Genetic diversity and population structure of A nastrepha obliqua in ...

S P E C I A L I S S U E – S T E R I L E I N S E C T T E C H N I QU E

DOI: 10.1111/eea.12613

Genetic diversity and population structure of Anastrepha obliqua in southwestern Colombia Elkin J. Aguirre-Ramirez* , Sandra M. Velasco-Cuervo, Jenny J. Gallo-Franco, Ranulfo Gonzales, Nancy S. Carrejo & Nelson Toro-Perea Department of Biology, Universidad del Valle, Cali, Colombia Accepted: 15 June 2017

Key words: cytochrome oxidase subunit 1, NADH dehydrogenase subunit 6, ecological differences, host plant preference, Diptera, Tephritidae, quarantine pest, West Indian fruit fly, demographic history

Abstract

The West Indian fruit fly, Anastrepha obliqua (Macquart) (Diptera: Tephritidae), is a quarantine pest in Colombia that infests economically important plants, although little is known about its population dynamics. In this study, cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 6 (ND6) mitochondrial genes were concatenated to characterise the genetic diversity and population structure of A. obliqua, associated with two factors: (1) ecosystem differences in two geographical regions of southwestern Colombia (the inter-Andean valley of the Cauca River and the mountain region), and (2) the host plants present in the area. Additionally, a first approach was made at understanding the species demographic history. Seven haplotypes were found with Kimura 2-parameter (K2P) genetic distances between 0.1 and 4%. Haplotype genealogies and demographic analyses suggest that the population of A. obliqua in southwestern Colombia is the result of introductory events of multiple populations of A. obliqua. However, the data indicate that the population genetic structure could be related to the ecological differences of the two regions being studied. Significant differences were also found among the distribution of haplotype frequencies of A. obliqua with regard to the diversity of host plants. This study is the first to provide an understanding of the population dynamics of A. obliqua in Colombia, which may ultimately contribute to strategies, such as sterile insect technique (SIT), for the management of the pest.

Introduction Many true fruit flies (Diptera: Tephritidae) are pests of global economic importance, limiting internal and external fruit trade. Among the fruit flies genera, Anastrepha represents the group of greater economic importance in tropical and subtropical areas of the American continent, with more than 250 species described (Norrbom et al., 2013). Anastrepha obliqua (Macquart), the West Indian fruit fly, is a polyphagous species of greater economic concern within the genus because it infests ca. 104 fruit species in 27 plant families (Norrbom, 2004), mainly mango

*Correspondence: Elkin J. Aguirre-Ramirez, Research Group in Molecular Biology and Ecogenetics, Department of Biology, Universidad del Valle, Valle del Cauca, Calle 13 # 100-00, Cali 760032, Colombia. E-mail: [email protected]

(Mangifera indica L.) and Spondias species in Mexico (Orozco-Davila et al., 2014), Colombia (Mangan et al., 2011), the Caribbean Islands (Mangan et al., 2011), and Brazil (Zucchi, 2000). Anastrepha obliqua is characterised by a wide geographic distribution ranging from northern Mexico to southern Brazil, including the Caribbean Islands (Fu et al., 2014). In Colombia, the species A. obliqua, Anastrepha fraterculus (Wiedemann), Anastrepha serpentina (Wiedemann), Anastrepha striata Schiner, and Ceratitis capitata (Wiedemann) are considered pests with the greatest negative effect on fruit crops. The larval stage of these flies causes direct physical damage to the fruit pulp, indirectly facilitating contamination with fungi and bacteria (CONPES, 2008). Losses between 30 and 40% of total fruit production are reportedly caused by A. obliqua, but losses may reach 70% if adequate pest management is not

© 2017 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 1–14, 2017

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2 Aguirre-Ramirez et al.

