Clarifying the source of Conicofrontia sesamoides ...

Clarifying the source of Conicofrontia sesamoides Hampson (Lepidoptera: Noctuidae) population in South African sugarcane using morphological identification and mitochondrial DNA analysis Y. Assefa, M. Goftishu, C. CapdevielleDulac & B. Le Ru Phytoparasitica ISSN 0334-2123 Phytoparasitica DOI 10.1007/s12600-017-0566-1

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Author's personal copy Phytoparasitica DOI 10.1007/s12600-017-0566-1

Clarifying the source of Conicofrontia sesamoides Hampson (Lepidoptera: Noctuidae) population in South African sugarcane using morphological identification and mitochondrial DNA analysis Y. Assefa & M. Goftishu & C. Capdevielle-Dulac & B. Le Ru Received: 11 October 2016 / Accepted: 25 January 2017 # Springer Science+Business Media Dordrecht 2017

Abstract Valid identification of a novel pest species and clarifying its origin are the primary steps in understanding population structure and development of biocontrol programs. In this study geographical populations of Conicofrontia sesamoides Hampson (Lepidoptera: Noctuidae) collected during surveys conducted in the years 2009, 2014 and 2015 were morphologically identified and their genetic diversity was analysed by using sequences of the mitochondrial cytochrome-c oxidase I (COI) gene in an attempt to examine host plant, or/and altitude

Y. Assefa (*) Department of Crop Production, University of Swaziland, Luyengo, Swaziland e-mail: [email protected] Y. Assefa : M. Goftishu Department of Zoology and Entomology, University ofFort Hare, Alice, South Africa M. Goftishu School of Plant Sciences, Haramaya University, P.O. Box 138, Dire Dawa, Ethiopia B. Le Ru icipe – African Insect Science for Food and Health, P. O. Box 30772, Nairobi, Kenya C. Capdevielle-Dulac : B. Le Ru IRD/CRNS UMR IRD 247 EGCE, Evolution GénomesComportementetEcologie, CNRS, Gif sur Yvette Cedex, France

: B. Le Ru

C. Capdevielle-Dulac Université Paris - Sud 11, 91405 Orsaycedex, France

associated differences among populations and determine the source of the newly recorded population of this species in the South African sugarcane. The C. sesamoides specimens in this study were collected from Miscanthus capensis (Nees) (Poaceae) and sugarcane (Saccharum officinarum L.) (Poaceae) in Eastern Cape and KwaZulu-Natal Provinces of South Africa. Analysis of Molecular Variance showed a moderate to highly significant genetic differentiation between C. sesamoides populations from different host plants (FST = 0.115, p = 0.14) and altitudinal range (FST = 0.159, p = 0.18). This result was however, contradictory to outcomes of phylogenetic analyses, haplotype networking and uncorrected sequence divergence (0.0–1.54%) which revealed no detectable genetic differentiation between populations from different host plants and altitudes. As it is difficult to measure FST accurately without a large data set, the very small sample used in the analysis might have resulted in inflation of the FST value in this study. After evaluation of the results, it was concluded that the sugarcane population of C. sesamoideshas originated from the population residing in wild host plants in the Eastern Cape and/or KwaZulu-Natal provinces of South Africa. Possible reasons for the host plant expansion and its implications to commercial sugarcane production in the country are discussed. Keywords Conicofrontia sesamoides . Cytochrome-c oxidase I . Morphological identification . South Africa . Sugarcane

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Introduction There are various sources for new crop pests, including the introduction of alien insect pests with their host to the environment (Assefa et al. 2014), host range expansion by pests known to attack related indigenous crops (Assefa et al. 2015) or a shift from the wild to newly introduced crops (Assefa 2015). The introduction of maize (Harris and Nwanze 1992) and sugarcane (Osborne 1964) and their increasing cultivation in commercial and subsistence farms of Africa has been enriching stem borer fauna in the continent’s farmland with mainly indigenous species which were feeding on related indigenous plants in the continent (Conlong 2001; Assefa et al. 2006; Calatayud et al. 2014). Indigenous stem borers (Such as. Busseola fusca Fuller (Lepidoptera: Noctuidae) and Eldana saccharina Walker (Lepidoptera: Pyralidae) represent the major group of organisms which cause economic impact both to maize and sugarcane in South Africa (Kfir et al. 2002; Assefa 2015). Of the total of four economically important species of stem borers of cereals and sugarcane in South Africa, three (75%) are indigenous to the continent (Kfir et al. 2002; Assefa 2015). Similarly, only two of the known cereal stem borers (Chilo partellus Swinehoe (Lepidoptera: Crambidae) and Chilo sacchariphagus Bojer (Lepidoptera: Crambidae) in the continent are exotic (Polaszek 1998) Noctuids represent one of the numerous groups of borers on the wild plants (Le Ru et al. 2006a, b; Ndemah et al. 2007; Matama-Kauma et al. 2008; Moolman et al. 2014) and cultivated crops (Polaszek 1998) in SubSaharan Africa. Sugarcane and maize growing in disturbed landscapes of subsistent farms and commercial plantations of the continent are mainly attacked by indigenous noctuid stem borers (Polaszek 1998). Host range expansion to sugarcane and maize by indigenous stem borers is becoming a frequent phenomenon (Calatayud et al. 2014). These exotic crops are characterized by fast growth and good yield, which contributed to their selection for cultivation in many parts of Africa (Hussain et al. 2001). The better performance of these exotic plants in subsistent farms and commercial plantations of the continent, although this pattern may not be universal (Parker et al. 2012), might have increasingly exposed them to damage by pest insects native to the area in which they have been cultivated (Brockerhoff et al. 2006). Development of new biotypes adapted to the crops could be the other reason for the observed host

