 40. Analyses of volatiles produced by the African fruit fly species complex (Diptera, Tephritidae)Zookeys2015540385404[PubMed][Google Scholar]
- 41. Comparative analysis of development and survival of two Natal fruit fly Ceratitis rosa Karsch (Diptera, Tephritidae) populations from Kenya and South AfricaZookeys2015540467487[PubMed][Google Scholar]
- 42. Niche partitioning among two Ceratitis rosa morphotypes and other Ceratitis pest species (Diptera, Tephritidae) along an altitudinal transect in Central TanzaniaZookeys2015540429442[PubMed][Google Scholar]
- 43. Dobzhansky, T. & Dobzhansky, T. G. Genetics and the origin of species. (Columbia University Press, 1937).
- 44. Inversions in the third chromosome of wild races of Drosophila pseudoobscura, and their use in the study of the history of the speciesProc. Natl. Acad. Sci.193622448450[PubMed][Google Scholar]
- 45. Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complexTrans. R. Soc. Trop. Med. Hyg.197973483497[PubMed][Google Scholar]
- 46. Ehrman, L., Powell, J. R., Ashburner, M., Carson, H. L. & Thompson, J. N. The genetics and biology of Drosophila. (Academic Press, 1982).
- 47. Krimbas, C. B. & Powell, J. R. Drosophila inversion polymorphism. (CRC Press, 1992).
- 48. Effects of inversions on within-and between-species recombination and divergenceGenome Biol. Evol.20113830841[PubMed][Google Scholar]
- 49. Chromosomal inversions and the reproductive isolation of speciesProc. Natl. Acad. Sci.2001981208412088[PubMed][Google Scholar]
- 50. Chromosomal rearrangements and speciationTrends Ecol. Evol.200116351358[PubMed][Google Scholar]
- 51. Chromosome inversions, local adaptation and speciationGenetics2006173419434[PubMed][Google Scholar]
- 52. Principles of genome evolution in the Drosophila melanogaster species groupPlos Biol.20075e152[PubMed][Google Scholar]
- 53. Chromosomal rearrangement inferred from comparisons of 12 Drosophila genomesGenetics200817916571680[PubMed][Google Scholar]
- 54. Polytene chromosomal maps of 11 Drosophila species: The order of genomic scaffolds inferred from genetic and physical mapsGenetics200817916011655[PubMed][Google Scholar]
- 55. The genomics of speciation in Drosophila: diversity, divergence, and introgression estimated using low-coverage genome sequencingPlos Genet.20095e1000550[PubMed][Google Scholar]
- 56. Chromosomal speciation revisited: rearranging theory with pieces of evidenceTrends Ecol. Evol.201025660669[PubMed][Google Scholar]
- 57. Genomic impacts of chromosomal inversions in parapatric Drosophila speciesPhil. Trans. R. Soc. B2012367422429[PubMed][Google Scholar]
- 58. Chromosome inversions, genomic differentiation and speciation in the African malaria mosquito Anopheles gambiaePLoS One20138e57887[PubMed][Google Scholar]
- 59. Mitotic karyotype of four species of fruit flies (Bactrocera) in ThailandKasetsart J Nat Sci199428142148[Google Scholar]
- 60. Metaphase karyotypes of fruit flies of Thailand. I. Five sibling species of the Bactrocera dorsalis complexGenome199538101522[PubMed][Google Scholar]
- 61. Cytological evidence for a complex of species within the taxon Bactrocera tau (Diptera: Tephritidae) in ThailandBiol. J. Linn. Soc.200069399409[PubMed][Google Scholar]