implemented (CONPES, 2008). Despite the negative effect of A. obliqua on agriculture, knowledge concerning the species in Colombia is only represented in some studies related to its distribution, host plants, associated parasitoids, and morphological variation (Carrejo & Gonzalez, 1994, 1999; Casta~ neda et al., 2010, 2015), ignoring such aspects as its population structure and dynamics as well as its genetic diversity, both of which are particularly relevant for the design of integrated pest management programmes (Meeyen et al., 2013). In southwestern Colombia, A. obliqua is found infesting several types of fruits. Southwestern Colombia is one of the most important agricultural areas of the country, characterised by three natural regions: the Pacific region, the inter-Andean valley of the Cauca River, and the mountain region. The Pacific region is characterised by flood-prone forests (mangroves) in the coastal area, and rainforests and humid tropical zones as one approaches the western slope of the western mountain range. The inter-Andean valley of the Cauca River primarily consists of areas dedicated to agricultural activities that have almost entirely replaced the natural formations of dry and very dry tropical forests. Finally, the mountain region includes the western and central mountain ranges of the northern Andes, which contain Andean forests that are characterised by high humidity, as they are covered with fog for several months of the year (Salazar et al., 2002; Kattan, 2003; Poveda et al., 2004). The ecological differences associated with natural regions and geographic barriers such as mountain ranges can prevent the flow of genes between populations, which may affect the evolution of these populations and any possible genetic differentiation (Hamilton, 2009; Ruiz-Arce et al., 2015). It can therefore be argued that the ecological differences found between the mountain ranges of the northern Andes and the inter-Andean valley of the Cauca River may play an important role in the genetic differentiation of populations of A. obliqua in this region, which may be revealed using molecular techniques. In addition to geographic barriers and ecological differences, the selective pressures caused by the presence of different host plants in a region may also be a key factor in the genetic differentiation of populations and sympatric speciation in phytophagous insects, particularly those that are polyphagous and oligophagous (Munday et al., 2004; Schwarz et al., 2005; Faucci et al., 2007). Saxena & Barrion (1987) demonstrated that haplotypes that be recovered from some species may be closely associated with the crop or plant they attack; therefore, there is evidence of co-specificity between them, which could be confirmation of an incipient speciation event. Knowledge of this co-specificity between

host plants and A. obliqua could lead to better pest control if the plant species where the fly is found are considered. Recently, mitochondrial markers have been used to explore the genetic diversity, population dynamics, and genetic population structure of species. The mitochondrial markers are characterised by uniparental inheritance, an absence of recombination, and a relatively high mutation rate (Galtier et al., 2009). Mitochondrial DNA sequences have been successfully used in Tephritidae to determine interspecific relationships (Han & McPheron, 1999; McPheron et al., 1999; Smith-Caldas et al., 2001; Barr et al., 2005; Barr & McPheron, 2006; Boykin et al., 2006), to evaluate population structure (McPheron et al., 1994; Alberti et al., 2008; Lanzavecchia et al., 2008), to develop methods for rapid species identification (Barr et al., 2006), and to analyse geneflow between populations (Lanzavecchia et al., 2008; Barr, 2009). Recent studies on the genetic diversity and population structure of A. obliqua have used mitochondrial DNA sequences. Smith-Caldas et al. (2001) analysed the cytochrome oxidase subunit I (COI) sequence of 15 Anastrepha species. They reported high genetic diversity in A. obliqua and postulated that this species may be composed of reproductively isolated populations. Ruiz-Arce et al. (2012) studied 54 A. obliqua populations in Latin America with mitochondrial gene (COI and ND6) sequences, identifying 61 A. obliqua haplotypes distributed among three phylogenetic clades. These two reports suggest the existence of a complex of cryptic species within the nominal species of A. obliqua. The objective of our study was to characterise the genetic diversity and population structure of A. obliqua associated with two regions of southwestern Colombia (inter-Andean valley of the Cauca River and the northern Andean mountain region) and with the host plants that this fly infests in both regions, using the concatenated sequences of mitochondrial genes COI and ND6. A second objective was to further our knowledge of the demographic history of A. obliqua in southwestern Colombia. The phylogenetic tree reported by Ruiz-Arce et al. (2012) was reconstructed to include the haplotypes found in our study.