range expansion by the indigenous borers (Berlocher and Feder 2002; Funk 1998; Nosil et al. 2002). Eldana saccharina which is the most important pest of sugarcane in South Africa was only recorded to feed on the crop in 1939 (Dick 1945), 100 years after the introduction of sugarcane to Africa (Osborne 1964). The success of this borer in sugarcane fields of South Africa is associated with lack of effective natural enemies in commercial sugarcane fields (Conlong 1997) and gradual increase in mean temperature of the sugarcane producing regions that improved E. saccharina’s mating success (Way and Turner 1999). Similarly, the maize stem borer, B. fusca, moved to maize (Fuller 1901) and sugarcane (Assefa et al. 2007; Assefa 2015) and got established in small scale farms of Ethiopia, Zimbabwe and South Africa (Assefa et al. 2015). In a recent surveys conducted in the sugarcane fields of subsistence farmers in Pondol and areas of South A f r i c a , a n i n d i g e n o u s n o c t u i d s t e m b o r e r, Conicofrontia sesamoides Hampson (Lepidoptera: Noctuidae), was recorded to be part of a borer complex attacking the crop (Assefa 2015). The borer was originally identified in 2013 from a wild host plant, Miscanthus capensis (Nees) (Poaceae), collected at three sites in KwaZulu-Natal province of South Africa (Moolman et al. 2014). The close taxonomic relation between Miscanthus capensis and sugarcane (Saccharum officinarum L.) (Poaceae) (both belong to the sub-family Andropogoneae) might have played a role in the host plant expansion. The insect has never been reported from cultivated crops anywhere and the report by Assefa (2015) is the first on host range expansion by C. sesamoides. Studies indicate that ecological specialization to different host plant species is a result of genetic differentiation and formation of biotypes (Abrahamson et al. 2003; Berlocher and Feder 2002; Funk 1998; Nosil et al. 2002; Sword et al. 2005). It is thus important to understand the source of the C. sesamoides population in the South African sugarcane and ascertain the degree of relatedness with the wild populations for effective use of biocontrol agents and habitat management control options to suppress its population and prevent a potential outbreak. Uniparentally inherited portions of the insect genome have important applications in a wide range of fields including evolutionary and population history (Sezonlin et al. 2006; Assefa et al. 2006), oviposition host-plant preference (Thomas and Singer 1987; Hanski and Singer 2001) genetic genealogy (Pons et al. 2006;

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Hurst and Jiggins 2005) as well as forensic science (Harvey et al. 2003; Tan et al. 2010). This study presents a phylogenetic analysis of C. sesamoides from wild and cultivated habitats to determine the origin of the sugarcane population of the species and to facilitate a better use of known mtDNA variation in the management of this insect in crop fields. The insect’s geographic distribution is confined to southern Africa (Le Ru et al. 2015) and this mtDNA analysis included specimens from most of the known geographic range of the species.

Material and methods

comparison with types and specimens housed by the Natural History Museum, London (BMNH), United Kingdom, the Museo Civico di Storia Naturale(MCSN), Milan, Italy and the Ditsong Museum: formerly Transvaal Museum of South Africa(TMSA), Republic of South Africa. Larvae collected from sugarcane were subjected to molecular analysis at South African Sugarcane Research Institute (SASRI) whereas only adult specimens of C. sesamoides sampled from wild hosts at eight localities in the KwaZulu-Natal and Eastern Cape provinces of South Africa were sequenced at Laboratoire Evolution Génome Comportement et Ecologie at Gifsur-Yvette in France.

Insect collection and morphological identification DNA extraction, PCR and sequencing Surveys and sampling of the noctuid stem borer larvae from damaged wild host-plants were conducted during November 2009 and during February to March in 2015. Similar surveys and collection of stem-borer larvae from infested sugarcane in small scale farms were done between April and June 2014 in Eastern Cape Province of South Africa (Table 1). A total of 16 C. sesamoides specimens, 12 from Kwazulu-Natal and four from Eastern Cape provinces, were collected. Locality names, host plant, GPS positions and altitude were recorded at each locality where borers were recovered. Host plant specimens were identified by Kathleen GordonGray (Pietermaritzburg University, South Africa), Xholani Simane (MSc, Department of Livestock and pasture, University of Fort Hare) and Simon Mathenge (PhD, Botany Department, University of Nairobi, Kenya). Larvae from wild host plants were reared on artificial diet (Onyango and Ochieng’Odero 1994) until pupation and pupae were kept in an empty vial with a perforated lead for aeration until adult emergence. Genitalia of the emerged adults were used for morphological identification. Larvae from sugarcane were kept in sugarcane stalks until they were used for molecular analysis. Genitalia of the wild populations were dissected after immersion of the end of the abdomen in a boiling 10% potash bath for a few minutes, then cleaned, immersed in absolute alcohol for a few minutes and mounted on slides in Euparal (after separating the aedeagus from the rest of the (male) genitalia. Collected insects were identified by