- 62. Heterochromatin accumulation and karyotypic evolution in some dipteran insectsZool. Stud.1998377588[Google Scholar]
- 63. Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera, Tephritidae)Cytogenet. Genome Res.20061147076[PubMed][Google Scholar]
- 64. Sex chromosomes in mitotic and polytene tissues of Anastrepha fraterculus (Diptera, Tephritidae) from Argentina: a reviewZookeys20155408394[PubMed][Google Scholar]
- 65. Polytene and mitotic chromosome analysis in Ceratitis capitata (Diptera; Tephritidae)Can. J. Genet. Cytol.198628180188[PubMed][Google Scholar]
- 66. Polytene chromosome mapping in Ceratitis capitata (Diptera: Tephritidae)Genome198729598611[PubMed][Google Scholar]
- 67. Polytene chromosome maps in the medfly Ceratitis capitataGenome199033184197[Google Scholar]
- 68. Mitotic and polytene chromosome analysis in Dacus oleae (Diptera: Tephritidae)Genome199235373378[PubMed][Google Scholar]
- 69. Cytogenetic analysis of Malpighian tubule and salivary gland polytene chromosomes of Bactrocera oleae (Dacus oleae)(Diptera: Tephritidae)Genome19953810701081[PubMed][Google Scholar]
- 70. Mitotic and polytene chromosome analyses in the Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae)Genome199841510526[PubMed][Google Scholar]
- 71. Genetic and cytogenetic analysis of the fruit fly Rhagoletis cerasi (Diptera: Tephritidae)Genome200851479491[PubMed][Google Scholar]
- 72. Mitotic and polytene chromosome analysis in the Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae)Genome2008522030[Google Scholar]
- 73. Genetic and cytogenetic analysis of the walnut-husk fly (Diptera: Tephritidae)Ann. Entomol. Soc. Am.201010310031011[PubMed][Google Scholar]
- 74. Genetic and cytogenetic analysis of the American cherry fruit fly, Rhagoletis cingulata (Diptera: Tephritidae)Genetica201113914491464[PubMed][Google Scholar]
- 75. Cytogenetic analysis of the Ethiopian fruit fly Dacus ciliatus (Diptera: Tephritidae)Genetica2011139723732[PubMed][Google Scholar]
- 76. Mitotic and polytene chromosomes analysis of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)Genetica20111397990[PubMed][Google Scholar]
- 77. Analysis of mitotic and polytene chromosomes and photographic polytene chromosome maps in Bactrocera cucurbitae (Diptera: Tephritidae)Ann. Entomol. Soc. Am.2011104306318[PubMed][Google Scholar]
- 78. Cytogenetic analysis of the South American fruit fly Anastrepha fraterculus (Diptera: Tephritidae) species complex: construction of detailed photographic polytene chromosome maps of the Argentinian Af. sp. 1 memberPLoS One201611e0157192[PubMed][Google Scholar]
- 79. Cytogenetic analysis of mitotic and salivary gland chromosomes in the medfly Ceratitis capitataGenome1987296771[PubMed][Google Scholar]
- 80. Cytogenetic and symbiont analysis of five members of the B. dorsalis complex (Diptera, Tephritidae): No evidence of chromosomal or symbiont-based speciation eventsZookeys2015540273298[PubMed][Google Scholar]
- 81. Avise, J. C. Phylogeography: the history and formation of species. (Harvard University Press, 2000).