Materials and methods Sample collection

Fruits of potential A. obliqua host plants were only collected in localities of the inter-Andean valley of the Cauca River and the mountain region of southwest Colombia – collection was not possible in the Pacific region despite the presence of potential host plants, as none were ever found to be fructified (Table 1). Fruits were taken from trees that

Population structure of Anastrepha obliqua in Colombia 3

did not belong to crops, i.e., trees adjacent to the road or in orchards of small farms. Once in the laboratory, the fruits were deposited into a plastic container with a mixture of non-sterile sawdust and sterilised fine sand. The containers were covered with a very fine porosity (white muslin) cloth (Carrejo & Gonzalez, 1999) and maintained at room temperature until the emergence of adult individuals. Each morphologically identified individual – based on the key of Steyskal (1977) – was preserved in 96% ethanol at 20 °C for subsequent DNA analysis. For each individual, a voucher specimen was prepared and deposited in the Entomology Museum at the University of Valle. DNA extraction, ampliﬁcation, and sequencing

DNA was extracted from the head and legs of adult individuals of A. obliqua using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), applying the insect protocol described by the supplier (Qiagen, 2006). The extracted DNA was maintained at 20 °C for the duration of the study. The COI gene was amplified using the primers LCO1490: 50 -GGTCAACAAATCATAAAGA TATTGG-30 and HCO2198: 50 -TAAACTTCAGGGT GACCAAAAAATCA-30 (Folmer et al., 1994), generating a fragment of ca. 710 bp. The reaction mixture was modified from Ruiz et al. (2010) for a total volume of 25 ll, containing 19 buffer (Qiagen), 0.05 mM dNTPs (Macrogen, Rockville, MD, USA), 2 mM MgCl2 (Qiagen), 1 U Taq DNA polymerase (Qiagen), 0.25 mM of each primer (Macrogen), and 30 ng of DNA. The thermal profile used was 5 min at 92 °C followed by 35 cycles of 30 s at 94 °C, 1 min at 52 °C, and 1 min at 72 °C, with a final extension of 5 min at 72 °C (modified from Folmer et al., 1994). For the ND6 gene, a ca. 725bp fragment was amplified with the primers TT-J-9886: 50 -TAAAAACATTGGTCTTGTAA-30 and ND6r: 50 TTATGATCCAAAATTTCATCA-30 (Ruiz-Arce et al., 2012), according to the protocol proposed by Ruiz-Arce et al. (2012). Additionally, a pair of primers was designed to amplify the ND6 gene in DNA samples, where no amplicon was obtained using the primers proposed by Ruiz-Arce et al. (2012). These primers were designed from an ND6 gene sequence of A. obliqua obtained from GenBank (accession number HM564065) and using the Primer-BLAST program (National Center for Biotechnology Information, NCBI; http://www.ncbi.nlm.nih. gov/tools/primer-blast/). PCR products were verified by 1.5% agarose gel electrophoresis in a 0.59 TBE buffer (0.0045 M tri-borate, 0.001 M EDTA), using EZ-Vision (Amresco, Solon, OH, USA) and visualised in an ultraviolet light transilluminator. For the purification and sequencing of mitochondrial DNA fragments, a specialised service (Macrogen) was contracted.

Data analysis

The sequences obtained from the COI and ND6 genes were edited manually using Sequencher v.4.1.4 (Gene Codes Corporation, Ann Arbor, MI, USA), obtaining final lengths of 658 and 541 bp, respectively. The COI and ND6 sequences were concatenated using DnaSP v.4.10 (Rozas et al., 2003), and subsequent analyses were performed with a 1 199-bp fragment (COI+ND6) (Table 2). All sequences were aligned using Clustal X 2.1 (Larkin et al., 2007). Detection of sequence variability in concatenated mitochondrial DNA. Haplotypes were identified with Mega 7 (Kumar et al., 2016). Standard genetic diversity indices, such as polymorphic sites, haplotype diversity (Hd) (Nei, 1987), and nucleotide diversity (p) (Lynch & Crease, 1990) were calculated using DnaSP v.4.10 (Rozas et al., 2003). Genetic distances between haplotype pairs were found using the Kimura 2-parameters (K2P) nucleotide substitution model in Mega 7 (Kumar et al., 2016), as Hebert et al. (2003) propose that this model is one of the best estimators of these distances when working with sequences of a single species. Genealogical relations among haplotypes. A haplotype network was constructed with the median joining (MJ) network algorithm, using Network 5 (Bandelt et al., 1999). Each haplotype was characterised according to the host plant and to the natural region where it was found. In addition, a phylogenetic reconstruction was made with the haplotypes found in this study and those reported by Ruiz-Arce et al. (2012), using the maximum likelihood (ML) and neighbour-joining (NJ) methods present in Mega 7 (Kumar et al., 2016). All COI and ND6 haplotypes reported by Ruiz-Arce et al. (2012) were edited to obtain a size similar to the fragment edited by us. An individual phylogenetic analysis of each gene was performed and compared against the others, later a phylogenetic reconstruction of the concatenated sequences was performed. The best nucleotide substitution model was determined using jModelTest v.2.1.7 (Guindon & Gascuel, 2003; Posada, 2008). The statistical support of the topology nodes was evaluated with 1 050 bootstraps, corresponding to a 95% confidence interval (Singh & Xie, 2010). An individual of A. striata collected and identified with the taxonomic key of Steyskal (1977) was included as an external group within the phylogenetic analyses. Determination of population genetic structure. The Arlequin v.3.5 program (Excoffier & Lischer, 2010) was used to calculate FST values (Wright, 1951; Weir &