DNA of Conicofrontia sesamoides was extracted from the thorax or hind legs of larvae/adult using a QiagenDNEasy extraction kit. Polymerase chain reaction (PCR) amplifications of the mitochondrial gene 658 bpfragment region of the Cytochrome Oxidase I (COI), and sequencing of specimens at Laboratoire Evolution, Genome and Speciation at Gif-sur-Yvette in France was done using the primers and settings detailed in Le Ru et al. (2015) whereas specimens sent to SASRI were sequenced following the method described in Assefa et al. (2015) and primer settings detailed in Le Ru et al. (2015). Sequences used in this study are accessible on GenBank under accessions numbers: GU549481 to GU549485 for C. sesamoides specimens sequences in France and KX572861 to KX572865 for specimens sequenced at SASRI. Molecular analysis Editing and assembling DNA sequence chromatograms was completed using the Staden package (Staden 1996). Sequences were aligned using ClustalX (Thompson et al. 1997) and manually corrected using BioEdit sequence alignment editor (Hall 1999). The resulting 527 bp fragment of the COI sequences of specimens from sugarcane were compared with specimens collected from Miscanthus capensis. A median-joining network was generated from the alignment using NETWORK 4.6 (Bandelt et al. 1999). Each haplotype was represented by a single sequence for phylogenetic analysis, which was performed by

Author's personal copy Phytoparasitica Table 1 Localities, host plants & Haplotypes of Conicofrontia sesamoides specimens used in the study Altitude

Province

Location

Geographic position Longitude

Latitude

Host Plant

Haplotype Name

HT-2

1069

KwaZulu-Natal

Oak Hotel

E 30°10′

S 29°49′

Miscanthus capensis

1462

KwaZulu-Natal

Minerva Forest 2

E 30°11′

S 29°48′

Miscanthus capensis

HT-9

348

KwaZulu-Natal

Slykraal

E 26°29′

S 29°22′

Miscanthus capensis

HT-3

1454

KwaZulu-Natal

Minerva Forest 2

E 30°11′

S 29°48′?

Miscanthus capensis

HT-10

1454

KwaZulu-Natal

MinervaForest 2

E 30°11′

S 29°48′

Miscanthus capensis

HT-8

1291

KwaZulu-Natal

Karkloof 2

E 30°21′

S 29°16′

Miscanthus capensis

HT-2

1128

KwaZulu-Natal

Karkloof River

E 30°02′

S 29°13′

Miscanthus capensis

HT-3

1053

KwaZulu-Natal

Shevers Farm

E 30°21′

S 29°10′

Miscanthus capensis

HT-6

348

KwaZulu-Natal

Slykraal

E 26°29′

S 29°22′

Miscanthus capensis

HT-3

1069

KwaZulu-Natal

Oak Hotel

E 30°10′

S 29°49′

Miscanthus capensis

HT-2

1128

KwaZulu-Natal

Karkloof River

E 30°02′

S 29°13′

Miscanthus capensis

HT-5

70

KwaZulu-Natal

Mtubatuba

E 32°15′

S 28°23′

Unknown

HT-4

106

Eastern Cape

Ferry Point Rd

E 29°59′

S 31°60′

Sugarcane

HT-7

213

Eastern Cape

Mkambati Rd

E 29°88′

S 31°21′

Sugarcane

HT-2

213

Eastern Cape

Mkambati Rd

E 29°88′

S 31°21′

Sugarcane

HT-2

98

Eastern Cape

Agate Terraces

E 29°35′

S 31°36′

Sugarcane

HT-1

neighbour joining (NJ) and maximum parsimony (MP) in PAUPversion4.0b10 (Swofford 1998). Conicofrontia bipartita Hampson (Lepidoptera Noctuidae) was used as an out-group in all the phylogenetic analyses. Maximum parsimony analyses were performed using a heuristic search strategy starting with stepwise addition trees replicated 100 times, with a random input order of sequences to get the initial tree for each replicate. Robustness of MP topologies was assessed by bootstrap analysis with 1000 replicates (tree-bisection–reconnection heuristic search) of 100 random stepwise addition replicates each. Bootstrap values for this analysis were obtained from 1000 replications. Analysis of molecular variance (AMOVA) and the extent of genetic differentiation between populations (FST) were performed with Arlequin 3.1 Software (Schneider et al. 2000).

Results Insect collection and morphological identification Conicofrontia sesamoides specimens collected from wild hosts were cross-checked against type specimens preserved in Museums to avoid coincidence of

synonymies and to provide a valid evidence for morphological identification of the species. After examination of morphological characters of genitalia of both sexes and cross-checking with the description by Hampson (1902) and further re-descriptions by Hampson (1910), Janse (1939), and Tams and Bowden (1953) it was confirmed that the specimens collected on wild hosts were Conicofrontia sesamoides (Fig. 1; Le Ru et al. 2015) These DNA sequences from these were then used as barcodes for identification of sequences of larval specimens collected from sugarcane in Eastern Cape Province. The DNA barcoding system for animal life based upon sequence diversity in cytochrome coxidase subunit 1 (COI) was first proposed by Hebert et al. (2003) and this system was proved to be effective in identification of field collected African cereal stem borers (Assefa et al. 2007). . We did not detect C. sesamoides on sugarcane in KwaZulu-Natal Province, and recorded the insect on sugarcane inonly two of the six sugarcane producing districts (Port St Johns and Flagstaff) of the Eastern Cape Province. In all the sugarcane fields, C. sesamoides larvae were recovered from young ratoons planted in low lying areas of the Indian Ocean coast with a percentage infestation ranging from 5% in Ferry point Rd to 75% in Agate Terraces (Assefa 2015).