- 82. Mitochondrial DNA under siege in avian phylogeographyMol. Ecol.20081721072121[PubMed][Google Scholar]
- 83. Molecular phylogenetic study of Tephritidae (Insecta: Diptera) using partial sequences of the mitochondrial 16S ribosomal DNAMol. Phylogenet. Evol.199771732[PubMed][Google Scholar]
- 84. Phylogenetic relationships among fruit flies, Bactrocera (Diptera, Tephritidae), based on the mitochondrial rDNA sequencesInsect Mol. Biol200110549559[PubMed][Google Scholar]
- 85. Phylogenetic relationships among species of the fraterculus group (Anastrepha: Diptera: Tephritidae) inferred from DNA sequences of mitochondrial cytochrome oxidase INeotrop. Entomol.200130565573[PubMed][Google Scholar]
- 86. Phylogenetic analysis of mitochondrial DNA supports the monophyly of Dacini fruit flies (Diptera: Tephritidae)Ann. Entomol. Soc. Am200295658664[PubMed][Google Scholar]
- 87. Phylogenetic relationships among Bactrocera species (Diptera: Tephritidae) inferred from mitochondrial DNA sequencesMol. Phylogenet. Evol.200326817[PubMed][Google Scholar]
- 88. New contributions towards the understanding of the phylogenetic relationships among economically important fruit flies (Diptera: Tephritidae)Bull. Entomol. Res.200696279288[PubMed][Google Scholar]
- 89. Molecular phylogeny of the family Tephritidae (Insecta: Diptera): New insight from combined analysis of the mitochondrial 12S, 16S, and COII genesMol. Cells2009275566[PubMed][Google Scholar]
- 90. African Dacus (Diptera: Tephritidae): Molecular data and host plant associations do not corroborate morphology based classificationsMol. Phylogenet. Evol.200951531539[PubMed][Google Scholar]
- 91. Molecular phylogeny of Bactrocera species (Diptera: Tephritidae: Dacini) inferred from mitochondrial sequences of 16S rDNA and COI sequencesFlorida Entomol.201093369377[PubMed][Google Scholar]
- 92. A molecular phylogeny for the Tribe Dacini (Diptera: Tephritidae): Systematic and biogeographic implicationsMol. Phylogenet. Evol.201264513523[PubMed][Google Scholar]
- 93. Developing diagnostic SNP panels for the identification of true fruit flies (Diptera: Tephritidae) within the limits of COI-based species delimitationBMC Evol. Biol.2013131[PubMed][Google Scholar]
- 94. Higher phylogeny of frugivorous flies (Diptera, Tephritidae, Dacini): Localised partition conflicts and a novel generic classificationMol. Phylogenet. Evol.201585171179[PubMed][Google Scholar]
- 95. Comparative genomics of mitochondrial DNA in members of the Drosophila melanogaster subgroupJ. Mol. Evol.2000514863[PubMed][Google Scholar]
- 96. Comparative genomics of mitochondrial DNA in Drosophila simulansJ. Mol. Evol.2000516475[PubMed][Google Scholar]
- 97. Significant levels of sequence divergence and gene rearrangements have occurred between the mitochondrial genomes of the wild mulberry silkmoth, Bombyx mandarina, and its close relative, the domesticated silkmoth, Bombyx moriMol. Biol. Evol.20021913851389[PubMed][Google Scholar]
- 98. Sequence and phylogenetic analysis of the complete mitochondrial genome of the flour beetle Tribolium castanaeumMol. Phylogenet. Evol.200326502512[PubMed][Google Scholar]
- 99. A mitochondrial genome phylogeny of Diptera: whole genome sequence data accurately resolve relationships over broad timescales with high precisionSyst. Entomol.2007324059[PubMed][Google Scholar]
- 100. Complete mitochondrial genome of the free-living earwig, Challia fletcheri (Dermaptera: Pygidicranidae) and phylogeny of PolyneopteraPLoS One20127e42056[PubMed][Google Scholar]
- 101. Beyond barcoding: A mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)Gene2012511131142[PubMed][Google Scholar]
- 102. Insect mitochondrial genomics: implications for evolution and phylogenyAnnu. Rev. Entomol.20145995117[PubMed][Google Scholar]
- 103. Complete mitochondrial genome of the atlas moth, Attacus atlas (Lepidoptera: Saturniidae) and the phylogenetic relationship of Saturniidae speciesGene201454595101[PubMed][Google Scholar]
- 104. The mitochondrial genome of the olive fly Bactrocera oleae: Two haplotypes from distant geographical locationsInsect Mol. Biol.200312605611[PubMed][Google Scholar]
- 105. Domestication of olive fly through a multi-regional host shift to cultivated olives: Comparative dating using complete mitochondrial genomesMol. Phylogenet. Evol.201057678686[PubMed][Google Scholar]
- 106. The complete nucleotide sequence of the mitochondrial genome of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae)Gene20073966674[PubMed][Google Scholar]
- 107. The mitochondrial genome of the peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae): Complete DNA sequence, genome organization, and phylogenetic analysis with other tephritids using next generation DNA sequencingGene2015569191202[PubMed][Google Scholar]
- 108. Complete mitochondrial genome of Bactrocera arecae (Insecta: Tephritidae) by next-generation sequencing and molecular phylogeny of Dacini tribeSci. Rep.2015515155[PubMed][Google Scholar]
- 109. Complete mitochondrial genome of three Bactrocera fruit flies of subgenus Bactrocera (Diptera: Tephritidae) and their phylogenetic implicationsPLoS One201611e0148201[PubMed][Google Scholar]
- 110. The first complete mitochondrial genome of Dacus longicornis (Diptera: Tephritidae) using next-generation sequencing and mitochondrial genome phylogeny of Dacini tribeSci. Rep.2016636426[PubMed][Google Scholar]
- 111. Mavragani-Tsipidou, P. et al. Tephritid fruit flies (Diptera) in Protocols for cytogenetic mapping of arthropod genomes 1–62 (CRC Press, 2014).