Table 1 Sampling sites and collection information of Anastrepha obliqua individuals included in this study

Geographic region

Location

Code

Coordinates

Altitude (m a.s.l.)

Collection date

Inter-Andean valley of the Cauca River

San Jer onimo El Frutal Chagres

SA FR CH

02°550 42.80″N, 76°300 37.50″W 02°550 57.60″N, 76°310 24.80″W 03°070 01.90″N, 76°360 19.10″W

1239 1151 1046

Guachinte Cali La Buitrera

GU CA BU

03°080 03.73″N, 76°360 15.96″W 03°220 29.64″N, 76°320 00.69″W 03°220 20.50″N, 76°340 11.30″W

1067 985 1129

Palmira Bolıvar La Uni on

PA BO UN

03°300 38.90″N, 76°190 36.50″W 04°260 08.40″N, 76°080 36.70″W 04°320 44.20″N, 76°050 37.90″W

982 940 936

Jan 2015 Jan 2015 Jan 2015 Jan 2015 Jan 2015 Jan 2015 Jan 2015 Jan 2015 Feb 2015 Jan 2015 Aug 2015

La Cumbre

CU

03°370 26.60″N, 76°320 08.20″W

1468

Dagua Sevilla

DA SE

03°390 27.44″N, 76°410 41.56″W 04°140 57.30″N, 75°560 49.30″W

849 1366

Mountain region

Mar 2015 Mar 2015 Sep 2015 Mar 2015

Host plant Mangifera indica M. indica M. indica Spondias purpurea M. indica Averrhoa carambola M. indica A. carambola A. carambola M. indica M. indica A. carambola S. purpurea M. indica S. purpurea M. indica Eugenia stipitata

Total

No. specimens 8 7 8 10 7 8 10 1 5 10 10 10 1 5 8 10 10 128

Table 2 Geographical distribution of the seven haplotypes of Anastrepha obliqua observed in southwestern Colombia

Natural region Inter-Andean valley of the Cauca River

Mountain region

Haplotype COI+ND6

GenBank number COI

ND6

Place of origin1

H1 H2 H3 H4 H5 H1 H4 H6 H7

KX869974 KX869972 KX869973 KX869973 KX869970 KX869974 KX869973 KX869969 KX869971

KX889123 KX889123 KX889123 KX889124 KX889121 KX889123 KX889124 KX889120 KX889122

SA, FR, CH, GU, CA, BU, PA, BO, UN SA CH SA, FR, CH, GU, CA, BU, UN BU SE, DA DA SE SE, DA, CU

1

See Table 1 for explanation of the codes.

Cockerham, 1984) between pairs of localities and to perform an analysis of molecular variance (AMOVA), considering two groups: the first group considered the genetic differences arising from the ecological characteristics of each of the regions studied (the mountain region and the inter-Andean valley of the Cauca River), and the second group considered the genetic differences between groups of populations from different host plants. A permutation method with 10 000 replicates was applied to test the significance of the tests. Both the AMOVA and FST were performed using the K2P substitution model. A v2 test was performed to evaluate differences in the distribution of haplotype frequencies from host plants using PAST v.3.13 (Hammer et al.,

2001). The relationship between the genetic distance of mitochondrial DNA (Arlequin FST) and the geographic distance (km) was evaluated with the Mantel test (Mantel, 1967) implemented in IBD v.1.52 (Bohonak, 2002) with 1 000 replicates. Demographic history. The demographic parameters Tajima’s D (Tajima, 1989) and Fu’s Fs (Fu, 1997) were calculated with DnaSP v.4.10 (Rozas et al., 2003) to determine the deviation from neutrality and thus identify bottlenecks or populations in expansion. The mismatched distribution was estimated using DnaSP v.4.10 as an additional indicator of demographic history, where a unimodal distribution may suggest an expanding


population and a bimodal distribution may suggest a stable population (Slatkin & Hudson, 1991).