Author's personal copy Phytoparasitica Fig. 1 Adults & genitalia of Conicofrontia sesamoides. a, Female, b, Male c, Female genitalia d, Aedeagus e, Male genitalia

Genetic differentiation

Haplotypes

We obtained sequences of 527 base pairs from 16 individuals of C. sesamoides used for molecular analysis. Levels of sequence divergence among the sampled specimens ranged from 0.0–1.54% (Table 2). Sequence divergences between C. sesamoides specimens and the outgroup taxa ranged from 9.97 to 10.98% (Table 2). There was no evidence of isolation by distance, altitude or host associated genetic differentiation observed. Specimens collected from M. capensis in inland Kwazulu-Natal (Oak Hotel and Karkloof 2) were identical to specimens collected from sugarcane in the coastal areas of Eastern Cape (Mkambati Rd) (Table 1). Similarly a specimen collected from M. capensis in Slykraal at an altitude of 348m.a.s.l. shared the same haplotype (HT-3) with a specimen from M. capensis in Karkloof River 1128m.a.s.l. (Table 1). Whereas specimens from high altitude and same host plant (HT 8 and HT-9) were separated by a sequence divergence of 0.77% (Table 2). Specimens from sugarcane in relatively closer distance to each other and within similar altitudinal range (HT-1, HT2 and HT-7) showed a sequence divergence ranging from 0.19 to 0.38% (Table 2).

Ten different haplotypes were identified of which eight were unique. The haplotypes were clustered in one clade with an overall mean distance of 0.58 ± 0.16% and a divergence ranging between 0.19% and 1.54% (Table 2). There was no clear association between geographic distance and genetic divergence between the 16 individuals of C. sesamoides in this study. The highest genetic divergence was recorded between specimens from Minerva Forest 2 (Haplotype 8) and Mtubatuba (Haplotype 4) in Kwazulu-Natal Province which are geographically closer to each other. Contrarily, aspecimen from Mkambati Rd in the coastal Eastern Cape shared the same haplotype with a specimen from Karkloof Forest 2 in the inland Kwazulu-Natal (Table 2). The most common haplotype (Haplotype 2) has six C. sesamoides specimens collected from M. capensis and sugarcane growing in high and low altitude areas of the two Provinces (Fig. 2). This haplotype differed from the second most common haplotype (Haplotype 3), which is represented in three of the sequenced individuals collected from

Author's personal copy Phytoparasitica Table 2 Percentage uncorrected sequence divergence between haplotypes of C. sesamoides HT-1

HT-2

HT-3

HT-4

HT-5

HT-6

HT-7

HT-8

HT-9

Haplotype-2

0.19

–

Haplotype-3

0.38

0.19

–

Haplotype-4

1.15

0.96

1.15

–

Haplotype-5

0.57

0.38

0.19

1.35

–

Haplotype-6

0.38

0.19

0.38

1.15

0.38

–

Haplotype-7

0.38

0.19

0.38

1.15

0.38

0.38

Haplotype-8

0.77

0.57

0.38

1.54

0.77

0.77

0.77

Haplotype-9

0.38

0.19

0.38

1.15

0.38

0.38

0.38

Haplotype-10

0.38

0.19

0.38

1.15

0.57

0.38

0.38

0.77

0.38

10.31

10.64

10.64

10.98

10.98

10.98

10.31

9.97

10.31

C. bipartita

Ht-10

– 0.77 10.64

M. capensis in different altitudinal ranges of the two Provinces, and from the five other haplotypes (Haplotypes 1, 5, 7, 9 and 10) by only one mutational step. One of the unique haplotype (Haplotype 8) collected on sugarcane in Eastern Cape Province differed from the most common haplotype by five mutational steps (Fig. 2).

Phylogenetic analysis

Fig. 2 Haplotype network showing the relationships between the sequences of C. sesamoides individuals from different a) host plants & b) Altitudinal range. Geographic origins, host plants & altitude of the localities from where specimens are collected are

indicated in Table 1. Each circle represents a haplotype & the sizes of the circles are proportional to the number of individuals represented in a particular haplotype. Numbers inside the circles correspond to the haplotype numbers in Tables 1 and 2

Parsimony reconstruction under equal weighting resulted in 25 most parsimonious trees that were 47 steps in length (CI = 0.8723, RI = 0.5000).Of the total 527 characters, 40 were variable and 9 were parsimony informative. The strict consensus of the

Author's personal copy Phytoparasitica Fig. 3 NJ tree showing the relationships among the 10 haplotypes of C. sesamoides. Haplotype numbers are as indicated in Table 1. Conicofrontia bipartita is used as an out-group

25 trees is represented in Fig. 3. Both neighbourjoining and parsimony analyses of the data set gave similar results, although only the neighbour joining tree is presented (Fig. 3). All nodes in Fig. 3 shown are also nodes that were recovered in the strict consensus of 25 trees resultingfrom parsimony analysis. Only one clade with few clusters that were not supported by bootstrap was present. Bootstrap values for all clusters within the clade were less than 50% or very close to zero that they are not shown (Fig. 3). Analysis of Molecular Variance revealed a moderate to highly significant genetic differentiation between C.sesamoides populations from Altitude (FST = 0.159, p = 0.18) and/or host plants (FST = 0.115, p = 0.14). These results were contradictory to the results from haplotype networking (Fig. 2) and the uncorrected genetic distances between geographic populations, altitudinal areas and/or host plants which show little or no genetic differentiation (Table 2).