- 112. Localization of ribosomal DNA in Rhagoletis pomonella (Diptera: Tephritidae) by in situ hybridizationInsect Mol. Biol.19932163174[PubMed][Google Scholar]
- 113. Frias, D., Barnes, B. N. & others. Importance of larval morphology and heterochromatic variation in the identification and evolution of sibling species in the genus Rhagoletis (Diptera: Tephritidae) in Chile. In Proceedings of the 6th International Symposium on fruit flies of economic importance, Stellenbosch, South Africa, 6–10 May 2002. 267–276 (2004).
- 114. Selivon, D. et al. New variant forms in the Anastrepha fraterculus complex (Diptera: Tephritidae). in Proceedings of the 6th International Symposium on fruit flies of economic importance, Stellenbosch, South Africa, 6–10 May 2002. 253–258 (2004).
- 115. Karyotype relationships among Anastrepha bistrigata, A. striata and A. serpentina (Diptera, Tephritidae)Genet. Mol. Biol.20073010821088[PubMed][Google Scholar]
- 116. Chromosomes of the Caribbean fruit fly (Diptera: Tephritidae)Florida Entomol.200487361364[PubMed][Google Scholar]
- 117. Polytene chromosomes as tools in the genetic analysis of the Mediterranean fruit flyCeratitis capitata. Genetica20021165971[PubMed][Google Scholar]
- 118. Patterns of puffing activity in the salivary gland polytene chromosomes of the medfly Ceratitis capitata, during larval and prepupal developmentGenome199942919929[PubMed][Google Scholar]
- 119. Ashburner, M. Transpositions and transposons in Drosophila: A laboratory handbook. 641–651 (Cold Spring Harbor Laboratory Press, 1989).
- 120. The white gene of Ceratitis capitata: a phenotypic marker for germline transformationScience199527020052008[PubMed][Google Scholar]
- 121. The heat shock 70 gene family in the Mediterranean fruit fly Ceratitis capitataInsect Mol. Biol.19987279290[PubMed][Google Scholar]
- 122. Muller, H. J. Bearing of the Drosophila work on systematics in The new Systematics 185–268 (Oxford University Press, 1940).