Results Fruits of different potential host plants of A. obliqua were collected: mango (Mangifera indica L., Anacardiaceae), red mombin (Spondias purpurea L., Anacardiaceae), carambola (Averrhoa carambola L., Oxalidaceae), araza (Eugenia

stipitata McVaugh, Myrtaceae), guava (Psidium guajava L., Myrtaceae), pomarrosa [Syzygium malaccense (L.) Merr. & LM Perry, Myrtaceae], orange (Citrus spec., Rutaceae), and coffee (Coffea arabica L., Rubiaceae). Of these seven plants, A. obliqua was only found to be infesting fruits of mango, red mombin, carambola, and araza. A total of 128 adult individuals of A. obliqua were analysed, collected from 12 sample locations (Table 1). Design of primers

Table 3 Genetic distances (%) according to K2P, between haplotype pairs of Anastrepha obliqua Haplotypes

H1

H2

H3

H4

H5

H6

H7

H1 H2 H3 H4 H5 H6 H7

0 0.1 1.7 3.9 2.4 2.3 2.5

0 1.8 4 2.5 2.4 2.6

0 2.1 3.4 3.4 3.5

0 3.9 3.4 3.6

0 1.3 1.3

0 0.2

0

Figure 1 Haplotype frequencies of Anastrepha obliqua in each sampled locality (see Table 1 for explanation of the codes). The symbols H1 to H7 represent the seven haplotypes found in southwestern Colombia. WMR, Western Mountain Range; IV, Inter-Andean Valley of the Cauca River; CMR, Central Mountain Range.

Thirty-three DNA samples, out of a total of 128, presented problems in the amplification of the ND6 gene sequence with the primers proposed by Ruiz-Arce et al. (2012); thus, it was necessary to design a new pair of primers (ND6F: 50 -AAACTTCAAGAAAATGAAATAACT-30 and ND6R: 50 -AATAATGGGTGTTGGGTTCG-30 ) generating a fragment of ca. 637 bp. The problematic DNA samples originated from individuals from different locations (BO, SA, CH, GU, SE, DA, and CU) and fruits (mango, red mombin, and araza), although most of them (64%) came from localities of the mountain region (Table 1). With these new primers, the 128 amplicons of the ND6 gene sequence were completed.


Variation in mitochondrial DNA concatenation sequence

A 1 199-bp fragment from the concatenated mitochondrial DNA (COI+ND6) was obtained after editing the sequences, and in total seven haplotypes were identified (Table 2). After alignment, 65 polymorphic sites (5.4%) and 58 parsimoniously informative sites (4.8%) were observed, and no indels (gaps) were detected. The COI (658 bp) and ND6 (541 bp) genes contributed 30 and 35 polymorphic sites, respectively. Haplotype and nucleotide diversity for the total dataset was 0.553 and 0.016, respectively. The range of genetic distances found between haplotypes varied between 0.1 and 4% (Table 3), according to the K2P substitution model. Due to the high levels of genetic distances found, we performed a detailed morphological characterisation of all individuals. This review verified that individuals corresponded to A. obliqua. Haplotype frequencies were determined for each locality (Figure 1). H1 was the most abundant haplotype found in

all localities (except CU) in a total of 81 individuals (65%). The second most abundant haplotype was H7 in a total of 23 individuals (19%), which were found in SE, DA, and CU. The third most abundant haplotype was H4 in a total of 17 individuals (14%), which were found in most of the localities except BO, SE, and CU. Haplotypes H2 (one individual), H3 (one individual), H5 (one individual), and H6 (four individuals) were localised for SA, CH, BU, and SE, respectively (Figure 1). Genealogical relations among haplotypes