Discussion Accurate identification and understanding of the genetic diversity of an insect pest are essential to develop and improve monitoring and biological control strategies. Recent investigations indicate that the identified species of noctuid borers in Africa are far fewer than the unknowns (Le Ru et al. 2006b, 2015) mainly because of the acute shortage of taxonomists and very little

attention to research on the diversity of these groups in the natural habitats in the continent. This made the use of molecular phylogenetic approaches in identification of new indigenous borers invading crop fields difficult. Fortunately, identification and clarifying the genetic relationship of C. sesamoides populations in this study was possible because of the valid morphological identification of the wild population by Le Ru et al. (2015) and the availability of COI sequences of the positively identified specimens. Observation on the genitalia of adult specimens from the wild hosts in Kwazulu-Natal province and the comparison of COI sequences of these with the specimens from sugarcane assisted in accurate identification of the new invasion and enabled us compare the genetic relation between the wild and sugarcane populations. Results of this study are of importance to answering C. sesamoides management questions in sugarcane fields. First, assist in understanding the factors responsible for host plant expansion by C. sesamoides and contribute towards preventing this pest from invadingcommercial sugarcane plantations. Furthermore, the quantification of intra and inter-population divergence among C. sesamoidespopulations is pivotal in its management in sugarcane fields. Studies show that highly divergent populations of pest insects can differently damage their hosts (Rugman-Jones et al. 2010), and can respond differentially to control methods (Assefa et al. 2006). Knowledge on geographic or host associated genetic differentiation is, therefore, crucial in evading erroneous employment of control techniques (Mills and Kean 2010).

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Despite the world-wide cultivation of sugarcane, stem borers attacking this crop are mostly indigenous to where the crop is cultivated (Pemberton and Williams 1969; Leslie 2004). Almost all sugarcane stem borers in Africa are indigenous to the continent (Leslie 2004). The evolution of preference for, and enhanced performance on sugarcane by the economically important African stem borer pests was reported several years after the introduction of maize and sugarcane in to the continent (Dick 1945 for Eldana saccharina, Assefa et al. 2010 for Busseola fusca). The host plant expansion by C. sesamoides to include sugarcane as a novel host is, therefore, not an isolated incident that came about through a set of entirely unique ecological circumstances. The incorporation of sugarcane as a novel host by this pest is likely to be a function of several factors. First, cultivation of this crop in small plots surrounded by natural habitats increases the likelihood of being encountered and colonized by native herbivores (Giffard et al. 2012). Second, the periodic burning and mowing of grasslands practiced in Eastern Cape which reduces M. capensis populations may have increased the relative availability of sugarcane and promoted sugarcane colonization and persistence. Third, the similarity in the secondary metabolites of these two plant species due to their close taxonomic relation probably facilitated this host expansion (Birkett et al. 2006). Fourth, the phenotypic plasticity in C. sesamoides may have resulted in rapid adaptation to this new host (Fox et al. 1997). This adaptation to a novel host is reported to result in the formation of host-associated subpopulations (Feder et al. 1988) that may lead to a sympatric speciation (Xue et al. 2016). Genetic divergences among hostassociated populations have evolved in several insects, including fruit flies (Feder et al. 1988), beetles (Xue et al. 2016) and stem borers (Silva-Brandão et al. 2015). Although one would expect adaptive genetic structure and formation of host associated biotypes of C. sesamoides (Mopper 1996), haplotype networking, phylogenetic tree reconstruction and uncorrected genetic distance analyses in this study showed lack of genetic diversification due to host plant use and altitude. Molecular analysis on C. sesamoides specimens, however, indicated that the COI sequences were moderately to highly variable within and between the samples collected on sugarcane and M. capensis from different altitudinal ranges indicating a moderate to high genetic differentiation within and between host and altitudinal groups. This is contradictory to results from

uncorrected genetic distance (Table 1), the haplotype networking (Fig. 2) and phylogenetic tree reconstruction (Fig. 3). The inflation in the FST value in this study might have resulted from the very few samples used in the analysis. Since FST is basically the ratio of two variances, it is difficult to measure it accurately without a large data set (Whitlock and McCauley 1999). For example, individuals from sugarcane and M. capensis shared a haplotype which is only one mutational step different from an individual from unknown host (Fig. 2). The analysis based on uncorrected genetic distance (Table 1), the haplotype networking (Fig. 2) and phylogenetic tree reconstruction (Fig. 3) confirmed that altitudes have no effect on the genetic diversity. Results show that the observed genetic difference is in the limit that is expected within a population of noctuid borers (Assefa et al. 2015; Sezonlin et al. 2006). Population of C. sesamoides in sugarcane is, therefore, likely part of the wild population in the two provinces. There may be two plausible reasons for the lack of host-associated genetic differentiation in C. sesamoides recorded in this study. First, the host range expansion and incorporation of sugarcane in the insect’s diet could be very recent that there is too little isolation for genetic d i v e rg e n c e t o b e d e t e ct e d ( R o u s s et 1 9 9 9 ) . Unfortunately, our understanding of how recent this invasion on sugarcane is challenged due to lack of previous stem borer diversity studies in small scale sugarcane fields (Assefa 2015). However, the results presented here are strikingly similar to the patterns in genetic differentiation observed in the studies conducted on Eldana saccharina (Assefa et al. 2006) and Bussueola fusca (Sezonlin et al. 2006; Assefa et al. 2015), where no host plant associated genetic differentiation were reported. Results of this study and previous reports may indicate that the period of time since the introduction of sugarcane into Africa in 1848 (Osborne 1964) was sufficient for the indigenous stem borers to develop preference for, and have enhanced performance on sugarcane (Dick 1945; Assefa et al. 2010) but not long enough for a detectable genetic differentiation in the COI gene of the mitochondrial DNA (Brower 1994). COI is a conventional marker for genetic study (Velasco‐Cuervo et al. 2016; Yang et al. 2016), but it may be highly conserved compared to other mitochondrial protein coding genes (Dowling et al. 2008) and may not provide full information about the evolutionary process in recently isolated populations. The use of multiple markers and larger number of individuals from