- 123. The homologies of the chromosome elements in the genus DrosophilaGenetics194126517[PubMed][Google Scholar]
- 124. Autosomal genetic maps of the Australian sheep blowfly, Lucilia cuprina dorsalis R.-D.(Diptera: Calliphoridae), and possible correlations with the linkage maps of Musca domestica L. and Drosophila melanogaster (Mg.)Genet. Res.1981375569[PubMed][Google Scholar]
- 125. Persistence of linkage relationships among enzyme loci in some dipteran speciesAtti Assoc Genet Ital198631121122[Google Scholar]
- 126. Genetic maps of the sheep blowfly Lucilia cuprina: linkage-group correlations with other dipteran generaGenome199336495506[PubMed][Google Scholar]
- 127. The genome of the Mediterranean fruit-fly Ceratitis capitata - Localization of molecular markers by in situ hybridization to salivary-gland polytene chromosomesChromosoma1992101448455[PubMed][Google Scholar]
- 128. The genome of the olive fruit fly Bactrocera oleae: localization of molecular markers by in situ hybridization to the salivary gland polytene chromosomesGenome199942744751[PubMed][Google Scholar]
- 129. Isolation, annotation and applications of expressed sequence tags from the olive fly, Bactrocera oleaeMol. Genet. Genomics20112853345[PubMed][Google Scholar]
- 130. The Bactrocera oleae genome: localization of nine genes on the polytene chromosomes of the olive fruit fly (Diptera: Tephritidae)Genome201457573576[PubMed][Google Scholar]
- 131. Extraordinary conservation of entire chromosomes in insects over long evolutionary periodsEvolution (N. Y)201670229234[Google Scholar]
- 132. The mitochondrial genome of the Mediterranean fruit flyCeratitis capitata. Insect Mol. Biol.2000913944[PubMed][Google Scholar]
- 133. The complete nucleotide sequence of the mitochondrial genome of Bactrocera minax (Diptera: Tephritidae)PLoS One20149e100558[PubMed][Google Scholar]
- 134. Zhang, K.-J. et al. The complete mitochondrial genome of Bactrocera diaphora (Diptera: Tephtitidae). Mitochondrial DNA27, doi:10.3109/19401736.2015.1089479 (2015).
- 135. Liu, J.-H., Xu, J., Li, Y.-H., Dan, W. & Pan, Y. Complete mitochondrial genome of the guava fruit fly, Bactrocera correcta (Diptera: Tephritidae). Mitochondrial DNA27, doi:10.3109/19401736.2015.1101559 (2015).
- 136. Tan, M., Zhang, R., Xiang, C. & Zhou, X. The complete mitochondrial genome of the pumpkin fruit fly, Bactrocera tau (Diptera: Tephritidae). Mitochondrial DNA27, doi:10.3109/19401736.2015.1036249 (2015).
- 137. tRNA punctuation model of RNA processing in human mitochondriaNature1981290470474[PubMed][Google Scholar]
- 138. Insect mitochondrial control region: A review of its structure, evolution and usefulness in evolutionary studiesBiochem. Syst. Ecol.19972599120[PubMed][Google Scholar]
- 139. Phylogenetic relationships of Ceratitis fruit flies inferred from nuclear CAD and tango/ARNT gene fragments: Testing monophyly of the subgenera Ceratitis (Ceratitis) and C. (Pterandrus)Mol. Phylogenet. Evol.200953412424[PubMed][Google Scholar]
- 140. A review of the current knowledge on Zeugodacus cucurbitae (Coquillett) (Diptera, Tephritidae) in Africa, with a list of species included in ZeugodacusZookeys2015540539557[PubMed][Google Scholar]
- 141. Isolation of plant DNA from fresh tissueFocus (Madison)1990121315[Google Scholar]
- 142. EMBOSS: the European molecular biology open software suiteTrends Genet.200016276277[PubMed][Google Scholar]
- 143. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequenceNucleic Acids Res.199725955964[PubMed][Google Scholar]
- 144. MITOS: Improved de novo metazoan mitochondrial genome annotationMol. Phylogenet. Evol.201369313319[PubMed][Google Scholar]
- 145. Tandem repeats finder: a program to analyze DNA sequencesNucleic Acids Res.199927573[PubMed][Google Scholar]
- 146. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasetsMol. Biol. Evol.20163318701874[PubMed][Google Scholar]
- 147. The white gene of the tephritid fruit fly Bactrocera tryoni is characterized by a long untranslated 5′ leader and a 12 kb first intronInsect Mol. Biol.19976343356[PubMed][Google Scholar]
- 148. Isolation and characterization of the Bactrocera oleae genes orthologous to the sex determining sex-lethal and doublesex genes of Drosophila melanogasterGene2005348111121[PubMed][Google Scholar]
- 149. Genetic and molecular markers of the Queensland fruit fly, Bactrocera tryoniJ. Hered.200394416420[PubMed][Google Scholar]