The haplotype network for the 128 sequences of A. obliqua revealed highly divergent haplotypes connected by long branches that reflect the considerable number of nucleotide changes found among them and the presence of several theoretical haplotypes (Figures 2 and 3). Haplotypes H2, H3, and H5 were specific to localities in the interAndean valley of the Cauca River, whereas haplotypes H6

Figure 2 Haplotype network of 128 sequences (concatenated COI+ND6) of Anastrepha obliqua in southwestern Colombia according to two natural regions (Inter-Andean Valley of the Cauca River and the mountain region). The circles labelled H1 to H7 represent haplotypes, and their size is relative to the number of individuals that share the haplotype. The numbers near the branches correspond to the number of nucleotide changes that exist between two connected haplotypes. The circles labelled mv1 and mv2 correspond to theoretical haplotypes that were not sampled.

Figure 3 Haplotype network of 128 sequences (concatenated COI+ND6) of Anastrepha obliqua in southwestern Colombia according to four host plants (mango, carambola, red mombin, and araza). The circles labelled H1 to H7 represent haplotypes, and their size is relative to the number of individuals that share the haplotype. The numbers near the branches correspond to the number of nucleotide changes that exist between two connected haplotypes. The circles labelled mv1 and mv2 correspond to theoretical haplotypes that were not sampled.


and H7 were specific to localities in the mountain region (Figure 2). Mango presented the highest diversity with five haplotypes (H1, H2, H4, H5, H7), the second highest was red mombin with four (H1, H3, H4, H7), followed by araza with three (H1, H6, H7) and carambola with two (H1, H4) (Figure 3). The haplotypes that were private were H3 for red mombin, H5 for mango, and H6 for araza (Figure 3). Phylogenetic constructions with ML and NJ (data not shown) provided similar results. For the individual phylogenetic analysis of each gene, we identified six haplotypes

Figure 4 Maximum likelihood (ML) tree of the mitochondrial concatenate (COI+ND6) showing the phylogeny of the Anastrepha obliqua haplotypes reported by Ruiz-Arce et al. (2012) (OB1–OB61) and the haplotypes identified in this study (H1–H7). The evolutionary history was based on the Hasegawa-Kishino-Yano model (+I: 0.6060, +G: 0.4620). The numbers near the branches correspond to the bootstrap value that supports the ML tree (1050 replicates). Each of the haplotypes was tagged according to the geographical region in which it was found (MA, Mesoamerica; CA, Central America; CR, Caribbean; WM, West of Mexico; ASA, Andes of South America; EB, Eastern Brazil). The tree was rooted with an individual of A. striata.

for COI (Figure S1) and five for ND6 (Figure S2). Both trees presented five phylogenetic clades with low and strong bootstrap supports and showed similarities between the origins of the haplotype sequences of each clade. The phylogenetic reconstruction of concatenated sequences with the ML method revealed five phylogenetic clades with bootstrap values >80% (Figure 4). A similar tree was obtained with NJ (data not shown). Some concatenated haplotype sequences identified in our study – haplotypes H1, H5, and H7 – were already reported by Ruiz-Arce et al. (2012) for populations found in Central America,


Table 4 FST values among the 12 sampling locations of Anastrepha obliqua in southwest Colombia1 Location

SA

FR

CH

GU

CA

BU

PA

BO

UN

SE

DA

CU

SA FR CH GU CA BU PA BO UN SE DA CU

0.000 0.049 0.131 0.049 0.177 0.208 0.342 0.468 0.224 0.447 0.245 0.706

0.000 0.078 0.166 0.065 0.027 0.102 0.234 0.029 0.446 0.220 0.758

0.000 0.078 0.079 0.039 0.013 0.089 0.042 0.504 0.324 0.737

0.000 0.065 0.027 0.102 0.234 0.029 0.446 0.220 0.758

0.000 0.101 0.068 0.029 0.092 0.558 0.331 0.857

0.000 0.057 0.027 0.053 0.503 0.298 0.794

0.000 0.000 0.029 0.691 0.432 1.000

0.000 0.039 0.762 0.537 1.000

0.000 0.551 0.375 0.772

0 0.039 0.233

0.000 0.259

0

1

Details of locations are presented in Table 1. Bold values indicate significant differences between locations (Mantel test: P