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different localities and host plants may disclose a hidden host/altitude associated genetic differentiation in C. sesamoides populations. The other plausible scenario for the absence of host plant associated genetic differentiation could be the presence of a sufficiently high gene flow between the wild and sugarcane populations of C. sesamoides. Populations of C. sesamoides could be better adapted to one of the two host species, in which the high-quality host plant (the source) produce an excess of insects, whereas the lower quality host plant (the sink) do not produce enough insects, such that populations may not persist without immigration from sources so one functions as a sink and the other acts as the source (Pulliam 1988; Bourguet 2000). If individuals encounter frequently changing selection pressures, migration over large geographical distances and colonisation of areas without primary hosts is likely to occur. Geographic range expansions due to climate change (Battisti et al. 2005) and human-caused habitat expansion (Gutiérrez and Thomas 2000) have been reported in a number of herbivorous insects. Surprisingly C. sesamoides has not yet been reported from commercial plantations despite its presence in wild host plants growing adjacent to the sugarcane fields. Evaluating the effects of agricultural landscape in host range expansion by this pest will increase our understanding on and contributes to the prevention of incursion. Conicofrontia sesamoides is a top borer that prefers to feed on young seedlings. Its potential incursion to the commercial farms, where E. saccharina, is already causing economic damage on old mature sugarcane, couldresult in the emergenceof a species complex attackingdifferent phonological stages, this may resultto devastating consequences. Therefore, the sugarcane plantations are warned of the danger and advised to take relevant measures to prevent incursion into the commercial sugarcane fields. Acknowledgements We express our appreciation to University of Fort Hare for providing the research fund.

References Abrahamson, W. G., Blair, C. P., Eubanks, M. D., & Morehead, S. A. (2003). Sequential radiation of unrelated organisms: the gall fly Eurosta solidaginis and the tumbling flower beetle Mordellistena convicta. Journal of Evolutionary Biology, 16, 781–789.

Assefa, Y. (2015). Potential new pests in the neighbourhood: diversity and abundance of sugarcane stem borers in the pondoland region of the Eastern Cape Province of South Africa. Proceedings of the South African Sugarcane Technologists’ Association, 88, 292–303. Assefa, Y., Conlong, D. E., & Mitchell, A. (2006). P h y l o g e o g r a p h y o f E l d a n a s a c c h a r i n a Wa l k e r (Lepidoptera: Pyralidae). Annales de la Sociètè entomologique de France, 42(3–4), 331–338. Assefa, Y., Conlong, D. E., & Mitchell, A. (2007). DNA identification of Busseola (Lepidoptera: Noctuidae) larvae in Ethiopian sugarcane. African Entomology, 15(2), 375–379. Assefa, Y., Conlong, D. E., van den Berg, J., & Mitchell, A. (2010). Distribution of sugarcane stem borers and their natural enemies in small-scale farmers' fields and adjacent wetlands of Ethiopia. International Journal of Pest Management, 56(3), 233–241. Assefa, Y., Tiroesele, B., Segwagwe, A., & Madisa, M. E. (2014). Lantana camara L. and its biocontrol agent, Teleonemia scrupulosa Stål, in Botswana. African Journal of Ecology, 53(3), 381–384. Assefa, Y., Conlong, D. E., van Den Berg, J., & Martin, L. A. (2015). Ecological genetics and host range expansion by Busseola fusca (Lepidoptera: Noctuidae). Environmental Entomology, 44(4), 1265–1274. doi:10.1093/ee/nvv079. Bandelt, H. J., Forster, P., & Rohl, A. (1999). Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution, 16, 37–48. Battisti, A., Stastny, M., Netherer, S., Robinet, C., Schopf, A., Roques, A., & Larsson, S. (2005). Expansion of geographic range in the pine processionary moth caused by increased winter temperatures. Ecological Applications, 15, 2084– 2096. Berlocher, S. H., & Feder, J. L. (2002). Sympatric speciation in phytophagous insects: moving beyond controversy? Annual Review of Entomology, 47, 773–815. Birkett, M. A., Chamberlain, K., Khan, Z. R., Pickett, J. A., Toshova, T., Wadhams, L. J., & Woodcock, C. M. (2006). Electrophysiological responses of the Lepidopterous stem borers Chilo partellus and Busseola fusca to volatiles from wild and cultivated host plants. Journal of Chemical Ecology, 32, 2475–2487. Bourguet, D. (2000). Host–plant diversity of the European corn borer Ostrinia nubilalis: what value for sustainable transgenic insecticidal Bt maize? Proceedings of the Royal Society B: Biological Sciences, 267(1449), 1177–1184. Brockerhoff, E. G., Liebhold, A. M., & Jactel, H. (2006). The ecology of forest insect invasions and advances in their management. Canadian Journal of Forest Research, 36(2), 263–268. Brower, A. V. Z. (1994). Rapid morphological radiation and convergence among races of the butterfly Heliconius erato inferred from patterns of mitochondrial DNA evolution. Proceedings of the National Academy of Sciences of the United States of America, 91, 6491–6495. Calatayud, P.-A., Le Ru, B., van den Berg, J., & Schulthess, F. (2014). Ecology of the African maize stalk borer, Busseola fusca (Lepidoptera: Noctuidae) with special reference to insect-plant interactions. Insects, 5(3), 539–563. doi:10.3390/insects5030539.

Author's personal copy Phytoparasitica Conlong, D. E. (1997). Biological control of Eldana saccharina Walker in South African sugarcane: constraints identified from 15 years of research. Insect Science and its Applications, 17, 69–78. Conlong, D. E. (2001). Biological control of indigenous African stem borers: what do we Know? Insect Science and its Applications, 21(4), 267–274. Dick, J. (1945). Some data on the biology of the sugarcane borer (Eldana saccharina Wlk.). Proceedings of the South African Sugarcane Technologists’ Association, 19, 75–79. Dowling, D. K., Friberg, U., & Lindell, J. (2008). Evolutionary implications of non-neutral mitochondrial genetic variation. Trends in Ecology and Evolution, 23(10), 546–554. Feder, J. L., Chilcote, C. A., & Bush, G. L. (1988). Genetic differentiation between sympatric host races of the apple maggot fly Rhagoletis pomonella. Nature, 336, 61–64. Fox, C. W., Nilsson, J. A., & Mousseau, T. A. (1997). The ecology of diet expansion in a seed-feeding beetle: Pre-existing variation, rapid adaptation and maternal effects? Evolutionary Ecology, 112, 183–194. Fuller, C. (1901). First Report of the Government Entomologist (pp. 1899–1900). Natal: Pietermaritzburg. Funk, D. J. (1998). Isolating a role for natural selection in speciation: host adaptation and sexual isolation in Neochlamisus bebbianae leaf beetles. Evolution, 52, 1744–1759. Giffard, B., Jactel, H., Corcket, E., & Barbaro, L. (2012). Influence of surrounding vegetation on insect herbivory: A matter of spatial scale and herbivore specialisation. Basic and Applied Ecology, 13(5), 458–465. Gutiérrez, D., & Thomas, C. D. (2000). Marginal range expansion in a host-limited butterfly species Gonepteryxr hamni. Ecological Entomology, 25, 165–170. Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/ NT. (http://www.mbio.ncsu.edu/BioEdit/bioedit.htm/). Hampson, G. F. (1902). The moths of South Africa (Part II). Annals of the South African Museum, 2, 255–446. Hampson, G. F. (1910). Catalogue of the Lepidoptera Phalaenae in the collection of the British Museum (Nat (Hist.). IX. Noctuidae). London: Taylor and Francis. 552 pp. Hanski, I., & Singer, M. (2001). Extinction–colonization dynamics andhost-plant choice in butterfly meta-populations. American Naturalists, 158, 341–353. Harris, K. M., & Nwanze, K. E. (1992). Busseola fusca (Fuller), the African maize stalk borer: a handbook of information. Patancheru: International Crops Research Institute for the Semi-Arid Tropics. Harvey, M. L., Dadour, I. R., & Gaudieri, S. (2003). Mitochondrial DNA cytochrome oxidase I gene: potential for distinction between immature stages of some forensically important fly species (Diptera) in western Australia. Forensic Science International, 131(2), 134–139. Hebert, P. D. N., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B, 270, 313–322. Hurst, G. D., & Jiggins, F. M. (2005). Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts. Proceedings of the Royal Society of London. Series B, Biological sciences Royal Society, 272(1572), 1525–1534.

Hussain, S. I., Khokhar, K. M., Mahmood, T., Laghari, M. H., Mahmud, M. M., Vegetable Crops, H. R. I., & HRI, N. (2001). Yield potential of some exotic and local tomato cultivars grown for summer production. Pakistan Journal of Biological Sciences, 10(4), 1215–1216. Janse, A. J. T. (1939). The Moths of South Africa.Vol. 3.Cymatophoridae, Callidulidae and Noctuidae. Durban: E.P. and Commercial Printing Co. Ltd. 435 pp. Kfir, R., Overholt, W. A., Khan, Z. R., & Polaszek, A. (2002). Biology and management of economically important lepidopteran cereal stem borers in Africa. Annual review of entomology, 47(1), 701–731. Le Ru, B. P., Ong’amo, G. O., Moyal, P., Muchungu, E., Ngala, L., Musyoka, B., et al. (2006a). Major ecological characteristics of East African noctuid stem borers. Annales de la Sociètè entomologique de France, 42, 353–361. Le Ru, B. P., Ong’amo, G. O., Moyal, P., Ngala, L., Musyoka, B., Abdullah, Z., et al. (2006b). Diversity of lepidopteran stem borers on monocotyledonous plants in eastern Africa and the islands of Madagascar and Zanzibar revisited. Bulletin of Entomological Research, 96, 555–563. Le Ru, B. P., Capdevielle-Dulac, C., Conlong, D. E., Pallangyo, B., van Den Berg, J., Ongamo, G. O., & Kergoat, G. J. (2015). A revision of the genus Conicofrontia Hampson (Lepidoptera, Noctuidae, Apameini, Sesamiina), with description of a new species: new insights from morphological, ecological and molecular data. Zootaxa, 3925(1), 056–074. Leslie, G. (2004). Pests of sugarcane. In G. James (Ed.), Sugarcane (2nd ed., pp. 78–100). Oxford/Britain: Blackwell Science. Matama-Kauma, T., Schulthess, F., Le Ru, B., Mueke, J. M., Ogwang, J. A., & Omwega, C. O. (2008). Abundance and diversity of lepidopteran stem borers and their parasitoids on selected wild grasses in Uganda. Crop Protection, 27, 505– 513. Mills, N. J., & Kean, J. M. (2010). Behavioral studies, molecular approaches, and modeling: Methodological contributions to biological control success. Biological Control, 52, 255–262. Moolman, J., van den Berg, J., Conlong, D. E., Cugala, D., Siebert, S., & Le Ru, B. (2014). Species diversity and distribution of lepidopteran stem borers in South Africa and Mozambique. Journal of Applied Entomology, 138, 52–66. Mopper, S. (1996). Adaptive genetic structure in phytophagous insect populations. Trends in Ecology and Evolution, 11, 235–238. Ndemah, R., Schulthess, F., Le Ru, B. P., & Bame, I. (2007). Lepidopteran cereal stem borers and associated natural enemies on maize and wild grass hosts in Cameroon. Journal of Applied Entomology, 131(9–10), 658–668. Nosil, P., Crespi, B. J., & Sandoval, C. P. (2002). Host-plant adaptation drives parallel evolution of reproductive isolation. Nature, 417, 440–443. Onyango, F. O., & Ochieng’Odero, J. P. R. (1994). Continuous rearing of the maize stem borer Busseola fusca on an artificial diet. Entomologia experimentalis et applicata, 73, 139–144. Osborne, R. F. (1964). Valiant harvest (The founding of the South African sugar industry 1848–1926). Durban: South African Sugar Association. Parker, J. D., Burkepile, D. E., Lajeunesse, M. J., & Lind, E. M. (2012). Phylogenetic isolation increases plant success despite

Author's personal copy Phytoparasitica increasing susceptibility to generalist herbivores. Diversity and Distributions, 18, 1–9. Pemberton, C. E., & Williams, J. R. (1969). Distribution, origin and spread of sugarcane insect pests. In J. R. Williams, J. R. Metcalfe, R. W. Mungomery, & R. Mathes (Eds.), Pests of Sugarcane (pp. 1–9). Amsterdam: Elsevier. Polaszek, A. (1998). African Cereal Stem Borers: Economic Importance, Taxonomy, Natural Enemies and Control. Wallingford: CABI. 530 pp. Pons, J., Barraclough, T. G., Gomez-Zurita, J., Cardoso, A., Duran, D. P., Hazell, S., et al. (2006). Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology, 55(4), 595–609. Pulliam, H. R. (1988). Sources, sinks, and population regulation. American Naturalists, 132, 652–661. Rousset, F. (1999). Genetic di¡erentiation within and between two habitats. Genetics, 151, 397–407. Rugman-Jones, P. F., Hoddle, M. S., & Stouthamer, R. (2010). Nuclear-mitochondrial barcoding exposes the global pest Western Flower Thrips (Thysanoptera: Thripidae) as two sympatric cryptic species in its native California. Journal of Economic Entomology, 103, 877–886. Schneider, S. D., Roessli, D., & Excoffier, L. (2000). ARLEQUIN (version 2000): A software for genetic data analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva. Sezonlin, M., Dupas, S., Le Ru, B. P., Le Gall, P., Moyal, P., Calatayud, P. A., Giffard, I., Faure, N., & Silvain, J. F. (2006). Phylogeography and population genetics of the maize stalk borer Busseola fusca (Lepidoptera, Noctuidae) in subSaharan Africa. Molecular Ecology, 15, 407–420. Silva-Brandão, K. L., Santos, T. V., Cônsoli, F. L., & Omoto, C. (2015). Genetic Diversity and Structure of Brazilian Populations of Diatraea saccharalis (Lepidoptera: Crambidae): Implications for Pest Management. Journal of Economic Entomology, 108(1), 307–316. Staden, R. (1996). The Staden sequence analysis package. Molecular Biotechnology, 5, 233–241. Swofford, D. L. (1998). PAUP phylogenetic analysis using parsimony (version 4.0). Sinauer Sunderland, MA. Tajima, F. 1983. Evolutionary relationship of DNA sequences in finite populations. Genetics, 105, 437–460. Sword, G. A., Joern, A., & Senior, L. B. (2005). Host plantassociated genetic differentiation in the snakeweed

grasshopper, Hesperotettix viridis (Orthoptera: Acrididae). Molecular Ecology, 14, 2197–2205. Tams, W. H. T., & Bowden, J. (1953). A revision of the African species of Sesamia guenée and related genera (AgrotidaeLepidoptera). Bulletin of Entomological Research, 43, 645– 678. http://dx.doi.org/10.1017/S0007485300026717. Tan, S. H., Rizman-Idid, M., Mohd-Aris, E., Kurahashi, H., & Mohamed, Z. (2010). DNA-based characterisation and classification of forensically important flesh flies (Diptera: Sa r c o p h a g i d a e ) i n M a l a y s i a . F o re n s i c S c i e n c e International, 199(1), 43–49. Thomas, C. D., & Singer, M. (1987). Variation in host preference affects movement patterns within a butterfly population. Ecology, 68, 1262–1267. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 24, 4876– 4882. Velasco‐Cuervo, S. M., Espinosa, L. L., Duque‐Gamboa, D. N., Castillo‐Cárdenas, M. F., Hernández, L. M., Guzmán, Y. C., & Toro‐Perea, N. (2016). Barcoding, population structure, & demographic history of Prodiplosis longifila associated with the & es. Entomologia Experimentalis Et Applicata, 158(2), 217–227. Way, M. J., & Turner, P. E. T. (1999). The spotted sugarcane borer, Chilo sacchariphagus (Lepidoptera: Pyralidae: Crambinae), in Mozambique. Proceedings of the South African Sugarcane Technologists’ Association, 73, 112–113. Whitlock, M. C., & McCauley, D. E. (1999). Indirect measures of gene flow and migration: FST ≠ 1/(4Nm + 1). Heredity, 82(2), 117–125. Xue, H. J., Wei, J. N., Magalhães, S., Zhang, B., Song, K. Q., Liu, J., & Yang, X. K. (2016). Contact pheromones of 2 sympatric beetles are modified by the host plant & affect mate choice. Behavioral Ecology, arv238. Yang, X., Ye, Q., Xin, T., Zou, Z., & Xia, B. (2016). Population genetic structure of Cheyletus malaccensis (Acari: Cheyletidae) in China based on mitochondrial COI & 12S rRNA genes. Experimental and Applied Acarology, 69(2), 117–128.